ORIGINAL RESEARCH published: 24 April 2019 doi: 10.3389/fvets.2019.00123 Age-Related Serum Biochemical Reference Intervals Established for Unweaned Calves and Piglets in the Post-weaning Period Kuai Yu 1*, Francesca Canalias 2 , David Solà-Oriol 3 , Laura Arroyo 1 , Raquel Pato 4 , Yolanda Saco 4 , Marta Terré 5 and Anna Bassols 1,4 1 Departament de Bioquímica i Biologia Molecular, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Departament de Bioquímica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Animal Nutrition and Welfare Service, Animal and Food Science Department, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain, 4 Servei de Bioquímica Clínica Veterinària, Facultat de Veterinària, Universitat Autònoma de Barcelona, Barcelona, Spain, 5 Departament de Producció de Remugants, Institut de Recerca i Tecnologia Agroalimentàries, Barcelona, Spain The purpose of the present study is to establish the influence of age on serum biochemistry reference intervals (RIs) for unweaned calves and recently-weaned piglets Edited by: using large number of animals sampled at different ages from populations under different Amy Louise Warren, University of Calgary, Canada season trials. Specifically, milk replacer (MR)-fed calves from April–July 2017 (n = 60); Reviewed by: from December 2016–March 2017 (n = 76) and from April–August 2018 (n = 57) and Stein Istre Thoresen, one group of healthy weaned piglets (n = 72) were subjected to the study. Serum Norwegian University of Life Sciences, Norway enzymes and metabolites of calves at age of 24 h (24 h after colostrum intake), 2, 5, Panagiotis Dimitrios Katsoulos, and 7 weeks from merged trials and piglets at 0, 7, and 14 days post-weaning (at 21, Aristotle University of Thessaloniki, 28, and 35 days of age) were studied. The main variable is age whereas no major trial- or Greece sex-biased differences were noticed. In calves, ALT, AST, GGT, GPx, SOD, NEFAs, *Correspondence: Kuai Yu triglycerides, glucose, creatinine, total protein, and urea were greatly elevated (p < 0.001) kuaiyu.vet@hotmail.com at 24 h compared with other ages; glucose, creatinine, total protein, and urea constantly decreased through the age; cholesterol’s lowest level (p < 0.001) was found in 24 h Specialty section: This article was submitted to compared with other ages and the levels of haptoglobin remained unchanged (p > 0.1) Veterinary Experimental and during the study. In comparison with the adult RIs, creatinine from 24 h, NEFAs from 2 w, Diagnostic Pathology, GGT from 5 w, and urea from 7 w are fully comparable with RIs or lie within RIs determined a section of the journal Frontiers in Veterinary Science for adult. In piglets, no changes were noticed on glucose (p > 0.1) and haptoglobin (p Received: 14 December 2018 > 0.1) and there were no major changes on hepatic enzymes (ALT, AST, and GGT), total Accepted: 02 April 2019 protein, creatinine and urea even though several statistical differences were noticed on 7 Published: 24 April 2019 days post-weaning. Cholesterol, triglycerides, NEFAs, cortisol and PigMAP were found Citation: Yu K, Canalias F, Solà-Oriol D, increased (p < 0.05) while TNF-alpha was found less concentrated (p < 0.001) at 0 days Arroyo L, Pato R, Saco Y, Terré M and post-weaning compared with other times. Moreover, the RIs of creatinine and GGT are Bassols A (2019) Age-Related Serum fully comparable with RIs or lie within RIs determined for adult. In conclusion, clinical Biochemical Reference Intervals Established for Unweaned Calves and biochemistry analytes RIs were established for unweaned calves and recently-weaned Piglets in the Post-weaning Period. piglets and among them some can vary at different ages. Front. Vet. Sci. 6:123. doi: 10.3389/fvets.2019.00123 Keywords: serum clinical biochemistry, reference interval, non-parametric, unweaned calf, weaning piglet, age Frontiers in Veterinary Science | www.frontiersin.org 1 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals INTRODUCTION have been calculated from small populations (<35), and this does not meet the requirement of non-parametrical RI. As it Serum biochemical reference intervals (RIs) play an important is recommended in Clinical and Laboratory Standards Institute role in assessing animal health. However, for farm animals, (CLSI) Guideline, a non-parametric method is recommended the majority of RIs are set for adult individuals. Similar to due to its simplicity and reliability (14), thus a sufficient human medicine, where pediatric RIs are readily available, there population for RI establishment could be more trustworthy. is a necessity of robust and specific RIs for young animals Second, many RIs are calculated from individual experiments, in veterinary science, since their physiology is different from and since factors like diet, geography, season, sex, and breed adults and it may be markedly affected by milk-feeding and may affect the RI, thus the obtained value may not applicable in weaning. The use of adult RIs intervals may be inadequate since other conditions. young animals may metabolize in a different way. Furthermore, In this study, the first objective is to determine clinical young animals must be closely monitored since they are more biochemistry values from a large set of healthy unweaned vulnerable to diseases, and their diets may have a bigger impact calf populations from 3 different nutritional programs and on the health condition in comparison with the adult. Thus, breeding seasons at 24 h, 2 weeks, 5 weeks, and 7 weeks many of the RIs publications in veterinary science established for of age; the second objective is to determine healthy recent- adults (1, 2) may be misleading (3) and the establishment of RIs weaned piglets’ clinical biochemistry values from both sexes in young animals is needed to adequately discriminate between at 0 days, 7 days, and 14 days post-weaning and the third healthy and diseased individuals. objective is to evaluate all the values and establish RIs. The In newborn calves, their growth involves several purpose is to obtain RIs useful for the veterinary practitioner and morphological, metabolic and physiological changes (4) for research. and these changes may be reflected by metabolites and enzymes’ levels in blood (5, 6). Calves undergo metabolic and digestive MATERIALS AND METHODS tract physical changes during the weaning process (in the dairy industry it occurs between 6 and 9 weeks of age) (7). At birth, Calf Housing and Diet Management most of the nutrients (glucose, amino acids, and long-chain fatty Male Holstein calves were managed according to the acids) from milk are digested in the abomasum and absorbed in recommendations of the Animal Care Committee of Institut the small intestine. Thus, young calves are non-ruminant. As calf de Recerca i Tecnologia Agroalimentàries (IRTA) under the starts to consume solid feeds (like concentrate feeds), the rumen approval research protocol FUE-2017-00587321, authorization starts to develop. At weaning, the rumen is mostly developed code 9733. Holstein male calves were obtained from Granja and it