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.

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.

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.

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.

Study of the stability of creatine kinase in control materials.

We examined the stability of twelve control materials from different suppliers containing creatine kinase. The stability was assessed storing preparations at 4 degrees C, 27 degrees C and 37 degrees C and periodically measuring creatine kinase catalytic concentration. The activity was significantly greater when the lyophilized material was reconstituted with water at 4 degrees C as compared to 27 degrees C and 37 degrees C. All preparations tested showed good stability at 4 degrees C over a 72 hour period. We considered the suitability of tested control materials for quality-control purposes.

Comparison of kits for the determination of creatine kinase activity in serum.

Thirteen kits from different suppliers for measurement of creatine kinase activity in human serum according to the IFCC recommendations were analyzed and compared. Concentrations of AMP, ADP, creatine phosphate, glucose, magnesium ion, NADP+, glucose-6-phosphate dehydrogenase, hexokinase and pH were measured in the reagents by various analytical techniques and compared with those recommended b the IFCC. We also compared by regression analysis the results of creatine kinase catalytic concentration obtained in human sera using commercial kits and in-house prepared reagents according tot he IFCC recommendation. Creatine kinase was also measured in a reference material using the different reagents. The overall results of the activity measurements and the composition of the majority of the kits agree well with one another and with the IFCC recommendation. Minor deviations were found in the evaluation of a few kits. One kit yielded creatine kinase activity values that were 17% lower. Results obtained in the reference material measurements showed differences with some kits which were not found using human sera.