Evaluation of blood biochemical parameters is critical to identify and monitor health status and disease incidence in dairy cattle farms. Evaluation of oxidative status is a relatively young field in ruminant medicine. Indeed, most studies in cattle, sheep and goats have been sporadic and dealt predominantly with the effects of diseases, including mastitis, pneumonia, sepsis, acidosis, ketosis, enteritis, joint diseases and retained placenta (Celi, Reference Celi2010). Increasingly, research has focused on metabolic diseases that affect dairy ruminants during the peripartum period (LeBlanc, Reference LeBlanc2010; Sepúlveda-Varas et al. Reference Sepúlveda-Varas, Huzzey, Weary and Von Keyserlingk2013). Exploration of metabolic profiles through the analysis of biochemical parameters in dairy cattle blood is well established (e.g., Payne et al. Reference Payne, Dew, Manston and Faulks1970). However, metabolic profile tests as an applicable approach can reveal which areas of nutrition and management require more attention in dairy herds (Kolver & Macmillan, Reference Kolver and Macmillan1994; Macrae et al. Reference Macrae, Whitaker, Burrough, Dowell and Kelly2006).
In recent years, there has been a growing concern for detection of free radical damage as a complementary tool in the evaluation of metabolic status. Under normal physiological conditions, the body has sufficient reserves of antioxidants to combat free radical accumulation. Production of reactive oxygen species (ROS) is the outcome of aerobic metabolic pathways in the body (Halliwell, Reference Halliwell2006). Achieving the balance between production and neutralisation of ROS is crucial for maintaining the homeostasis (Poljsak et al. Reference Poljsak, Šuput and Milisav2013). Oxygen free radicals are produced during the productive phases of domestic animals (Piccione et al. Reference Piccione, Borruso, Fazio, Grasso and Caola2006; Celi, Reference Celi2010). Oxidative stress results from increased exposure to, or production of, oxidants, and can be monitored with a number of biomarkers. Several methods have been developed to assess total antioxidant capacity (TAC; Del Rio et al. Reference Del Rio, Stewart and Pellegrini2005); TAC, for example, describes the dynamic equilibrium between pro-oxidants and antioxidants in the plasma compartment (Castillo et al. Reference Castillo, Hernandez, Bravo, Lopez-Alonso, Pereira and Benedito2005). Monitoring of the efficiency of antioxidative systems against ROS is made by the estimation of TAC. The primary antioxidant capacity of serum is derived from non-enzymatic antioxidants, such as glutathione, α-tocopherol, β-carotene and uric acid (Halliwell & Gutteridge, Reference Halliwell and Gutteridge2007). A sensitive, reliable and useful indicator for measurement of cumulative action of all the antioxidants present in plasma is TAC. TAC is also a useful tool for measuring stress in transported calves (Pregel et al. Reference Pregel, Bollo, Cannizzo, Biolatti, Contato and Biolatti2005), and could potentially be used to evaluate the general nutritional status of animals (Mandebvu et al. Reference Mandebvu, Castillo, Steckley and Evans2003; Zdunczyk et al. Reference Zdunczyk, Flis, Zielinski, Wróblewska, Antoszkiewicz and Juskiewicz2006).
Evaluation of TAC along with measuring thiols groups as the major portion of the total body antioxidants, and plasma levels of malondialdehyde (MDA), a degradation product of lipid peroxidation are useful in specify their relationship with different lactational stage and dry period in dairy cow.
Plasma total thiols as a marker of oxidative protein damage constitute the major portion of the total body antioxidants and play a significant role in defence against reactive oxygen species. The antioxidant capacity of thiol compounds is due to the sulphur atom, which can easily accommodate the loss of a single electron (Karoui et al. Reference Karoui, Hogg, Fréjaville, Tordo and Kalyanaraman1996). Decreased levels of total thiols are reported in various physiological and pathological conditions, including diabetes mellitus, cardiovascular disorders, and disorders related to kidney, neurological disorders due to excess free radical generation (Mungli et al. Reference Mungli, Shetty, Tilak and Anwar2009). The entire antioxidant system seems to be under homeostatic control.