supports part of the nutrient absorption from solid Murucuc (Gurb, Barcelona, Spain) and housed individually feed fermentation. This different nutrient supply at weaning at IRTA in Torre Marimon (Barcelona, Spain). Plastic ear tag also entails a shift in the hepatic function from a glycolytic identification with the animal’s number was used. In all cases, to glucogenic liver altering metabolites and enzymes levels animals were under veterinary control and no health problems in blood. were observed during the experimental period. The total number In swine industry, an “early weaning” is commonly used with of individuals included in the study was n = 193. Animals the objective of reducing and improving the sow productive cycle. belonged to three trials. Normally weaning occurs abruptly at 21–28 days of age and the In Trial 1 (T1), 60 new-born calves (age = 0 h) were fed with piglet needs to be adapted to eat a novel food (usually solid colostrum within the first 6 h after birth. At 24 h of age, blood diet) after the separation from the sow. In contrast to the natural samples were taken. Then, at age of 3 ± 1.3 days old, animals weaning which gradually process until 17 weeks of age (8), early were fed with a MR and blood samples were collected at 2 and 5 weaning may become stressful for piglets. The immediate post- weeks of age 4 h after the morning feeding. The T1 was carried weaning period is critical for the piglet health since at that out between April and July 2017. moment they are very susceptible to gastrointestinal problems In Trial 2 (T2), 76 calves (average age = 3 ± 1.7 d) were fed and infections. with MR and blood samples were collected at 2, 5, and 7 weeks In both species, the early stage age involves several of age 4 h after the morning feeding. The T2 started in December biochemical and metabolic changes that may be fundamentally 2016 and ended in March 2017. different from the adult ones. Moreover, since young animals In Trial 3 (T3), 57 calves (average age = 5 ± 2.2 d) were fed are more vulnerable to certain pathogens, basic health with MR and blood samples were taken at 2, 5, and 7 weeks of age laboratory routine check based on serum biochemical analytes 4 h after the morning feeding. The T3 was carried out between is essential. April and August 2018. In the past 20 years, neonatal clinical biochemistry gradually All animals followed the same MR feeding program that has been established and the biochemical RIs on unweaned consisted on feeding 2-L of MR (Nukamel, Weert, The cattle calves of different breeds and ages up to 3 months Netherlands) twice a day containing at 12.5% concentration of old (3, 9–11) and for piglets younger than 1 month of of MR powder (500 g/d) for the first 4 days, subsequently age (12, 13) have been published. In all these studies, several MR was increased to 5 L/d of MR at 12.5% concentration noticeable biochemical changes along the age were reported. of MR powder (625 g/d) for the next 3 days, then MR was Nevertheless, these studies have two main drawbacks. First, RIs increased to 6 L/d of MR at 12.5% concentration of MR powder Frontiers in Veterinary Science | www.frontiersin.org 2 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals (750g/d) for the next 7 days and then 2 meals of 3 L at 15% piglets, two commercial ELISA kits were used: Cortisol ELISA Kit concentration (900 g/d) until 49 d of age. Then, MR was (DRG, Marburg, Germany) and Porcine TNF-alpha Quantikine limited to a single offer of 3 L also at 15% (450 g/d) until end ELISA Kit (R&D Systems, Abingdon, UK). The analytes, of the study. Animals also had access to water and chopped methods, reagents, and coefficients of variation (CV%) are shown barley straw ad libitum. Chemical composition and ingredient in Table S3. of the MRs and the starter intake information are shown in Tables S1, S2. RI Establishment and Statistics The RI establishment was in accordance with American Society Piglet Housing and Sampling for Veterinary Clinical Pathology (ASVCP) guidelines (18). A total of 336 commercial crossing piglets ([Large White x These guidelines mirror the CLSI recommendations. All RIs Landrace] x Pietrain), male and female 21-d old piglets of 4– were generated by Reference Value Advisor (19), an add-in 7 kg of body weight (BW) were weaned. The animals were program in Excel (Office 365, Microsoft, Redmond, WA, USA). moved from the farrowing to the nursery unit (within the For population amounts over 120, non-parametric RIs were same commercial farm without transport) in the morning on directly used since this method doesn’t assume any specific shape the day of weaning. Plastic ear tag identification with the for data distribution. For populations <120 (24 h of age for animal’s number was used. In the nursery room, pigs were calves and piglet samples), non-parametric RIs were not able then allocated in 24 pens (14 piglets/pen). Each pen has had a to be computed. The criteria of outlier removal are following: commercial non-lidded hopper and a nipple waterer to ensure (a) For interquartile range (IQR) = Q3 (third quartiles)—Q1 (first ad libitum feeding and free water access. The experimental quartiles), values that exceed interquartile fence set at Q1 −3∗ IQR procedure has been approved by ethical committee (CEEAH) of and Q3 +3∗ IQR are considered outliers; (b) Without removing Universitat Autònoma de Barcelona (Registration n◦ :1406 of the certain outliers, if Reference Value Advisor computes result Departament de Medi Ambient i Habitatge of the Generalitat with good distribution and/or symmetricity, then the outliers de Catalunya). are retained; (c) If no normal distribution and/or symmetricity At weaning, animals were offered the same basal pre-starter are observed even outliers are removed and data are power diets following the same specification. The pre-starter and starter transformed, such population is calculated with Cook’s distance diets were fed ad libitum for fourteen consecutive days (P1) and Cook’s outliers are detected and deleted. After the removal and 21 consecutive days (P2), respectively. A basal diet was of outliers, populations were recalculated for RIs. Once the formulated to contain 2,470 kcal NE/kg; 20.5% CP/kg and 1.35% RIs were established, one-way analysis of variance (ANOVA) Dig Lys for P1 and 2,400 kcal NE/kg; 18.1% CP/kg and 1.20% and Tukey’s HSD with Bonferroni correction test, boxplots Dig Lys for P2. Diets were formulated to meet the requirements with difference annotations and distribution histograms were for growth of newly weaned piglets (15). No antibiotics and ZnO made by “R” (20) under RStudio environment (21) using the were included in the basal diets. combinations of “multcompView” (22), “nortest” (23), “ggplot2” Blood samples from 3 piglets per pen (a total of 72 animals) (24), “gridExtra” (25), and “ggpubr” (26) packages and values that were collected avoiding cross-contamination between pens at exceed interquartile fence set at Q1 −3∗ IQR and Q3 +3∗ IQR were 8 am at week 3 of age (day 0 after weaning), week 4 (day considered outliers. 