Influence of the circadian cycle, sex and production stage on the reference values of parameters related to stress and pathology in porcine saliva.

BACKGROUND: The concentration of biomarkers in saliva could be influenced by several factors not related to the specific condition under analyses, which should be considered for proper clinical interpretation. In the present study, the circadian rhythm of C-reactive protein (CRP), haptoglobin (Hp), Pig-MAP, S100A12, Cu, Zn, Adenosine deaminase (ADA), total protein (TP), total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI), cortisol and α-amylase in saliva of 20 female and 20 male pigs was investigated. Moreover, the influence of sex and production phase (post-weaning, fattening and finishing) on the concentrations of biomarkers in a total of 414 healthy pigs was studied and the reference intervals for all salivary biomarkers were calculated accordingly. RESULTS: All parameters except Pig-MAP, OSI and α-amylase varied significantly along the daytime, and most of them peak around early afternoon (13-15 h). The cosinor analysis described the temporal dynamics of circadian rhythms for all parameters. The range values showed differences between male and female pigs in 8 out of the 13 biomarkers, with higher concentrations in females in comparison to male pigs. The influence of the production phase on the salivary concentrations was observed for all the biomarkers. The highest concentrations were observed for Pig-MAP, S100A12 and α-amylase in post-weaning animals, for TP in growing pigs and for OSI in finishing animals. Most of the sex-influenced biomarkers showed the highest concentrations at growing stages with some exceptions such as ADA or Hp that showed the peak at finishing and post-weaning stages respectively. CONCLUSIONS: It is necessary to establish the optimal daytime for routine saliva sampling to avoid circadian variations and for that end, the time interval between 10:00 a.m. to 12:00 a.m. is highly recommended. The factors sex and production phase influence the concentration of biomarkers and should be considered for proper biomarker interpretation. The reference intervals presented here for each salivary biomarker will help to correctly interpret the results of these analytes and contribute to the use of saliva as a non-invasive sample for the diagnosis and monitoring of the health status of swine farms.

The effects of colostrum consumption and feed restriction during marketing and transportation of male dairy beef calves: Impact on pre-transport nutritional status and on farm recovery.

The aim of this study was to evaluate the effects of colostrum consumption and feed restriction on biomarkers of stress, nutritional and health status, gut functionality, and behavior in male dairy beef calves being marketed and transported. A total of 82 male Holstein calves (42 ± 1.2 kg of body weight and 14 ± 0.9 d of age) were used to study the amount of colostrum given at birth at the dairy farm of origin, the degree of feed restriction suffered at an assembly center simulation (d -4 to d -1), and the effects of a 19 h transportation (d -1). Treatments were as follows: control calves (CTRL; n = 16) were fed 10 L of colostrum at the dairy farm of origin, milk replacer (MR) and concentrate at the assembly center, and were not transported; calves fed high colostrum and milk replacer (HCMR; n = 17) were given 10 L of colostrum at the dairy farm of origin, MR at the assembly center, and transported; calved fed high colostrum and rehydrating solution (HCRS; n = 16) were given 10 L of colostrum at the dairy farm of origin, a rehydrating solution (RS) at the assembly center, and transported; calves fed low colostrum and milk replacer (LCMR; n = 17) were given 2 L of colostrum at the dairy farm of origin, MR at the assembly center, and transported; and calves fed low colostrum and rehydrating solution (LCRS; n = 16) were given 2 L of colostrum at the dairy farm of origin, RS at the assembly center, and transported. Transported calves mimic a 19-h transportation. After transport, all calves were fed 2.5 L of MR twice daily and had ad libitum access to concentrate, straw, and water. Calves' recovery was followed for 7 d. Concentrate intake and health records were collected daily from d -4 until d 7 and body weight (BW) and blood samples were collected on d -4, -1, 0, 1, 2, and 7 of the study. Results showed that the feeding regimen provided at the assembly center reduced BW for the HCRS and LCRS calves compared with the CTRL, HCMR, and LCMR calves. Concentrate intake peaked on d 0 in the transported calves, followed by a reduction of intake on d 1 after transportation. Concentrate intake recovery was lower for the LCRS and LCMR calves. On d -1, nonesterified fatty acids and ß-hydroxybutyrate concentrations were greater for the HCRS and LCRS calves compared with the CTRL, HCMR, and HCRS calves. After transportation, serum Cr-EDTA concentration was greater for the HCRS and LCRS calves than the HCMR, LCMR, and CTRL calves. The LCRS calves had the lowest serum concentration of citrulline. Finally, health scores were greater for the LCRS calves from d 0 to 7. In summary, both the greatest degree of feed restriction during the assembly center and the low colostrum consumption at birth negatively affected the recovery of concentrate consumption and BW, gut functionality, health status, and behavior in calves after arrival at the rearing farm.