It is clear that primiparous cows, and cows in early lactation, are more sensitive than multiparous cows to disease (metabolic & infectious disease). Therefore, the aim of this current study was to carry out an assessment of metabolic and oxidative markers in order to find a sensitive test that revealed the health condition of at-risk cows (especially primiparous or in early lactation). This was conducted using three parameters: (1) TAC; (2) total thiols, as markers of oxidative protein damage, and; (3) plasma levels of MDA.
Material and methods
Animals, husbandry and nutrition
A total of 69 Holstein breed dairy cows were used in the study, and each animal was observed once. All animals were held in intensive farm breeding premises, divided into groups according to level of production. Feed was adjusted to production intensity, and consisted of alfalfa hay, corn silage, wheat bran and concentrates with energy and protein supplements (Tables 1 and 2). For each animal the clinical history was reviewed with the farm manager, physical examinations were made by the first author. Cows with suspected clinical disease were excluded from the study and an alternate animal chosen from the list previously selected by random numbers. Cows were body condition scored on a five-point scale (1 = thin, 5 = obese). Maximum 0·25 differences in body condition score (BCS) were accepted between cows in each group. Group allocation was as thus: Group E (fresh): dairy cows from the 3rd to 21st day of lactation, (E1: primiparous, E2: multiparous). Group M (peak milk): dairy cows from the 45th to 100th day of lactation, (M1: primiparous, M2: multiparous). Group L: dairy cows from the 260th day of lactation until the end of milk production, (L1: primiparous, L2: multiparous). Group F: dairy cows in the far-off dry period (from the 305th–345th day). Group C: dairy cows in the close-up dry period (from the 346th–360th day).
The composition of the diets of different groups (% DM)
Group E: dairy cows from the 3th to 21th day of lactation, (E1: primiparous, E2: multiparous); Group M: dairy cows from the 45th to 100th day of lactation, (M1: primiparous, M2: multiparous); Group L: dairy cows from the 260th day of lactation until the end of milk production, (L1: primiparous, L2: multiparous); Group F: dairy cows in the far-off dry period (from the 305th–345th day); Group C: dairy cows in the close-up dry period (from the 346th–360th day).
Estimated daily nutrients fed to the cows in different groups
† Dry matter (DM), net energy lactation (NEl), crude protein (CP), rumen degradable protein (RDP), rumen undegraded protein (RUP), neutral detergent fiber (NDF), non-fibrous carbohydrates (NFC), ether extract (EE). Group E: dairy cows from the 3th to 21th day of lactation, (E1: primiparous, E2: multiparous); Group M: dairy cows from the 45th to 100th day of lactation, (M1: primiparous, M2: multiparous); Group L: dairy cows from the 260th day of lactation until the end of milk production, (L1: primiparous, L2: multiparous); Group F: dairy cows in the far-off dry period (from the 305th–345th day); Group C: dairy cows in the close-up dry period (from the 346th–360th day).
Sample collection and analytical procedures
Blood samples were collected by coccygeal venepuncture between 8 and 9 A.M. on three consecutive days. Samples were collected in plain tubes and vacationers containing EDTA (10 : 1 ratio), centrifuged (750 g , 15 min, room temperature), and stored at −80C until analysis. Samples exhibiting haemolysis were discarded.
Measurement of TAC
The ferric reducing antioxidant potential (FRAP) assay was used to measure TAC, using the DetectX® Ferric Reducing Antioxidant Power (FRAP™) Detection Kit (Company Arbor Assays, USA). FRAP is expressed as mmol/l.
Measurement of total thiols
Total thiols in plasma were determined according to the spectrometric method of Hu using the Measure-iT™ Thiol Assay Kit (Invitrogen European Headquarters Company, Paisley PA4 9RF, UK). Plasma total thiols are expressed as mmol/l (Hu, Reference Hu1994).