7 after weaning), and week 5 (day 14 after weaning). Piglets were individually weighted before weaning and distributed for RESULTS a balanced body weight within each pen. Piglets were sorted by descending BW within each pen and the one in the middle For calves and piglets, the frequency of distribution histograms of (median), the ones immediately above and below the median all analytes from all the individuals (both trial-wise and merged- were picked out to be sampled. The same animals per pen were data-wise) were visualized before the RIs were established (shown sampled on weeks 4 and 5, respectively. in Figures S1, S2). The distribution from the different sets of animals overlapped and differences between groups were not Sample Collection and Analysis significant, indicating that no year season, experimental or sex- Blood samples obtained from jugular venipuncture were biased distributions were found and in consequence, all the collected in 10 ml Vacutainer tubes without anticoagulant. Sera individuals of the same age were included in the same reference were obtained by centrifugation at 1,500 g for 10 min and population. There were only two exceptions identified in calves: transferred to new tubes to be stored in aliquots at −80◦ C AST and cholesterol at week 2, where T3’s population displayed a until further analysis. At the day of analysis, aliquots were non-overlapping distribution curve. thawed. Measurement of serum clinical biochemistry analytes For calves, the methods of RI calculations of 24 h was was performed on the Olympus AU400 analyser except cortisol parametric since population consisted in 30 individuals and from and TNF-alpha. The protocols for the use of Olympus System 2 w non-parametric method was used as the populations are Reagents (OSRs) and other commercial reagents were in over 120. reference of previous studies (16, 17). Quality control protocols For piglets, since the population size didn’t reach 60, were based on the daily quantification of two control sera parametric method was used for all ages and analytes except one of low and high concentration (Control serum I and Control non-parametric method was suggested using by the software on serum II, Beckman Coulter). For cortisol and TNF-alpha in AST at 7 d due to unfixable normality of distribution. Frontiers in Veterinary Science | www.frontiersin.org 3 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals A summary of the results of both species at different ages SOD and GPx also showed rather stable levels from 2 to 7 w. are shown in Tables 1, 2, respectively. Figures 1, 2 show the Unfortunately, we didn’t find any RIs of GPx for adult; and for pattern of age-related differences for all parameters. The post-hoc SOD only RI of cow during transition period (60 d before until multi-comparison results are shown in Tables S4, S5. Missing 100 d after milk) was found. Both mineral dependent enzymes and undetectable samples were discarded in this study. [Mn, Cu and Zn for SOD (37) and Se for (2, 38)] are important enzymes whereas SOD can dismutate two O− 2 molecules to H2 O2 and O2 while GPx can catalyze H2 O2 to H2 O (2). Study of SOD DISCUSSION in RBC suggested that there is no such variation in the activity between cattle and human (39). Even though there are very A table with RI for calves and piglets has been designed for few information about SOD and GPx, a better characterization veterinary use with comparison of results in adults obtained from may contribute to the monitor of minerals and oxidation in the literature (Tables 3, 4). animal’s body. NEFAs and TGs were also elevated at 24 h because colostrum RIs for Unweaned Calves is rich in fat (40); their concentrations dropped at 2 w compared It was noticed that the all enzymes (ALT, AST, GGT, GPx, and to 24 h and started increasing moderately with age until 7 w. SOD) not only showed the highest concentrations at 24 h of While the level of NEFAs is almost comparable with adult’s RI age, but also significantly higher than any other times of age. roughly from 5 w, TG’s level is still beyond the adult’s RI upper The high concentration in newborn animals is an indication that limit at 7 w. TGs can richly appear in chylomicrons, which are these enzymes are absorbed from colostrum, at least in the case secreted by the intestine after fat-containing meal in preruminant of GGT and AST (9, 32). The most extreme elevation occurred calves. The TGs from chylomicrons can be hydrolyzed to NEFAs with GGT, which was 20 folds higher at 24 h than at 2 w and and then be transported to tissue for fat storage or for oxidation further decreases at 5 w and 7 w. Even though the GGT level is to produce energy (41); on the other hand, serum NEFAs can relatively low in adult cow’s serum, its level in colostrum can increase in case of fasting or negative energy balance. Indeed, peak-up to ∼800-fold greater than in the serum of the same cow the growth of calf requires a large amount of energy from fat (33). GGT can increase its activity in calves’ serum as early as 6 h and evidences have already shown that extra fat can increase after colostrum intake (intake at birth) (34) and similar to our BW gains (42, 43). An adult’s-like RI in NEFAs from 5 w and a results, it has been described that it takes approximately up to rather higher levels of TGs throughout the study indicate that 5 weeks for GGT to decline to low level (adult’s level) after the the calves demanded high amount of energy for growth and no intake of colostrum (3, 11, 33). Other studies found that lower negative energy balance was noticed. Another form of TGs is concentrations of GGT in new-born calves are linked with much very-low-density lipoprotein (VLDL) but it is generally low in lower levels of globulins/total protein and higher risk of death bovine compared with in human (41). (33, 35); moreover, one study showed 0% sensitivity of GGT when Cholesterol had a very similar trend with one study (9) it comes to the detection of hepatic diseases before 6 w old (36) where the lowest concentration was found at 24 h and the since the highly concentrated colostrum-derived GGT presented concentrations started to increase smoothly. Even though in the serum can mask the level of GGT that is derived from it was previously described that colostrum contains higher liver damage. Indeed, a better characterization of GGT levels for cholesterol concentration compared with milk (44) and the new-born calves may help to monitor calves’ health to prevent serum cholesterol concentration is proportional to the colostral early-age death but not used to detect hepatic diseases. It is also cholesterol concentration (45), the increase in total cholesterol relevant to notice the high individual variability of GGT values along with increases in NEFAs and TGs probably indicate a major in 24 h old calves, probably indicating differences in colostrum lipid mobilization within tissues after 24 h. intake, absorption and/or metabolization. Glucose showed significant drop throughout age. The glucose After 24 h of age, ALT’s level reached its lowest at 2 w and supply from mother in