Evaluation of potential biomarkers to determine adequate colostrum provision in male dairy-beef calves upon arrival at the rearing facility beyond 14 days of age.

Colostrum consumption is crucial for passive immunization and development of the newborn calf. However, the incidence on failed transfer of passive immunity in male calves destined to dairy-beef production remains high to date. In addition, the lack of an automated procedure to validate the immunization status upon arrival at rearing facilities in calves beyond 14 d of age impedes the identification of failed transfer of passive immunity, and therefore, of those calves at high risk of suffering diseases. For this study, 82 newborn male Holstein calves (43.3 ± 0.86 kg of body weight; mean ± standard error) from a commercial dairy farm were used to investigate potential serum biomarkers of colostrum provision. The potential biomarkers selected were IgG, IgG1, cholesterol, alkaline phosphatase, gamma-glutamyl transferase (GGT), and total protein (TP). Treatments were as follows: high-colostrum (HC; n = 49), in which calves received 4 L of colostrum within the first 2 h after birth and 2 L of colostrum in the next 3 feedings within the first 24 h after birth, for a total of 10 L of colostrum; and low-colostrum (LC; n = 33), in which calves received only 2 L of colostrum within the first 2 h after birth. After colostrum consumption, calves were allocated to individual hutches and fed 2 L of milk replacer twice daily at a concentration of 125 g/L as fed. Starter feed and water were offered ad libitum. At approximately 14 d of age (14.2 ± 0.81 d of age; mean ± standard error) calves were transported 2.5 h to a research unit at IRTA (Torre Marimon, Spain) simulating the arrival to a rearing facility. Blood samples were collected before feeding at birth, 48 h after birth, and at arrival to the rearing facility. Results on the serum concentrations of the potential biomarkers at arrival to the rearing facility showed that IgG, IgG1, GGT, and TP were greater for the HC calves compared with the LC calves. Serum concentrations of cholesterol and alkaline phosphatase did not show differences between treatment groups. Additionally, body weight losses from birth until arrival to the rearing facility were greater for the LC treatment compared with the HC. Because of their low cost, quickness, and ease of measurement, GGT and TP were good indicators of colostrum intake in calves arriving at rearing facilities beyond 14 d of age.

Plasmatic B-esterases as potential biomarkers of exposure to marine plastics in loggerhead turtles.

Sea turtles are particularly vulnerable to plastic exposures, and the associated chemical additives, due to their feeding strategies. The species Caretta caretta is a proposed sentinel of plastic pollution worldwide. Thus, there is a need to find adequate biomarkers of plastic exposure through non-invasive protocols for this IUCN protected species. Plasmatic acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and carboxylesterase (CE) which participate in xenobiotic and endogenous metabolic reactions could all serve as biomarkers, as they are responsive to plasticizers and have already proved adequate for identifying organophosphorus esters exposures. Here we measured plasmatic B-esterases in wild specimens captured as accidental by-catch. Measurements were taken in each individual either at entry into the rehabilitation program or immediately before release after a recovery period. For CE measurements, 4 commercial substrates were used as potentially indicative of distinct enzyme isoforms. Increased activity was seen with the butyrate-derived substrates. Plasmatic CE activities were over one order of magnitude higher than AChE and BuChE substrates. Moreover, an in vitro protocol with the inclusion of plastic additives such as tetrabromobisphenol A (TBBPA), bisphenol A and some of its analogues was considered a proxy of enzymatic interactions. A clear inhibition by TBBPA was found when using commercially purified AChE and recombinant CE proteins. Overall, from in vitro and in vivo evidences, CEs in plasma are sensitive and easily measurable and have been shown to significantly increase after turtles have been rehabilitated in rescue centres. Nevertheless, the inclusion of plastic (or plasticizers) characterisation would help to confirm its association with plasmatic enzyme modifications before they can be adopted as biomarkers of plastic contamination.