Measurement of MDA
To evaluate lipid peroxidation in serum, a modified HPLC method was used based on the reaction of MDA with thiobarbituric acid (TBA), to form a coloured MDA–TBA adduct (Lykkesfeldt, Reference Lykkesfeldt2007). Forty μl of sample was diluted with 100 µl of H2O and mixed with 20 µl of 2·8 mmol/l butylated hydroxyl toluene (BHT) in ethanol, 40 µl of 81 g/l sodium dodecyl sulphate and 600 µl of TBA reagent (8 g/l TBA diluted 1 : 1 with 200 ml/l acetic acid adjusted to pH 3·5 with NaOH). The mixture was immediately heated (60 min at 95 °C) and cooled with running water; 200 µl of H2O and 1000 µl of butanol–pyridine (15 : 1, v/v) were then added. After vigorous mixing, the organic layer was separated by centrifugation (3 min at 16 000 g ). The supernatant was analysed on a UV-visible spectrophotometer fitted with an 80 µl flow cell. The absorbance was measured at 532 nm (the mobile phase consisted of 300 ml/l methanol in 50 mmol/l potassium dihydrogen phosphate buffer, pH 7·0). 1, 1, 3, 3-tetraethoxypropane was used as a standard, and MDA-TBA reactive substances’ values were expressed as MDA (mmol/l). The HPLC system consisted of a solvent delivery pump (JASCO 980-PU, Tokyo, Japan), a reversed-phase column (Luna C18, 250 × 4·6 mm, Phenomenex, CA, USA), and a UV–Vis detector (Jasco, UV-975, Tokyo, Japan) operated at 532 nm.
Statistical analyses
The results were analysed using SPSS (version 21.0) software. The data are presented as mean ± standard error of mean (sem). Residuals were tested for normality using the Kolmogorov–Smirnov method. Parity (multiparous vs primiparous) was included in initial models, and body condition score was included as a covariate. Body condition score did not significantly affect any of the models, and was excluded from further analysis. Statistical analysis was performed using one-way ANOVA, followed by Bonferroni-corrected post-hoc comparisons. Effect sizes (Partial-eta2; η2 p) are reported for all significant ANOVAs. The alpha level for all statistical tests was set at P < 0·05.
Results
There was a significant main effect of group on total antioxidant capacity (TAC), F (4, 64) = 3·35, P > 0·05, η 2 p = 0·17. Bonferroni-corrected post-hoc tests revealed that there were no detectable significant differences between the treatments at the adjusted alpha-level, but there was a suggestion of a difference between groups M (446·44 ± 23·75) and C (550·55 ± 28·41; P = 0·056). There were no main effects of group on malondialdehyde (MDA; F < 1), nor total thiols (F < 1).
With the inclusion of multiparity in the model, a significant effect of group on TAC resulted for groups E, M and L; F (2, 43) = 3·8, P < 0·05, η2p = 0·15.
Figure 1 displays the data for TAC according to group with parity included in the model. For TAC, including parities in the model for groups E, M and L revealed a significant effect of group, F (2, 43) = 3·8, P < 0·05, η 2 p = 0·15. Here, the Bonferroni-corrected post-hoc test revealed group M < group L (P < 0·05). In addition, there was a significant main effect of number of pregnancies on TAC, with primiparous < multiparous, F (1, 43) = 4·46, P < 0·05, η 2 p = 0·09. There was no group x parity interaction (F < 1). For MDA, there was no effect of group (F < 1) nor parities (F < 1) nor their interaction, F (2, 43) = 2·04, P < 0·05. Finally, there was no main effect of group on total thiols (F < 1). There was, however, a main effect of parities, with multiparous cows (13·27 ± 0·82) having lower total thiols than primiparous cows (15·63 ± 0·73), F (1, 43) = 4·63, P < 0·05, η 2 p = 0·1.
Total antioxidant capacity (mmol/l) for cows in groups E, M and L, and for primiparous and multi parous cows; Group E (fresh): dairy cows from the 3th to 21th day of lactation, Group M (peak milk): dairy cows from the 45th to 100th day of lactation, Group L: dairy cows from the 260th day of lactation until the end of milk production; Note: *P < 0·05 (Bonferroni corrected).