neonate stops abruptly immediately after gradually went uphill afterwards while AST stayed relatively birth and the suckling neonate is entirely dependent on glucose steady and low levels from 2 to 7 w. However, at 7 w of age, both supply via colostrum/milk, meanwhile the neonate needs to levels of ALT and AST were still lower compared with adult’s RI. rapidly turn on pathways of endogenous glucose production as Other studies with longer terms showed that AST reaches adult’s well as for fatty acid oxidation to maintain its organs and cells’ level around 8–10 w of life (9, 10). According to previous study functionality (46). The decrease of glucose and increase of NEFAs along with GGT and other enzymes (36), AST’s sensitivity to liver and TGs during the first 7 w may be the consequence of the high damage is 80% for calves <6 w old thus AST alone should not fat and relatively low carbohydrate content of colostrum/milk be considered clinical useful in diagnosing hepatic diseases. In (47, 48). Despite the constant fall of serum glucose throughout addition, despite the statistical differences in AST throughout 2– the study, the RIs obtained from last measurement was still 7 w, none of their pair-wise comparison fold changes were larger higher than the adult’s one. than 1.15 or <0.85, thus biologically these were not considered Moreover, creatinine, TP and urea also decreased throughout major differences. For ALT, its characterization in cow is seldom age until 7 w. Creatinine is a breakdown product of creatine studied because is not so commonly used for hepatic disease (Cr) and creatine phosphate (PCr) in muscle and is cleared by diagnosis since its activity is low and isn’t massively liberated into the kidney. In human neonatology, it was well explained that serum during hepatic diseases in large animal (2). the kidneys in fetus don’t play a major role in maintaining Frontiers in Veterinary Science | www.frontiersin.org 4 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals TABLE 1 | Summary of statistical values for clinical biochemistry analytes from calves of 24 h (24 h after colostrum taking), 2, 5, and 7 weeks of age. Analyte ALT AST Cholesterol Creatinine GGT Glucose GPx Haptoglobin NEFAs TGs TP SOD Urea Unit U/L U/L mg/dL mg/dL U/L mg/dL U/L mg/mL mmol/L mg/dL g/dL U/mL mg/dL 24 h Mean 12.1 87.5 36.1 1.22 1459.8 125.8 737.1 0.16 0.41 39.1 6.02 0.90 19.4 Median 10.8 85.0 33.6 1.20 1303.0 119.4 632.0 0.14 0.40 38.2 6.15 0.53 19.3 SD 4.4 20.4 11.1 0.17 831.3 28.5 347.4 0.05 0.15 19.7 0.71 0.89 5.6 Minimum 6.8 53.0 20.6 0.96 288.0 68.5 343.1 0.09 0.19 13.5 4.42 0.05 8.7 Maximum 22.3 134.0 67.4 1.64 3170.0 211.1 1650.4 0.32 0.77 87.0 7.43 2.79 34.9 N 29 30 30 30 28 30 28 28 30 30 30 30 30 Method Trans. Trans. Trans. Trans. Trans. Untrans. Trans. Trans. Untrans. Trans. Trans. Trans. Trans. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. 2w Mean 6.8 36.6 71.5 1.00 100.8 110.6 505.0 0.14 0.10 19.6 5.11 0.31 15.2 Median 6.6 38.0 78.3 0.99 76.0 110.6 452.4 0.13 0.09 16.0 5.10 0.25 13.9 SD 1.7 9.3 29.3 0.20 70.6 22.1 194.0 0.12 0.06 12.1 0.63 0.21 7.9 Minimum 3.3 0.0 11.5 0.54 12.0 65.4 231.6 0.06 0.03 5.5 3.75 0.02 6.3 Maximum 12.1 60.0 145.0 1.71 348.0 222.5 1610.7 1.48 0.31 81.8 7.14 1.33 92.7 N 188 188 188 188 188 188 186 190 188 188 188 185 188 Method NP NP NP NP NP NP NP NP NP NP NP NP NP 5w Mean 8.3 42.2 82.1 0.83 24.5 101.8 421.1 0.14 0.15 21.9 4.99 0.23 13.9 Median 8.1 42.0 77.0 0.82 23.0 101.4 383.0 0.12 0.14 18.1 4.99 0.20 13.7 SD 2.1 15.0 29.4 0.16 9.5 18.4 157.6 0.18 0.06 12.0 0.50 0.14 3.9 Minimum 4.3 19.0 30.6 0.50 7.0 43.8 242.2 0.06 0.05 7.4 3.77 0.02 6.3 Maximum 19.5 178.0 187.7 1.40 54.0 157.9 1525.3 2.21 0.30 71.7 6.49 0.94 27.3 N 193 193 193 193 193 193 192 193 193 193 193 191 193 Method NP NP NP NP NP NP NP NP NP NP NP NP NP 7w Mean 9.5 37.5 65.5 0.67 16.8 93.0 437.1 0.13 0.18 24.1 4.80 0.23 11.2 Median 9.4 36.5 64.9 0.66 16.0 93.3 413.2 0.12 0.17 21.5 4.70 0.20 10.9 SD 2.4 10.8 23.0 0.12 4.4 15.3 128.0 0.06 0.07 14.3 0.54 0.13 2.9 Minimum 4.7 16.0 18.8 0.43 8.0 59.2 262.4 0.07 0.05 6.1 3.66 0.01 5.8 Maximum 19.0 77.0 133.4 1.03 31.0 147.8 1330.8 0.56 0.40 101.9 6.47 0.66 18.8 N 134 134 134 134 134 134 133 134 134 134 134 130 134 Method NP NP NP NP NP NP NP NP NP NP NP NP NP SD, Standard Deviation; N, Number of animals; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; GGT, Gamma-Glutamyl Transferase; GPx, Glutathione Peroxidase; NEFAs, Non-Esterified Fatty Acids; TGs, Triglycerides; TP, Total Proteins; SOD, Superoxide Dismutase. Methods: NP, Non-parametric; Trans. Std., Transformed data using standard method; Untrans. Std., Untransformed data using standard method. fetal homeostasis (49, 50); also, the high blood concentration excreted by the kidney, which is directly influenced by the total of creatinine comes from maternal transfer and endogenous Cr protein level. and PCr degradation and it does not indicate fetal renal failure. A special attention was paid to markers of stress since It was noticed that at 2 and 5 w the median creatinine levels assessing a good welfare status is one of the main problems in were around the bottom line of the adult’s RI while at 7 w it was calf production. The levels of haptoglobin didn’t change along below [median and SD at 7 w was 0.66 ± 0.12 mg/dL and adult’s the time, similar to one previous study (54) and are lying within RIs are 1.0–2.0 mg/dL according to Kaneko et al. (2) and 0.9– the health adult’s RI range (10 mg/dL) (27). It is important to have 1.4 mg/dL according to Pérez-Santos et al. (11)], comparing to specific RIs for haptoglobin in young calves since it is an acute- another similar study (11), where mean creatinine laid within phase protein used as biomarker of inflammatory and infectious normal adult’s RI from 6 d until 90 d of age. Yet we didn’t find any diseases (55), very common in young calves. The individuals with other issues related to the muscle or health in these calves and a high haptoglobin values identified as outliers may be individuals possible reason of this is due to diet/season/genetic variabilities with subclinical diseases, which were recognized as healthy in between the farms. TP was also elevated at 24 h due to passive a clinical inspection. The antioxidant enzymes GPx and SOD, transfer of protein which mainly contains immunoglobulins markers of oxidative stress (56) had higher values at 24 h, but the from colostrum (32, 51, 52). This result is similar to those RIs are similar afterwards. were reported in previous studies (11, 53). Urea had a similar As it was previously mentioned, on week 2 we found trial- change pattern to the TP along the time. It is the product of biased distributions (Figure S1) on AST and cholesterol, where protein breakdown (52) and is synthesized by the liver and finally T3 showed lower concentration range compared with others. Frontiers in Veterinary Science | www.frontiersin.org 5 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals TABLE 2 | Summary of statistical values for clinical biochemistry analytes from piglets at 0, 7, and 14 days post-weaning (at 21, 28, and 