Using compositional mixed-effects models to evaluate responses to amino acid supplementation in milk replacers for calves.

The consequences of supplementing Lys, Met, and Thr in milk replacers (MR) for calves have been widely studied, but scarce information exists about potential roles of other AA (whether essential or not). The effects on growth performance of supplementation of 4 different AA combinations in a mixed ration (25.4% crude protein and 20.3% fat) based on skim milk powder and whey protein concentrate were evaluated in 76 Holstein male calves (3 ± 1.7 d old). The 4 MR were as follows: CTRL with no AA supplementation; PG, supplying additional 0.3% Pro and 0.1% Gly; FY, supplying additional 0.2% Phe and 0.2% Tyr; and KMT, providing additional 0.62% Lys, 0.22% Met, and 0.61% Thr. All calves were fed the same milk allowance program and were weaned at 56 d of study. Concentrate intake was limited to minimize interference of potential differences in solid feed intake among treatments. Animals were weighed weekly, intakes recorded daily, and blood samples obtained at 2, 5, and 7 wk of study to determine serum urea and plasma AA concentrations. Plasma AA concentrations were explored using compositional data analysis, and their isometric log-ratio transformations were used to analyze their potential influence on ADG and serum urea concentration using a linear mixed-effects model. We detected no differences in calf performance and feed intake. Plasma relative concentration of the AA supplemented in the KMT and PG treatments increased in their respective treatments, and, in PG calves, a slight increase in the proportion of plasma Gly, Glu, and branched-chain AA was also observed. The proportions of plasma branched-chain AA, His, and Gln increased, and those of Thr, Arg, Lys, and Glu decreased with calves' age. A specific log-contrast balance formed by Arg, Thr, and Lys was found to be the main driver for lowering serum urea concentrations and increasing calf growth. The use of compositional mixed-effects models identified a cluster formed by the combination of Arg, Thr, and Lys, as a potential AA to optimize calf growth.

Effects of a high-fat-diet supplemented with probiotics and ω3-fatty acids on appetite regulatory neuropeptides and neurotransmitters in a pig model.

The pig is a valuable animal model to study obesity in humans due to the physiological similarity between humans and pigs in terms of digestive and associated metabolic processes. The dietary use of vegetal protein, probiotics and omega-3 fatty acids is recommended to control weight gain and to fight obesity-associated metabolic disorders. Likewise, there are recent reports on their beneficial effects on brain functions. The hypothalamus is the central part of the brain that regulates food intake by means of the production of food intake-regulatory hypothalamic neuropeptides, as neuropeptide Y (NPY), orexin A and pro-opiomelanocortin (POMC), and neurotransmitters, such as dopamine and serotonin. Other mesolimbic areas, such as the hippocampus, are also involved in the control of food intake. In this study, the effect of a high fat diet (HFD) alone or supplemented with these additives on brain neuropeptides and neurotransmitters was assessed in forty-three young pigs fed for 10 weeks with a control diet (T1), a high fat diet (HFD, T2), and HFD with vegetal protein supplemented with Bifidobacterium breve CECT8242 alone (T3) or in combination with omega-3 fatty acids (T4). A HFD provoked changes in regulatory neuropeptides and 3,4-dihydroxyphenylacetic acid (DOPAC) in the hypothalamus and alterations mostly in the dopaminergic system in the ventral hippocampus. Supplementation of the HFD with B. breve CECT8242, especially in combination with omega-3 fatty acids, was able to partially reverse the effects of HFD. Correlations between productive and neurochemical parameters supported these findings. These results confirm that pigs are an appropriate animal model alternative to rodents for the study of the effects of HFD on weight gain and obesity. Furthermore, they indicate the potential benefits of probiotics and omega-3 fatty acids on brain function.

Evaluating the potential role of tryptophan in calf milk replacers to facilitate weaning.

