All parts of the experiment including the evaluation were performed in a double-blinded manner. This is not a randomized study. Clinical, hematological, and clinical chemistry examinations of all animals were performed in a blinded fashion. After completion of all examinations, associations between clinical parameters and hematological/clinical chemistry parameters were examined.
Animals and experimental design
The animal experiment was carried out in the conventional stables of the State Institution for Swine Breeding (Landesanstalt für Schweinezucht, LSZ) Baden-Wurttemburg in Boxberg, Germany. The experiments were performed with offspring of 40 Baden-Wurttemburg Genetics hybrid sows artificially inseminated with Pietrain semen. Sows were not randomly selected. Instead, two groups of sows, one with the least and one with the most severe alterations in claws and teats, were selected and the experiment was carried out in two successive repetitions with different husbandry conditions. Because sows’ quality and husbandry are not the topic of the present paper, more information about sow selection and husbandry can be found in Reiner et al. [2].
A total of 360 pigs were used in the experiment. Nine offspring per sow from the 40 sows were successively examined in three age groups: Three piglets per sow as suckling piglets on the third day of life, three others per sow as weanlings 11 days after weaning and the remaining three piglets per sow as fattening pigs during slaughter. All non-slaughtered pigs were euthanized immediately after the clinical examination and sampled for further examination [2].
Clinical scoring
Piglets were clinically scored for SINS on the third day of life as described by Reiner et al. [2]. Due to time constraints and to minimize the animal load, clinical signs were recorded using a digital camera (Lumix, Panasonic Corporation) according to a standardized scheme for later detailed evaluation of the images (Windows Media Player, Version 12, Microsoft GmbH, Germany). The tail base and tip, ears, teats and navel, coronary bands, wall horn, balls and soles of the feet along with the face were all initially assessed individually. The following clinical characteristics were considered and scored with 0 if the sign was not visible or 1 if the sign was visible. The tail base was screened for the presence of bristles (0 = present; 1 = absent), swelling of the tail base (0/1), redness of the tail base (0/1), exudation (0/1) and clinical signs of necrosis (0/1). The tail, including the whole tail except the tail base was scored for swelling (0/1), scab formation (0/1), rhagades (0/1), exudation (0/1), necrosis (0/1), bleeding (0/1) and ring lacings (0/1). Ears were scored for the presence of bristles (0 = present; 1 = absent), ear vein combustion (0/1) and necrosis of the ears (0/1). The face was scored for lid oedema (0/1) and nasal oedema (0/1). Teats were scored for scab formation (0/1), swelling (0/1), redness (0/1), necrosis (0/1) and venous combustion (0/1). Each claw was individually scored for redness of coronary band (0/1), exudation of coronary band (0/1) and necrosis of coronary band (0/1), for wall bulging (0/1) and wall bleeding (0/1), for sole redness (0/1) and detachment of sole from heel (0/1), and for swelling (0/1), redness (0/1) and detachment of heels (0/1). In the end, mean scores of the eight claws, resp. 4 ft were used for the calculation of the organ scores. For the other body parts, all detailed scores were added to produce organ scores. All organ scores were unweighted summed up for the SINS-Score. SINS-Scores between 0 and 31 were theoretically possible.
Sample collection
After the clinical scoring, the piglets were sampled by collection of 4.5 ml serum (Monovette Serum 4.5 ml, Sarstedt AG), 2.5 ml citrate whole blood (S-Monovette® 2.9 ml 9NC, Citrate, Sarstedt AG) and 2.5 ml EDTA whole blood (S-Monovette® 2.7 ml K3E, EDTA, Sarstedt AG) from the cranial vena cava using a 1.2 × 4.0 mm cannula (Agani, Terumo Corporation).
Sample processing and examination
The collected EDTA whole blood was processed approx. 4–6 h after collection. First, 1000 μl were pipetted into EDTA sample tubes (Sarstedt AG, Germany) using a volumetric pipette (Eppendorf AG, Germany). The haematological examination was performed with a VetScan HM5 (Abaxis Europe GmbH) using a VetScan HM5 Reagent Pack (Abaxis Europe GmbH). The examination profile animal species pig was selected for a 3-fold differentiation. The parameters determined included the red blood count with the individual values RBC, HGB, HCT, MCV, MCH, MCHC, RDW, PLT, PCT, MPV and PDW, and the white blood count with WBC, LYM, MON, NEU, LYM in %, MON in % and NEU in %.
The collected citrated whole blood was centrifuged approx. 4–6 h after collection at 1500 rpm for 30 min with a Labofuge 400 (Heraeus, Thermo Fisher Scientific Inc.). The plasma supernatant was pipetted into microcentrifuge tubes (Sarstedt AG) using a volumetric pipette (Eppendorf AG) and stored at − 21 °C for the investigation of the coagulation profile.
The coagulation parameters were determined with a STA Compact (Stago Deutschland GmbH). The profile included values for fibrinogen, prothrombin time (PT), activated partial thromboplastin time (APTT), prothrombin time – international normalized ratio (PTINR), antithrombin III (AT-III) and D-dimers.
The collected serum-blood samples were centrifuged approx. 4–6 h after collection at 3000 rpm for 5 min with a Labofuge 400 (Heraeus, Thermo Fisher Scientific Inc.). The serum was pipetted with a volumetric pipette (Eppendorf AG) into microcentrifuge tubes (Sarstedt AG) and stored at − 21 °C for further investigations.
For the CRP (C-reactive protein) analysis, the samples were sent to Biocheck GmbH in Leipzig. There, the analysis was carried out using an ELISA (Phase Porcine CRP Assay Kit, Tridelta Development Ltd.) according to the manufacturer’s instructions.
The clinical blood chemistry parameters were determined using an ABX Pentra 400 (Horiba Europe GmbH). Urea, creatinine, total protein, albumin, globulin, glucose, bilirubin, cholesterol, triglycerides, alkaline phosphatase (AP), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), aspartate aminotransferase (AST), creatine kinase (CK) and haptoglobin were measured. The electrolyte values sodium, chloride, potassium, calcium, phosphate and magnesium were determined using the Nova Electrolyte Analyzer (Nova Biomedical GmbH).
Statistical analysis
All data were analysed with the Program Packagge IBM-SPSS, V.26, Munich, Germany.
For the present study, all individuals were classified according to their SINS score. The 20% individuals with the lowest SINS scores were assigned to the SINS low group, the 20% individuals with the highest SINS scores to the SINS high group, and the remaining individuals (60%) to the SINS med (medium) group. Clinical, haematological and clinical-chemical characteristics were compared between these groups. Because several animals had the same SINS score, the exact proportions of 20% could not be kept. Because the fatteners hadsignificantly lower SINS scores, the group with low SINS scores admitted considerably more animals than the group with high SINS scores in this age group. Means ± standard deviations of the three groups were given for all parameters (0/1) and scores. Frequencies of alterations in the parameters were calculated by chi2-Test, Scores were analysed by univariate analysis of variance. Correlation coefficients were calculated for all parameters with the SINS-Score and significances for these correlations were given as the distribution independent Spearman’s correlation coefficient. An α-level of < 0.003 was considered as statistically significant for clinical parameters, after Bonferroni adjustment (original: α < 0.1). Haematological and clinical chemical parameters were considered as statistical significant at α < 0.1, respectively at α < 0.002 after Bonferroni correction. P values in Tables are the original values (no Bonferroni correction).