35 days of age). Analyte ALT AST Cholesterol Cortisol Creatinine GGT Glucose Haptoglobin NEFAs PigMAP TGs TNF-alpha TP Urea Unit U/L U/L mg/dL ng/L mg/dL U/L mg/dL mg/mL mmol/L g/L mg/dL pg/mL g/dL mg/dL 0d Mean 32.8 78.4 125.5 74.9 0.80 46.0 84.9 0.50 0.40 3.26 43.2 54.3 3.78 12.1 Median 31.3 63.0 122.0 60.2 0.79 41.0 80.8 0.41 0.40 2.88 40.9 48.8 3.68 11.7 SD 9.4 47.5 41.8 50.1 0.15 16.5 17.8 0.39 0.14 1.22 13.1 22.3 0.64 3.6 Minimum 10.2 20.0 51.4 2.0 0.57 11 49.0 0.05 0.15 1.22 22.6 23.1 2.34 6.7 Maximum 63.7 242.0 272.2 202.2 1.16 89 141.4 1.68 0.76 5.88 85.0 110.6 5.42 25.2 N 66 68 61 71 54 65 57 61 61 71 59 67 58 57 Method Trans. Trans. Trans. Trans. Untrans. Trans. Untrans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Std. Std. Std. Std. Rob. Std. Rob. Std. Std. Std. Std. Std. Std. Std. 7d Mean 24.8 45.8 53.1 19.0 0.94 40.9 79.1 0.62 0.18 0.99 35.5 103.2 3.49 18.2 Median 22.8 37.0 52.0 15.6 0.93 40.0 78.9 0.49 0.15 0.94 31.7 101.7 3.46 17.2 SD 8.8 22.3 14.3 12.4 0.16 13.9 12.4 0.52 0.11 0.31 11.2 27.7 0.48 6.9 Minimum 8.2 15.0 27.0 1.1 0.66 13 55.3 0.07 0.04 0.43 18.8 41.4 2.39 4.3 Maximum 53.3 125.0 95.5 52.5 1.51 74 107.6 2.39 0.58 2.24 73.3 165.7 4.57 41.3 N 67 67 65 68 65 67 64 66 62 69 65 72 65 64 Method Trans. NP* Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. 14 d Mean 35.5 65.6 69.7 21.2 0.85 50.9 83.0 0.57 0.11 0.74 38.8 98.1 3.84 14.7 Median 28.7 48.0 62.4 18.9 0.82 49.0 85.8 0.30 0.10 0.70 38.0 92.5 3.74 12.9 SD 23.6 45.7 36.9 16.7 0.17 17.4 16.5 0.66 0.03 0.28 10.0 42.8 0.69 7.5 Minimum 11.4 23.0 37.5 1.6 0.53 17 34.5 0.05 0.05 0.32 20.2 43.6 2.76 4.6 Maximum 177.9 260.0 303.9 83.8 1.26 112 126.9 3.05 0.21 2.13 68.3 292.0 6.36 38.5 N 64 62 62 65 57 66 60 61 55 63 60 71 59 57 Method Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Trans. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. Std. SD, Standard Deviation; N, Number of animals; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; GGT, Gamma-Glutamyl Transferase; NEFAs, Non-Esterified Fatty Acids; PigMAP, Pig Major Acute Phase Protein; TGs, Triglycerides; TNF-alpha, Tumor Necrosis Factor alpha; TP, Total Proteins. Methods: NP, Non-parametric method; Trans. Std., Transformed data using standard method; Untrans. Rob., Untransformed data using robust method. *Non-parametric method was used due to unfixable distribution [p-value (Anderson-darling) < 0.05 and p-value (symmetry test) < 0.01] both before and after transformation. This method was recommended by the software. This reason may be because the average age in T3 is ∼2 d and 30 d, different from our opinion, the author’s explanation of older than other two. This difference indicates that only 2-days the increases was due to the increase of muscle mass and helped age difference can cause mean concentration difference in some by the gradual supplementation of enriched solid diet. parameters, thus special care should be taken when comparing Special interest has been devoted to markers of stress and local clinical values with RIs in literature. inflammation. Thus, cortisol was measured as the paradigmatic stress hormone and found higher just after weaning, similar to RIs for Post-weaning Piglets other studies (58–60), this probably is due to the stress associated No major changes were noticed on hepatic enzymes (ALT, AST, to separation of the mother and change of diet; it’s also interesting and GGT), glucose and TP. Cholesterol, NEFAs and TGs showed to notice that from 7 d post-weaning, the mean concentrations their highest values just after weaning (0 d). This may be due of piglets are comparable to healthy adults’ one (31), which to the sudden dietary change from high fat content of milk suggests that in this study the diet change only caused a transient to less-digestible, more-complex solid feed that piglets eat after stress to the animals. The acute phase proteins haptoglobin and weaning (57). Pig-MAP were also measured; whereas haptoglobin RIs were Throughout the ages, all medians of urea were within adult’s not influenced by age and weaning, Pig-MAP was higher at 3 RI while all medians of creatinine were <0.8 folds to the adult’s weeks-old just after weaning. Absolute values for haptoglobin RI lower fence. Both analytes showed highest concentrations at were similar to those reported at 3-week-old (13). In that study, 7 d post-weaning. The increase in urea and creatinine only at 7 reference values for both acute phase proteins were established d post-weaning may be due to transient dehydration after the for pigs older than 4 weeks. Pig-MAP is also considered a stress dietary change. These pattern changes are very similar to one marker, which increases in stressful conditions like transport study between 5-day-old unweaned piglets and 30-day-old post- (61–63). Finally, the cytokine TNF-alpha is frequently used as a weaning piglets (weaned at 28 d of life) supplemented with iron marker of infection, especially in gastrointestinal disorders which in solid diet (12). However, since this report only measured 5 d often affect piglets at weaning (31, 64, 65). For this reason, it is Frontiers in Veterinary Science | www.frontiersin.org 6 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals FIGURE 1 | Boxplots of analytes with Tukey’s HSD posthoc with Bonferroni correction from calves’ sera at 24 h, 2 w, 5 w, and 7 w of age. Annotations over the boxes with different superscripts are significantly different (p < 0.05). Interquartile fence was set between Q1-3*IQR and Q3+3*IQR. 24 h, calves at 24 hours of age; 2 w, calves at 2 weeks of age; 5 w, calves at 5 weeks of age; 7 w, calves at 7 weeks of age. Analytes were alphabetically plotted according to the orders of discussion. interesting to have RIs available in healthy animals. A moderate management of the individuals. Even in farm animals, where increase in TNF-alpha was shown at 7 and 14 days after weaning, collective evaluation of health is frequent instead of an individual probably due to the change of diet. This increase in serum may assessment, the availability of specific RIs for young ages is be secondary to the transient response in gene expression of essential to for an accurate diagnosis of health problems. In inflammatory cytokines in the gut after weaning (66). this sense, most important life periods in these two farm Similar to the calves, creatinine from 0 d post-weaning (3 w animal species: in cattle, colostrum uptake and suckling is a of age) was also comparable to the adult’s RIs. GGT’s RI is also critical period during which the calf starts growing and obtains comparable to adult’s one from 7 d of age while for calves it takes the immune defenses necessary for a sustained growth. In 5 weeks to drop into adult’s RI (2). piglets, the post-weaning period is the most critical due to the high incidence of gastrointestinal and respiratory disorders. CONCLUSIONS In this work, we have used a large number of individuals to obtain RIs specific for calves and piglets at these critical Specific information about reference intervals for young animals stages, and the most relevant parameters