Tryptophan is a precursor of serotonin, a neurotransmitter that participates in the control of the affective state of an animal. We hypothesized that Trp supplementation could help dairy calves to cope with weaning stress. Twenty-seven Holstein male calves (48 ± 0.8 d old; 82 ± 2.6 kg of body weight) were used to evaluate the effects of Trp supplementation at a rate of 4.5 g/d via milk replacer (MR) on performance and behavioral parameters around weaning. All calves received the same feeding program (6 L/d at 15% dry matter from d 1 to 7, 4 L/d at 15% dry matter from d 8 to 14, and 2 L/d at 15% dry matter in one feeding until d 21 of study) and were completely weaned 22 d after the beginning of the study (around 70 d of life). Calves were fed a starter feed (19.3% crude protein and 16.2% neutral detergent fiber, on a dry matter basis) and chopped straw ad libitum. Animals were weighed weekly, dry matter intakes were recorded daily, lying behavior was recorded using accelerometers throughout the study, and scan sampling was performed twice a week, 1 h after the morning feeding, to record behavioral activity (nonnutritive oral behaviors, suckling a neighbor calf, standing, resting, rumination, vocalizations, eating, and drinking). Tryptophan supplementation did not affect calf performance or concentrate and MR intake, but straw intake tended to be greater in nonsupplemented compared with Trp-supplemented calves (153 vs. 129 ± 9.0 g/d, respectively). Lying time, lying bouts, and lying duration decreased when changes in the MR feeding program occurred, independent of treatment. Similarly, differences in behavioral observations occurred along days of study, with no effect of Trp supplementation. The main changes observed in calf behavior were an increase in vocalizations and standing time 1 h after the morning feeding at weaning, but again these changes were independent of treatment. Parameters measured in serum and plasma indicated an increase in Trp, kynurenine, and the kynurenine/Trp ratio after feeding in the Trp calves. A tendency for lower plasma glucose concentration after feeding was observed in the Trp group. No changes in stress markers such as cortisol and haptoglobin in serum were detected. In conclusion, supplementing 4.5 g/d of Trp via MR between 48 and 62 d of life had no effect on performance or behavior in calves around weaning.

Short communication: Performance, intestinal permeability, and metabolic profile of calves fed a milk replacer supplemented with glutamic acid.

The objective of this study was to evaluate the potential benefits of supplementing glutamic acid in milk replacers (MR) with respect to calf performance, intestinal permeability, and metabolism. Sixty Holstein male calves (3 ± 1.3 d old and 45 ± 5.9 kg body weight) were individually housed and fed a control MR without AA supplementation (24.8% crude protein and 19.1% fat, dry matter basis), or MR supplemented with 0.3% glutamic acid (25.1% crude protein and 20.3% fat, dry matter basis). Animals followed the same MR feeding program and were weaned at 56 d of the study. The amount of starter concentrate offered was restricted to limit the effect of concentrate intake on calf metabolism. Individual daily consumption and weekly body weight were measured, and 4 h after the morning feeding, blood samples were obtained at 14 and 35 d to determine general biochemical parameters and plasma AA concentrations. On d 10 of the study, we conducted an intestinal permeability test by including 21 g of lactulose and 4.2 g of d-mannitol as markers in the MR. We found no differences in calf performance or in intestinal permeability (measured as lactulose:mannitol ratio). Serum glucose concentration was greater in unsupplemented calves than in Glu-supplemented calves. At 14 d, the proportion of plasma Leu was greater in Glu-supplemented calves; the proportion of Ile tended to be greater in Glu-supplemented calves; and the proportion of Met tended to be greater in unsupplemented calves. We observed no other differences. Small changes occurred in AA metabolism when supplementing calf MR with 0.3% glutamic acid, without leading to improvements in calf performance or changes in intestinal permeability.

Pig cognitive bias affects the conversion of muscle into meat by antioxidant and autophagy mechanisms.