Discussion
In the present study, cows in the dry period (C group) had the highest level of TAC and postpartum and high yielding cows (E and M groups, respectively) had the lowest levels of TAC when controlling for parities. Primiparous cows have lesser TAC when compared with multiparous cows, supporting Albera & Kankofer (Reference Albera and Kankofer2010), who found significantly higher TAC values in plasma of second lactation cows. Social and maintenance behaviour of primiparous and multiparous cows were compared by González et al. (Reference González, Yabuta and Galindo2003). They concluded that cows giving birth for the first time had to face more intensive stress not only because of new physiological experience but also because of new procedures in herd management. Based on our data, it appears that multiparous cows may become accustomed to diverse circumstances more readily than primiparous cows (Liesegang et al. Reference Liesegang, Risteli and Wanner2007).
Immediately after parturition, MDA levels increased, while blood TAC and total thiols began to decrease. The main source of plasma total thiols is albumin, which is synthesised in the liver. Lower plasma total thiols levels observed in the E and M groups might explain the reduction in liver function that is usually observed in the early postpartum period (Celi, Reference Celi2010). Lower levels of total thiols were seen in multiparous cows compared to primiparous cows. Increase of metabolic rate after calving in accordance with increase of milk production in multiparous cows might be responsible for the elevation of respiratory electron transfer and use of total thiols as efficient antioxidants for reacting with free radicals to protect against damages (Rahman, Reference Rahman2007).
MDA is an end product formed via the decomposition of certain primary and secondary lipid peroxidation products (Janero, Reference Janero1990). In the present study, comparison among different groups and between multiparous and primiparous cows found no differences in MDA level. Despite there being no detectable differences between the oxidative measures in the groups, we reasoned that variation in oxidative parameters may be dependent on cumulative impacts of multiple pregnancies in groups E, M and L. Previous studies in dairy cows have found contrasting results; some findings show dramatic changes in plasma MDA concentrations (Bernabucci et al. Reference Bernabucci, Ronchi, Lacetera and Nardone2005), whilst others don't show any significant changes (Castillo et al. Reference Castillo, Hernandez, Bravo, Lopez-Alonso, Pereira and Benedito2005, Reference Castillo, Hernandez, Valverde, Pereira, Sotillo, Alonso and Benedito2006). Discrepancies are due to the great individual variation in MDA concentrations measured in the various studies or different methodologies employed to assess MDA (Castillo et al. Reference Castillo, Hernandez, Bravo, Lopez-Alonso, Pereira and Benedito2005, Reference Castillo, Hernandez, Valverde, Pereira, Sotillo, Alonso and Benedito2006; Celi, Reference Celi2011). Similarly, studies in transported cattle have failed to report a consistent change in MDA concentration (Celi, Reference Celi2011). It should also be mentioned that in the present study, all of the animals were clinically healthy. Our results reveal for the first time that the increase in TAC is not parallel to an increase in MDA levels especially in primiparous cows. This means that the metabolism of these cows produces peroxides faster than the capacity of their antioxidant system.
Conclusion
Any attempts to reduce prevalence of production diseases should rely on a continuous and comprehensive monitoring of appropriate indicators on the farm level and accounting for the ability of dairy cows in different stages of lactation to cope with unbalanced situations. Many studies revealed consumption of antioxidants and induction of oxidative stress in disease and disorders conditions. The present study demonstrates for the first time that apparently clinically healthy dairy cows may have lower total antioxidant capacity (especially in primiparous cows) and that this condition might make them more sensitive to stress, disease or health problems. Furthermore, we suggest that measurement of total antioxidant capacity maybe a useful addition to traditional metabolic profile tests for evaluating the herd health status.
The authors would like to acknowledge the University of Birjand for support of this study. Also, we highly appreciate the assistance of Professor Zarban for measurement of laboratory parameters and staff of Animal Health Management Department of Shiraz University for their valuable comments.