related to these is a requirement for the adequate clinical and nutritional stages evaluated. Frontiers in Veterinary Science | www.frontiersin.org 7 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals FIGURE 2 | Boxplots of analytes with Tukey’s HSD posthoc with Bonferroni correction from piglets’ sera at 0, 7, and 14 days post-weaning (age: 21, 28, and 35 days, respectively). Annotations over the boxes with different superscripts are significantly different (p < 0.05). Interquartile fence was set between Q1-3*IQR and Q3+3*IQR. 0d, piglets at the 0 day of weaning (right after weaning); 7d, piglets at the 7th day of weaning; 14d, piglets at the 14th day of weaning. Analytes were alphabetically plotted according to the orders of discussion. Frontiers in Veterinary Science | www.frontiersin.org 8 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals TABLE 3 | Reference intervals (RIs) of clinical biochemistry analytes from calves of 24 h of age (24 h after colostrum taking) 2, 5, and 7 weeks of age and RIs of adult cattle from literature. Analyte Unit 24 h 2w 5w 7w Adult ALT U/L 6.3–24.7 4.0–11.1 4.8–13.3 5.2–15.3 11–40 (2) AST U/L 49.6–133.4 19.5–55.0 22.0–63.3 19.0–63.3 78–132 (2) Cholesterol mg/dL 20.8–67.5 20.8–128.9 33.9–136.4 25.4–118.3 80–120 (2) Creatinine mg/dL 0.93–1.65 0.64–1.51 0.56–1.20 0.47–0.99 1.0–2.0 (2) GGT U/L 0.0–3201.8 16.5–262.5 12.0–48.6 11.0–27.6 6.1–17.4 (2) Glucose mg/dL 75.3–193.3 70.3–152.9 63.0–144.1 63.4–123.0 45–75 (2) GPx U/L 363.7–2116.5 270.5–1023.7 255.5–842.3 288.7–711.0 - Haptoglobin mg/dL 0.09–0.31 0.07–0.36 0.07–0.46 0.08–0.38 < 1.0 § (27) NEFAs mmol/L 0.09–0.72 0.04–0.27 0.07–0.29 0.06–0.35 0.07–0.46 (28) SOD U/mL 0.05–4.57 0.04–0.84 0.05–0.62 0.03–0.57 (12.71 ± 0.34) * (29) TGs mg/dL 11.3–93.7 6.6–57.5 8.2–58.8 6.7–58.5 0–14 (2) TP g/dL 4.58–7.52 3.95–6.54 3.94–5.93 3.87–6.13 6.74–7.46 (2) Urea mg/dL 9.2–32.5 7.5–30.4 7.7–23.8 6.8–18.3 20–30 (2) ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; GGT, Gamma-Glutamyl Transferase; GPx, Glutathione Peroxidase; NEFAs, Non-Esterified Fatty Acids; TGs, Triglycerides; TP, Total Proteins; SOD, Superoxide Dismutase. § Mean concentration obtained from 10 healthy cows. *Cow in transition period (60 d before until 100 d after milk). TABLE 4 | Reference intervals (RIs) of clinical biochemistry analytes from piglets at 0, 7, and 14 days post-weaning (PW) (at 21, 28, and 35 days of age) and RIs of adult pig from literature. Analyte Unit 0 d PW 7 d PW 14 d PW Adult ALT U/L 16.5–54.2 11.4–47.1 13.6–92.7 31–58 (2) AST U/L 27.5–230.4 15.7–122.9 26.6–223.9 32–84 (2) Cholesterol mg/dL 58.9–224.3 28.7–85.5 39.8–141.2 36–54 (2) Cortisol ng/mL 6.9–206.5 1.4–50.8 1.0–66.7 (29.7 ± 1.0) (2) Creatinine mg/dL 0.50–1.09 0.69–1.34 0.55–1.22 1.0–2.7 (2) GGT U/L 18.7–85.1 15.3–70.8 23.1–92.7 10–60 (2) Glucose mg/dL 44.0–117.8 56.4–106.3 46.6–113.6 85–150 (2) Haptoglobin mg/dL 0.03–1.55 0.08–2.34 0.05–0.18 0.02–3.00 § (30) NEFAs mmol/L 0.15–0.71 0.04–0.48 0.05–0.18 - PigMAP g/L 1.33–6.14 0.51–1.73 0.36–1.39 0.46–2.36 § (30) TGs mg/dL 23.9–76.5 21.0–65.9 22.2–62.0 - TNF-alpha pg/mL 22.0–108.4 51.9–162.4 46.3–202.6 < 10 * (31) TP g/dL 2.67–5.27 2.59–4.52 2.79–5.57 7.90–8.90 (2) Urea mg/dL 7.1–21.6 6.1–33.7 5.1–36.0 10–30 (2) ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; GGT, Gamma-Glutamyl Transferase; NEFAs, Non-Esterified Fatty Acids; PigMAP, Pig Major Acute Phase Protein; TGs, Triglycerides; TNF-alpha, Tumor Necrosis Factor alpha; TP, Total Proteins. § RIs made for mixed-age pigs at 5 w old obtained from 10 commercial farms. *Undetectable TNF-alpha level for 6 healthy pigs. The lower limit for detection is > 10 pg according to manufacturer. The first set of analytical parameters are related to susceptibility of neonates to infection, and limited antioxidant nutrition and metabolism, i.e., indicators of carbohydrate, protection (68). In piglets, several diseases with severe lipid, and protein metabolism. Many health problems consequences on health occur concomitantly with oxidative in young animals arise from nutritional unbalances and stress (56). management procedures. Third, markers of stress (cortisol) and inflammation/infection Secondly, enzymatic markers of oxidative stress were have been also evaluated. In this sense, the TNF-alpha cytokine also evaluated. Oxidative stress is a relevant issue in cattle and the acute phase proteins Pig-MAP and haptoglobin have (67) and, specifically, neonates are particularly susceptible been analyzed for piglets and calves, respectively. because of the challenge due to the transition from the This information will help veterinarians and hypoxic intrauterine environment to extrauterine life, animal science researchers to better practice their Frontiers in Veterinary Science | www.frontiersin.org 9 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals professions by providing them with more adequate revised the manuscript. All authors read and approved the diagnostic tools. final manuscript. ETHICS STATEMENT FUNDING Male Holstein calves were managed according to the This research was supported by the Spanish Ministry of recommendations of the Animal Care Committee of Economy and Competitivity (AGL2015-68463-C2-1-P to Institut de Recerca i Tecnologia Agroalimentàries (IRTA) MT and AGL2015-68463-C2-2-P to AB). The author KY under the approval research protocol FUE-2017-00587321, acknowledges China Scholarship Council (CSC) for the graduate authorization code 9733. Piglets’ procedure has been scholarship program’s funding. approved by ethical committee (CEEAH) of Universitat Autònoma de Barcelona (Registration no: 1406 of the ACKNOWLEDGMENTS Departament de Medi Ambient i Habitatge of the Generalitat de Catalunya). The authors gratefully thank all the staff of the IRTA (Torre Marimon, Caldes de Montbui, Spain) and pig farm (Els AUTHOR CONTRIBUTIONS Campassos, Monistrol de Calders, Spain) for their assistance in feeding and care of the animals. KY performed the manuscript writing, RI establishment and statistics analyses. FC provided statistical advices. SUPPLEMENTARY MATERIAL Handling of animals and sample collection was performed by DS-O for piglets and MT for calves. LA, RP, and YS The Supplementary Material for this article can be found performed sample analyses and AB conducted in the online at: https://www.frontiersin.org/articles/10.3389/fvets. conceptualization, supervised the analytical work, and 2019.00123/full#supplementary-material REFERENCES 13. Perri AM, O’Sullivan TL, Harding JCS, Wood RD, Friendship RM. Hematology and biochemistry reference intervals for Ontario commercial 1. Kessell A. Bovine haematology and biochemistry. In: Bovine Medicine. nursing pigs close to the time of weaning. Can Vet J. (2017) 58:371–6. Adelaide, SA: John Wiley & Sons (2015). 159 p. doi: 10.1002/9781118948538 14. CLSI. Defining, Establishing, and Verifying Reference Intervals in the Clinical 2. Kaneko JJ, Harvey JW, Bruss M, editors. Clinical Biochemistry of Domestic Laboratory. 