Slaughter is a crucial step in the meat production chain that could induce psychological stress on each animal, resulting in a physiological response that can differ among individuals. The aim of this study was to investigate the relationship between an animal's emotional state, the subsequent psychological stress at slaughter and the cellular damage as an effect. In all, 36 entire male pigs were reared at an experimental farm and a cognitive bias test was used to classify them into positive bias (PB) or negative bias (NB) groups depending on their decision-making capabilities. Half of the animals, slaughtered in the same batch, were used for a complete study of biomarkers of stress, including brain neurotransmitters and some muscle biomarkers of oxidative stress. After slaughter, specific brain areas were excised and the levels of catecholamines (noradrenaline (NA) and dopamine (DA)) and indoleamines (5-hydroxyindoleacetic acid and serotonin (5HT)) were analyzed. In addition, muscle proteasome activity (20S), antioxidant defence (total antioxidant activity (TAA)), oxidative damage (lipid peroxidation (LPO)) and autophagy biomarkers (Beclin-1, microtubule-associated protein I light chain 3 (LC3-I) and LC3-II) were monitored during early postmortem maturation (0 to 24 h). Compared with PB animals, NB pigs were more susceptible to stress, showing higher 5HT levels (P<0.01) in the hippocampus and lower DA (P<0.001) in the pre-frontal cortex. Furthermore, NB pigs had more intense proteolytic processes and triggered primary muscle cell survival mechanisms immediately after slaughter (0 h postmortem), thus showing higher TAA (P<0.001) and earlier proteasome activity (P<0.001) and autophagy (Beclin-1, P<0.05; LC3-II/LC3-I, P<0.001) than PB pigs, in order to counteract the induced increase in oxidative stress, that was significantly higher in the muscle of NB pigs at 0 h postmortem (LPO, P<0.001). Our study is the first to demonstrate that pig's cognitive bias influences the animal's susceptibility to stress and has important effects on the postmortem muscle metabolism, particularly on the cell antioxidant defences and the autophagy onset. These results expand the current knowledge regarding biomarkers of animal welfare and highlight the potential use of biomarkers of the proteasome, the autophagy (Beclin-1, LC3-II/LC3-I ratio) and the muscle antioxidant defence (TAA, LPO) for detection of peri-slaughter stress.

Fetal growth-retardation and brain-sparing by malnutrition are associated to changes in neurotransmitters profile.

The present study assesses possible changes in the levels of different neurotransmitters (catecholamines and indoleamines) in fetuses affected by nutrient shortage. Hence, we determined the concentration of catecholamines and indoleamines at the hypothalamus of 56 swine fetuses obtained at both 70 and 90days of pregnancy (n=33 and 23 fetuses, respectively). The degree of fetal development and the fetal sex affected the neurotransmitters profile at both stages. At Day 70, there were found higher mean concentrations of l-DOPA in both female and male fetuses with severe IUGR; male fetuses with severe IUGR also showed higher concentrations of TRP than normal male littermates. At Day 90 of pregnancy, the differences between sexes were more evident. There were no significant effects from either severe IUGR on the neurotransmitter profile in male fetuses. However, in the females, a lower body-weight was related to lower concentrations of l-DOPA and TRP and those female fetuses affected by severe IUGR evidenced lower HVA concentration. In conclusion, the fetal synthesis and use of neurotransmitters increase with time of pregnancy but, in case of IUGR, both catecholamines and indoleamines pathways are affected by sex-related effects.

Sex and intrauterine growth restriction modify brain neurotransmitters profile of newborn piglets.

The current study aimed to determine, using a swine model of intrauterine growth restriction (IUGR), whether short- and long-term neurological deficiencies and interactive dysfunctions of Low Birth-Weight (LBW) offspring might be related to altered pattern of neurotransmitters. Hence, we compared the quantities of different neurotransmitters (catecholamines and indoleamines), which were determined by HPLC, at brain structures related to the limbic system (hippocampus and amygdala) in 14 LBW and 10 Normal Body-Weight (NBW) newborn piglets. The results showed, firstly, significant effects of sex on the NBW newborns, with females having higher dopamine (DA) concentrations than males. The IUGR processes affected DA metabolism, with LBW piglets having lower concentrations of noradrenaline at the hippocampus and higher concentrations of the DA metabolites, homovanillic acid (HVA), at both the hippocampus and the amygdala than NBW neonates. The effects of IUGR were modulated by sex; there were no significant differences between LBW and NBW females, but LBW males had higher HVA concentration at the amygdala and higher concentration of 5-hydroxyindoleacetic acid, the serotonin metabolite, at the hippocampus than NBW males. In conclusion, the present study shows that IUGR is mainly related to changes, modulated by sex, in the concentrations of catecholamine neurotransmitters, which are related to adaptation to physical activity and to essential cognitive functions such as learning, memory, reward-motivated behavior and stress.

Proteomics and the search for welfare and stress biomarkers in animal production in the one-health context.

Stress and welfare are important factors in animal production in the context of growing production optimization and scrutiny by the general public. In a context in which animal and human health are intertwined aspects of the one-health concept it is of utmost importance to define the markers of stress and welfare. These are important tools for producers, retailers, regulatory agents and ultimately consumers to effectively monitor and assess the welfare state of production animals. Proteomics is the science that studies the proteins existing in a given tissue or fluid. In this review we address this topic by showing clear examples where proteomics has been used to study stress-induced changes at various levels. We adopt a multi-species (cattle, swine, small ruminants, poultry, fish and shellfish) approach under the effect of various stress inducers (handling, transport, management, nutritional, thermal and exposure to pollutants) clearly demonstrating how proteomics and systems biology are key elements to the study of stress and welfare in farm animals and powerful tools for animal welfare, health and productivity.

C-reactive protein, haptoglobin and Pig-Major acute phase protein profiles of pigs infected experimentally by different isolates of porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) is the etiologic agent of PRRS, one of the most important diseases in swine worldwide. In the present work, the effects of different PRRSV strains were tested on a piglet experimental model to study the induced acute phase response. For this purpose, pigs (n=15 for each group) were intranasally inoculated with one of five PRRSV strains (isolates EU10, 12, 17, 18 from genotype 1 and isolate JA-142 from genotype 2). The acute phase response was monitored by measuring acute phase proteins (APPs). Specifically, the serum concentration of haptoglobin (Hp), C-reactive protein (CRP) and Pig-Major Acute Protein (Pig-MAP) was determined at 0, 3, 6, 9, 12, 15, 18 and 21 days p.i. Clinical signs and growth performance were also monitored during the experiment. All animals became viremic after inoculation during the study period. The APP response was heterogeneous and dependent on the strain, being strains EU10, EU 18 and JA-142 those that induced the highest response and the strongest clinical signs. In general, Hp was the most sensitive biomarker for PRRSV infection, CRP behaved as moderate and Pig-MAP was the less responsive during the course of PRRSV experimental infection. Hp and CRP were significantly discriminatory between infected and control pigs, but not Pig-MAP.

Animal board invited review: advances in proteomics for animal and food sciences.

Animal production and health (APH) is an important sector in the world economy, representing a large proportion of the budget of all member states in the European Union and in other continents. APH is a highly competitive sector with a strong emphasis on innovation and, albeit with country to country variations, on scientific research. Proteomics (the study of all proteins present in a given tissue or fluid - i.e. the proteome) has an enormous potential when applied to APH. Nevertheless, for a variety of reasons and in contrast to disciplines such as plant sciences or human biomedicine, such potential is only now being tapped. To counter such limited usage, 6 years ago we created a consortium dedicated to the applications of Proteomics to APH, specifically in the form of a Cooperation in Science and Technology (COST) Action, termed FA1002--Proteomics in Farm Animals: www.cost-faproteomics.org. In 4 years, the consortium quickly enlarged to a total of 31 countries in Europe, as well as Israel, Argentina, Australia and New Zealand. This article has a triple purpose. First, we aim to provide clear examples on the applications and benefits of the use of proteomics in all aspects related to APH. Second, we provide insights and possibilities on the new trends and objectives for APH proteomics applications and technologies for the years to come. Finally, we provide an overview and balance of the major activities and accomplishments of the COST Action on Farm Animal Proteomics. These include activities such as the organization of seminars, workshops and major scientific conferences, organization of summer schools, financing Short-Term Scientific Missions (STSMs) and the generation of scientific literature. Overall, the Action has attained all of the proposed objectives and has made considerable difference by putting proteomics on the global map for animal and veterinary researchers in general and by contributing significantly to reduce the East-West and North-South gaps existing in the European farm animal research. Future activities of significance in the field of scientific research, involving members of the action, as well as others, will likely be established in the future.

Immunohistochemical investigation of extracellular matrix components in the lymphoid organs of healthy pigs and pigs with systemic disease caused by circovirus type 2.