3rd ed. (2010). Available online at: https://clsi.org/standards/ Animals. Elsevier/Academic Press (2008). products/method-evaluation/documents/ep28/ 3. Knowles TG, Edwards JE, Bazeley KJ, Brown SN, Butterworth A, Warriss 15. NRC. Nutrient Requirements of Swine. Washington, DC: National Academies PD. Changes in the blood biochemical and haematological profile of neonatal Press (2012). doi: 10.17226/13298 calves with age. Vet Rec. (2000) 147:593–8. doi: 10.1136/vr.147.21.593 16. Saco Y, Fina M, Giménez M, Pato R, Piedrafita J, Bassols A. Evaluation 4. Baldwin RL, McLeod KR, Klotz JL, Heitmann RN. Rumen of serum cortisol, metabolic parameters, acute phase proteins and faecal development, intestinal growth and hepatic metabolism in the corticosterone as indicators of stress in cows. Vet J. (2008) 177:439–41. pre- and postweaning ruminant. J Dairy Sci. (2004) 87:E55–65. doi: 10.1016/j.tvjl.2007.05.019 doi: 10.3168/jds.S0022-0302(04)70061-2 17. Marco-Ramell A, Arroyo L, Peña R, Pato R, Saco Y, Fraile L, et al. Biochemical 5. Roy JHB, Huffman CF, Reineke EP. The basal metabolism of the newborn calf. and proteomic analyses of the physiological response induced by individual Br J Nutr. (1957) 11:373. doi: 10.1079/BJN19570058 housing in gilts provide new potential stress markers. BMC Vet Res. (2016) 6. Le Huerou I, Guilloteau P, Wicker C, Mouats A, Chayvialle JA, Bernard C, 12:265. doi: 10.1186/s12917-016-0887-1 et al. Activity distribution of seven digestive enzymes along small intestine 18. Szladovits B, Walton RM, Barnhart KF, Friedrichs KR, Blanco-Chavez in calves during development and weaning. Dig Dis Sci. (1992) 37:40–6. J, Harr KE, et al. ASVCP reference interval guidelines: determination doi: 10.1007/BF01308340 of de novo reference intervals in veterinary species and other 7. Jones C, Heinrichs A. Calf Care. State College, PA: W. D. Hoard (2006). related topics. Vet Clin Pathol. (2012) 41:441–53. doi: 10.1111/vcp. 8. Jensen P. Maternal behaviour and mother-young interactions during lactation 12006 in free-ranging domestic pigs. Appl Anim Behav Sci. (1988) 20:297–308. 19. Geffré A, Concordet D, Braun JP, Trumel C. Reference value advisor: doi: 10.1016/0168-1591(88)90054-8 a new freeware set of macroinstructions to calculate reference 9. Egli CP, Blum JW. Clinical, haematological, metabolic and endocrine traits intervals with Microsoft Excel. Vet Clin Pathol. (2011) 40:107–12. during the first three months of life of suckling simmentaler calves held doi: 10.1111/j.1939-165X.2011.00287.x in a cow-calf operation. Zentralbl Veterinarmed A. (1998) 45:99–118. 20. R Project. R: The R Project for Statistical Computing. Available online doi: 10.1111/j.1439-0442.1998.tb00806.x at: https://www.r-project.org/ (accessed December 14, 2018). 10. Mohri M, Sharifi K, Eidi S. Hematology and serum biochemistry of Holstein 21. RStudio Team. RStudio: Integrated Development Environment for R. RStudio, dairy calves: age related changes and comparison with blood composition in Inc. (2016). Available online at: http://www.rstudio.com/ adults. Res Vet Sci. (2007) 83:30–9. doi: 10.1016/j.rvsc.2006.10.017 22. Graves S, Piepho H-P, Selzer L, Dorai-Raj S. Package “multcompView”. 11. Pérez-Santos M, Castillo C, Hernández J, Abuelo Á. Biochemical variables Visualizations of Paired Comparisons. (2015). Available at: https://cran.r- from Holstein-Friesian calves older than one week are comparable to those project.org/web/packages/multcompView/multcompView.pdf (accessed July obtained from adult animals of stable metabolic status on the same farm. Vet 25, 2015). Clin Pathol. (2015) 44:145–51. doi: 10.1111/vcp.12218 23. Tests for Normality [R Package Nortest Version 1.0-4]. Available online 12. Ventrella D, Dondi F, Barone F, Serafini F, Elmi A, Giunti M, et al. at: https://cran.r-project.org/web/packages/nortest/index.html (accessed The biomedical piglet: establishing reference intervals for haematology and December 14, 2018). clinical chemistry parameters of two age groups with and without iron 24. Hadley Wickham. ggplot2: Elegant Graphics for Data Analysis. (2016). supplementation. BMC Vet Res. (2017) 13:23. doi: 10.1186/s12917-017-0946-2 Available online at: https://www.springer.com/gp/book/9783319242750 Frontiers in Veterinary Science | www.frontiersin.org 10 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals 25. Auguie B. gridExtra: Miscellaneous Functions for “Grid” Graphics. Available in neonatal calves fed either colostrum or milk replacer at two levels. J Anim online at: https://cran.r-project.org/web/packages/gridExtra/index.html Sci. (2000) 78:609–20. doi: 10.2527/2000.783609x (accessed December 14, 2018). 46. Girard J, Ferré P, Pégorier JP, Duée PH. Adaptations of glucose and fatty acid 26. Kassambara A. ggpubr: “ggplot2” Based Publication Ready Plots. Available metabolism during perinatal period and suckling-weaning transition. Physiol online at: https://cran.r-project.org/web/packages/ggpubr/index.html Rev. (1992) 72:507–62. doi: 10.1152/physrev.1992.72.2.507 (accessed December 14, 2018). 47. Hammon HM, Steinhoff-Wagner J, Flor J, Schönhusen U, Metges CC. 27. Smith BI, Kauffold J, Sherman L. Serum haptoglobin concentrations in Lactation biology symposium: role of colostrum and colostrum components dairy cattle with lameness due to claw disorders. Vet J. (2010) 186:162–5. on glucose metabolism in neonatal calves. J Anim Sci. (2013) 91:685–95. doi: 10.1016/j.tvjl.2009.08.012 doi: 10.2527/jas.2012-5758 28. Chemistry Reference Intervals | New York State Veterinary Diagnostic Center. 48. Girard J. Metabolic adaptations to change of nutrition at birth. Neonatology. Available online at: https://ahdc.vet.cornell.edu/sects/clinpath/reference/ (1990) 58:3–15. doi: 10.1159/000243294 chem.cfm (accessed December 14, 2018). 49. Guignard JP, Drukker A. Why do newborn infants have a high plasma 29. Machado VS, Oikonomou G, Lima SF, Bicalho MLS, Kacar C, Foditsch C, creatinine? Pediatrics. (1999) 103:e49. doi: 10.1542/PEDS.103.4.E49 et al. The effect of injectable trace minerals (selenium, copper, zinc, and 50. Bueva A, Guignard J-P. Renal function in preterm neonates. Pediatr Res. manganese) on peripheral blood leukocyte activity and serum superoxide (1994) 36:572–7. doi: 10.1203/00006450-199411000-00005 dismutase activity of lactating Holstein cows. Vet J. (2014) 200:299–304. 51. Rauprich AB, Hammon HM, Blum JW. Effects of feeding colostrum doi: 10.1016/J.TVJL.2014.02.026 and a formula with nutrient contents as colostrum on metabolic and 30. Piñeiro C, Piñeiro M, Morales J, Andrés M, Lorenzo E, Pozo M del, endocrine traits in neonatal calves. Biol Neonate. (2000) 78:53–64. et al. Pig-MAP and haptoglobin concentration reference values in swine doi: 10.1159/000014247 from commercial farms. Vet J. (2009) 179:78–84. doi: 10.1016/j.tvjl.2007. 52. Blum JW, Hammon H. Colostrum effects on the gastrointestinal tract, 08.010 and on nutritional, endocrine and metabolic parameters in neonatal 31. Fossum C, Wattrang E, Fuxler L, Thorleif Jensen K, Wallgren P. Evaluation calves. Livest Prod Sci. (2000) 66:151–9. doi: 10.1016/S0301-6226(00)0 of various cytokines (IL-6, IFN-α, IFN-γ, TNF-α) as markers for acute 0222-0 bacterial infection in swine -a possible role for serum interleukin-6. Vet 53. Wilm J, Costa JHC, Neave HW, Weary DM, von Keyserlingk MAG. Technical Immunol Immunopathol. (1998) 64:161–72. doi: 10.1016/S0165-2427(98) note: serum total protein and immunoglobulin G concentrations in neonatal 00126-3 dairy calves over the first 10 days of age. J Dairy Sci. (2018) 101:6430–6. 