The extracellular matrix (ECM) provides a scaffold for cell growth, impacts on cellular behaviour and plays an important role in pathological conditions. Several components of the ECM of lymphoid tissues have been shown to be crucial in the maturation, differentiation and migration of lymphocytes and other immune cells and, therefore, in the development of immune responses. Little is known of the composition and function of the ECM in porcine lymphoid tissues. The present study characterizes immunohistochemically the expression of several ECM-related molecules (i.e. hyaluronan [HA] and its receptor CD44, tenascin-C [TN-C] and versican) in primary and secondary lymphoid organs of healthy pigs and animals affected by porcine circovirus type 2-systemic disease (PCV2-SD). These ECM molecules displayed a highly defined expression pattern in healthy animals, suggesting that they may have a role in the compartmentalization of immune cells within lymphoid tissues. HA was abundant in the medulla of the thymus and follicles of secondary organs; CD44 and TN-C were present in the thymic medulla and parafollicular areas of secondary lymphoid organs; however, there was minimal expression of versican in healthy tissues. In PCV2-SD-affected animals, HA and CD44 showed a similar but more diffuse distribution. TN-C was increased in the T-cell-dependent areas and in tonsillar crypts, and versican was more abundantly expressed, with expression restricted to vascular structures and trabeculae and also surrounding tonsillar crypts. The altered expression in PCV2-SD-affected pigs was most probably related to a higher content of connective tissue secondary to tissue destruction and remodelling attempts as part of the disease process.

Effects of trailer design on animal welfare parameters and carcass and meat quality of three Pietrain crosses being transported over a long distance.

This study aimed at evaluating the effects of trailer design on stress responses and meat quality traits of 3 different pig crosses: 50% Pietrain breeding with halothane (HAL)(Nn) (50Nn); 50% Pietrain breeding with HAL(NN) (50NN); and 25% Pietrain breeding with HAL(NN) genotype (25NN). Over a 6-wk period, pigs (120 pigs/crossbreed) were transported for 7 h in either a pot-belly (PB) or flat-deck (FD) trailer (10 pigs/crossbreed(-1)·trailer(-1)·wk(-1)). Temperature (T) and relative humidity (RH) were monitored in each trailer. Behaviors during loading and unloading, time to load and unload, and latency to rest in lairage were recorded, whereas a sub-population of pigs (4 pigs/crossbreed(-1)·trailer(-1)·wk(-1)) was equipped with gastro-intestinal tract (GIT) temperature monitors. Blood samples were collected at exsanguination for measurement of cortisol, creatine kinase (CK), lactate, haptoglobin, and Pig-MAP concentrations. Meat quality data were collected at 24 h postmortem from the LM and semimembranosus (SM) and adductor (AD) muscles of all 360 pigs. Greater T were recorded in the PB trailer during transportation (P = 0.006) and unloading (P < 0.001). Delta GIT temperature was greater (P = 0.01) in pigs unloaded from the PB. At loading, pigs tended to move backwards more (P = 0.06) when loaded on the FD than the PB trailer. At unloading, an interaction was found between trailer type and crossbreed type, with a greater (P < 0.01) frequency of overlaps in 50NN and 25NN pigs and slips/falls in 50Nn and 50NN pigs from the FD than the PB trailer. Cortisol concentrations at slaughter were greater (P = 0.02) in pigs transported in the PB than FD trailer. Greater lactate concentrations were found in 50Nn and 50NN pigs (P = 0.003) and greater CK concentrations (P < 0.001) in 50Nn pigs. As expected, 50Nn pigs produced leaner (P < 0.001) carcasses, with greater (P = 0.01) dressing percentages, as well as lower (P < 0.001) ultimate pH values and greater (P < 0.001) drip loss percentages in the LM and greater (P = 0.002) drip losses and a paler color (greater L* values, P = 0.02) in the SM than 50NN pigs. When used for long distance transportation under controlled conditions, the PB trailer produced no detrimental effects on animal welfare or pork quality. Pigs with 50% Pietrain crossbreeding appear to be more responsive to transport stress, having the potential to produce acceptable carcass and pork quality, provided pigs are free of the HAL gene.

[Safety and efficacy of a new preservative-free levocabastine ophthalmic solution (Levofree®) using the conjunctival provocation test]. / Efficacité et tolérance d'une nouvelle formulation oculaire de lévocabastine sans conservateur (Levofree®) évaluées lors d'un test de provocation conjonctivale.

OBJECTIVES: To evaluate the safety and efficacy of preservative-free levocabastine 0.05 % ophthalmic solution compared to placebo (vehicle) and to preserved levocabastine 0.05 % ophthalmic suspension in the prevention of allergic conjunctivitis induced by a conjunctival provocation test. METHODS: Ninety-two subjects (18-50 years) with a previous history of allergic conjunctivitis to pollen were randomised to receive either preservative-free levocabastine solution in one eye and preserved levocabastine suspension in the fellow eye (n=69), or preservative-free levocabastine in one eye and placebo in the fellow eye (n=23). One drop of each product was administered 10 minutes (visit 3) and 4 hours (visit 4) prior to the provocation test. The primary efficacy criterion was the sum of the itching and conjunctival hyperemia scores assessed at 3, 5 and 10 minutes after the provocation test. The safety evaluation included adverse events, visual acuity, intra-ocular pressure and study drug drop sensation. RESULTS: The efficacy of the preservative-free solution was significantly higher than that of placebo at all time points (P≤0.01) with one exception at visit 4 (3 minutes after the provocation test). It was significantly higher than that of the preserved suspension at visit 3, and equivalent at visit 4. The incidence of adverse events was lower with the preservative-free solution than with the preserved suspension. 94.2 % and 95.7 % subjects rated preservative-free levocabastine drop sensation as "good" or "very good" at visits 3 and 4 respectively, whereas these rates were 68.1 % and 63.8 % with preserved levocabastine. This difference between the two formulations was highly statistically significant (P<0.001). CONCLUSIONS: The efficacy of preservative-free levocabastine was superior to that of the placebo and of the preserved suspension at visit 3, at least as effective as the preserved suspension at visit 4, and better tolerated at each visit.

Identification of serum stress biomarkers in pigs housed at different stocking densities.

Eight Duroc×(Landrace×Large White) male pigs housed at a stocking rate of 0.50m(2)/pig were subjected to a higher stocking rate of 0.25m(2)/pig (higher density, HD) for two 4-day periods over 26 days. Using biochemical and proteomic techniques serum and plasma samples were examined to identify potential biomarkers for monitoring stress due to HD housing. HD housed pigs showed significant differences (P<0.001) in total cholesterol and low density lipoprotein-associated cholesterol, as well as in concentrations of the pig-major acute phase protein (Pig-MAP) (P=0.002). No differences were observed in serum cortisol or other acute phase proteins such as haptoglobin, C-reactive protein or apolipoprotein A-I. HD-individuals also showed an imbalance in redox homeostasis, detected as an increase in the level of oxidized proteins measured as the total plasma carbonyl protein content (P<0.001) with a compensatory increase in the activity of the antioxidant enzyme glutathione peroxidase (P=0.012). Comparison of the serum proteome yielded a new potential stress biomarker, identified as actin by mass spectrometry. Cluster analysis of the results indicated that individuals segregated into two groups, with different response patterns, suggesting that the stress response depended on individual susceptibility.

Haptoglobin serum concentration is a suitable biomarker to assess the efficacy of a feed additive in pigs.

Levels of haptoglobin and Pig-major acute phase protein (MAP) were analysed in animals from a commercial herd receiving or not a diet enriched with an additive. The group receiving the additive exhibited a decrease in haptoglobin after 3 weeks, suggesting that a better health status has been established, together with an improvement in total body weight and average daily gain. In contrast, Pig-MAP does not significantly change under these conditions. Aujeszky live modified vaccination, which is compulsory in Spain, did cause a significant increment in haptoglobin serum concentration although it did not affect Pig-MAP. The response of acute phase proteins to vaccination was similar in both control and additive-treated groups. Interleukins (IL)-1ß and IL-6 was below the detection limits in most of the animals. In conclusion, this study shows that haptoglobin serum concentration, but not Pig-MAP, is a good biomarker to monitorize production parameters and for monitoring Aujeszky modified live vaccine in pigs reared under standard commercial conditions.