32. Hadorn U, Blum JW. Effects of feeding colostrum, glucose or water on the doi: 10.3168/jds.2017-13553 first day of life on plasma immunoglobulin G concentrations and gamma- 54. Orro T, Jacobsen S, LePage JP, Niewold T, Alasuutari S, Soveri T. glutamyltransferase activities in calves. Zentralbl Veterinarmed A. (1997) Temporal changes in serum concentrations of acute phase proteins in 44:531–7. doi: 10.1111/j.1439-0442.1997.tb01139.x newborn dairy calves. Vet J. (2008) 176:182–7. doi: 10.1016/j.tvjl.2007. 33. Thompson JC, Pauli J V. Colostral transfer of gamma glutamyl transpeptidase 02.010 in calves. N Z Vet J. (1981) 29:223–6. doi: 10.1080/00480169.1981.34851 55. Murray CF, Windeyer MC, Duffield TF, Haley DB, Pearl DL, Waalderbos KM, 34. Kurz MM, Willett LB. Carbohydrate, enzyme, and hematology et al. Associations of serum haptoglobin in newborn dairy calves with health, dynamics in newborn calves1. J Dairy Sci. (2016) 74:2109–18. growth, and mortality up to 4 months of age. J Dairy Sci. (2014) 97:7844–55. doi: 10.3168/jds.S0022-0302(91)78383-5 doi: 10.3168/jds.2014-8465 35. Parish SM, Tyler JW, Besser TE, Gay CC, Krytenberg D. Prediction 56. Lykkesfeldt J, Svendsen O. Oxidants and antioxidants in disease: oxidative of serum IgG1 concentration in Holstein calves using serum gamma stress in farm animals. Vet J. (2007) 173:502–11. doi: 10.1016/j.tvjl.2006. glutamyltransferase activity. J Vet Intern Med. (1997) 11:344–7. 06.005 doi: 10.1111/j.1939-1676.1997.tb00478.x 57. Hurley WL. 9. Composition of sow colostrum and milk. In: The Gestating and 36. Pearson EG, Dirksen G, Meyer J, Seitz A, Rowe KE. Evaluation of liver Lactating Sow. Urbana-Champaign, IL: Wageningen Academic Publishers function tests in neonatal calves. J Am Vet Med Assoc. (1995) 207:1466–9. (2015). p. 193–230. 37. Andrieu S. Is there a role for organic trace element supplements in transition 58. Moeser AJ, Klok C Vander, Ryan KA, Wooten JG, Little D, Cook VL, cow health? Vet J. (2008) 176:77–83. doi: 10.1016/J.TVJL.2007.12.022 et al. Stress signaling pathways activated by weaning mediate intestinal 38. Thomson CD, Rea HM, Doesburg VM, Robinson MF. Selenium dysfunction in the pig. Am J Physiol Liver Physiol. (2007) 292:G173–81. concentrations and glutathione peroxidase activities in whole blood of doi: 10.1152/ajpgi.00197.2006 New Zealand residents. Br J Nutr. (1977) 37:457. doi: 10.1079/BJN19770049 59. Spreeuwenberg MAM, Verdonk JMAJ, Gaskins HR, Verstegen MWA. Small 39. Suzuki T, Agar NS, Suzuki M. Red cell metabolism: a comparative study intestine epithelial barrier function is compromised in pigs with low of some mammalian species. Comp Biochem Physiol B. (1984) 79:515–20. feed intake at weaning. J Nutr. (2001) 131:1520–7. doi: 10.1093/jn/131. doi: 10.1016/0305-0491(84)90358-4 5.1520 40. Nardone A, Lacetera N, Bernabucci U, Ronchi B. Composition of colostrum 60. Wu G, Meininger CJ, Kelly K, Watford M, Morris SM. A cortisol from dairy heifers exposed to high air temperatures during late pregnancy surge mediates the enhanced expression of pig intestinal pyrroline- and the early postpartum period 1. J Dairy Sci. (1997) 80:838–44. 5-carboxylate synthase during weaning. J Nutr. (2000) 130:1914–9. doi: 10.3168/jds.S0022-0302(97)76005-3 doi: 10.1093/jn/130.8.1914 41. Bauchart D. Lipid absorption and transport in ruminants. J Dairy Sci. (1993) 61. Piñeiro M, Piñeiro C, Carpintero R, Morales J, Campbell FM, Eckersall 76:3864–81. doi: 10.3168/jds.S0022-0302(93)77728-0 PD, et al. Characterisation of the pig acute phase protein response 42. Jaster EH, McCoy GC, Spanski N, Tomkins T. Effect of extra to road transport. Vet J. (2007) 173:669–74. doi: 10.1016/j.tvjl.2006. energy as fat or milk replacer solids in diets of young dairy calves 02.006 on growth during cold weather. J Dairy Sci. (1992) 75:2524–31. 62. Saco Y, Docampo MJ, Fàbrega E, Manteca X, Diestre A, Lampreave F, et al. doi: 10.3168/jds.S0022-0302(92)78014-X Effect of transport stress on serum haptoglobin and pig-map in pigs. Anim 43. Jaster EH, McCoy GC, Fernando RL. Dietary fat in milk or milk replacers Welf. (2003) 12:403–9. for dairy calves raised in hutches during the winter. J Dairy Sci. (1990) 63. Alava MA, González-Ramón N, Heegaard P, Guzylack S, Toussaint 73:1843–50. doi: 10.3168/jds.S0022-0302(90)78864-9 MJM, Lipperheide C, et al. Pig-MAP, porcine acute phase proteins and 44. Ontsouka EC, Albrecht C, Bruckmaier RM. Invited review: growth-promoting standardisation of assays in Europe. Comp Haematol Int. (1997) 7:208–13. effects of colostrum in calves based on interaction with intestinal cell surface doi: 10.1007/BF02658691 receptors and receptor-like transporters. J Dairy Sci. (2016) 99:4111–23. 64. Pauli U. Porcine TNF: a review. Vet Immunol Immunopathol. (1995) 47:187– doi: 10.3168/JDS.2015-9741 201. doi: 10.1016/0165-2427(94)05405-H 45. Kühne S, Hammon HM, Bruckmaier RM, Morel C, Zbinden Y, Blum JW. 65. Van Reeth K, Van Gucht S, Pensaert M. In vivo studies on cytokine Growth performance, metabolic and endocrine traits, and absorptive capacity involvement during acute viral respiratory disease of swine: troublesome Frontiers in Veterinary Science | www.frontiersin.org 11 April 2019 | Volume 6 | Article 123 Yu et al. Young Animal Reference Intervals but rewarding. Vet Immunol Immunopathol. (2002) 87:161–8. Conflict of Interest Statement: The authors declare that the research was doi: 10.1016/S0165-2427(02)00047-8 conducted in the absence of any commercial or financial relationships that could 66. Pié S, Lallès JP, Blazy F, Laffitte J, Sève B, Oswald IP. Weaning is associated be construed as a potential conflict of interest. with an upregulation of expression of inflammatory cytokines in the intestine of piglets. J Nutr. (2004) 134:641–7. doi: 10.1093/jn/134.3.641 Copyright © 2019 Yu, Canalias, Solà-Oriol, Arroyo, Pato, Saco, Terré and Bassols. 67. Sordillo LM, Aitken SL. Impact of oxidative stress on the health and immune This is an open-access article distributed under the terms of the Creative Commons function of dairy cattle. Vet Immunol Immunopathol. (2009) 128:104–9. Attribution License (CC BY). The use, distribution or reproduction in other forums doi: 10.1016/J.VETIMM.2008.10.305 is permitted, provided the original author(s) and the copyright owner(s) are credited 68. Rajabian F, Mohri M, Heidarpour M. Relationships between oxidative stress, and that the original publication in this journal is cited, in accordance with accepted haematology and iron profile in anaemic and non-anaemic calves. Vet Rec. academic practice. No use, distribution or reproduction is permitted which does not (2017) 181:265. doi: 10.1136/vr.104179 comply with these terms. Frontiers in Veterinary Science | www.frontiersin.org 12 April 2019 | Volume 6 | Article 123