Oxidative stress resulting from an imbalance between oxidant and reductant (antioxidant) substances in a living organism plays a pivotal role in several pathological events associated with animal production. The majority of the evidence attests to the fact that high-yielding dairy cows experience such stress during the periparturient period (4, 6, 7). Dairy cows with high body condition scores and high body condition losses are more prone to oxidative stress during this period (4), and increased lipid peroxidation around parturition has been reported (6). Sordilo and Aitken (15) observed that high concentrations of reactive oxygen species (ROS) during this period could expose cows to increased oxidation status. Elevated levels of reactive oxygen metabolites (ROM) at calving have been reported (12). An increase in ROS, also, occurs immediately after parturition (1). The oxidative stress index (OSi), determined as the ratio of the reactive oxygen species (ROS) to serum antioxidant capacity (SAC), has been proposed as a more accurate index for estimating the antioxidant status in a living organism (1). According to the same authors (1), OSi levels were higher in cows post-partum compared to cows in late lactation. Another blood parameter that has been widely associated with oxidative stress in dairy cows is
Mastitis is a severe infection in both heifers and multiparous cows caused by environmental pathogens, especially coliforms, and is considered as one of the most prevalent and costly production diseases in the dairy sector. The disease lowers milk production and milk quality, resulting in higher production costs. One factor making mastitis a major challenge during the transition from the dry period to lactation, when its incidence is higher (3, 12, 14), is the fact that dairy cows undergo many metabolic and physiological changes including immunosuppression (13, 14, 15). Oxidative stress is considered a major underlying cause of these changes (2, 11). However, as no specific clinical symptoms are directly connected with oxidative stress (in a cause-effect relationship), oxidation biomarkers are used to determine the levels of oxidation and inform the choice of measures to alleviate its impact on cow health (2, 8). Our working hypothesis is that cows with higher oxidation load might be more prone to developing mastitis. Thus, the objectives of the present study were (a) to determine the effect of udder condition (healthy or mastitic) on the levels of the oxidative stress indicators ROS, SAC, and OSi at dry-off, calving, and 30 days (30 d) post-partum in dairy cows; (b) to determine whether differences occur in
A total of 110 dairy cows from two commercial farms located in the northern part of Greece participated in this observational field study. Healthy animals were designated as group A (n = 90), and mastitic cows were group B (n = 20). Diets (based on dry matter basis) during the dry period were 42% corn silage, 40% straw hay, 9.6% soybean meal, and 8.4% molasses. After calving, the diet was 48.5% corn silage, 16.5% soybean meal, 14% alfalfa hay, 12% corn, 3.7% molasses, 1.7% rumen-protected fat, and 3.5% vitamin and mineral premix.
Blood samples from all cows were collected at dry-off, calving, and 30 d post-partum. Blood serum was obtained after centrifugation at 820 × g for 10 min at 4°C, and it was stored at −80°C for further analysis. The determination of the
Cases of clinical mastitis during the dry period and the subsequent lactation period were diagnosed and recorded by the veterinarian personnel of the farms. Mastitis was initially identified based on the occurrence of gross swelling, heat, redness, pain, disturbed function characterised by a decrease in milk production, or changes in milk composition. The animals were also examined for systemic symptoms (fever, depression, shivering, loss of appetite, and loss of weight) or lesser symptoms (fever or milder depression). Lastly, the milk was checked for watery appearance and the presence of clots. Milder cases of mastitis that occurred during middle and late lactation were left untreated but were recorded. A cow was classified as having mastitis only once, regardless of how many quarters were mastitic or whether the infection recrudesced. Of the 110 cows, 20 developed clinical mastitis. Fifteen cows developed mastitis in early lactation (days in milk less than 45), four cows developed mastitis during middle to late lactation (days in milk 124–245), and one cow did during the dry period. Cows in early lactation and the dry period were treated, while all cows in middle and late lactation were left untreated because they developed milder symptoms. Milk samples from all cows (except that at dry period), which developed symptoms were collected and bacteriological analyses were performed following the methods previously described (9).
Animals were divided into two groups based upon udder health. Group A comprised healthy cows (n = 90) and group B (n = 20) cows that developed mastitis. Α general linear model (GLM) was used for analysing the associations between the determined blood parameters (ROS, SAC, OSi and
statistical model was:
Yij: the mean value of the examined parameter, μ: the general mean,
Gi: the fixed effect of animal group based on udder health (i = 1 – group A, i = 2 – group B),
Lj: the fixed effect of the sampling period (j = 1 – dry-off, 2 – calving, 3 – 30 d post-partum),
Gi*Lj: the effect of interaction between udder health and sampling period, and
eij: the random error.
Receiver operating characteristic curve (ROC) analysis was further implemented to test whether the determined oxidative biomarker concentrations (ROS, SAC, OSi, and
Comparisons of ROS, SAC, and OSi values between group A and group B are presented in Table 1. There were no differences in all three parameters between the two groups at both dry-off and 30 d postpartum. In contrast, at calving time, ROS and OSi levels were higher (P < 0.001) by 13.9% and 25.7%, while SAC levels were lower (P < 0.001) by 10% in group B than in group A. In addition, Table 1 allows for a comparison of ROS, SAC and OSi values between the three sampling points within each group (A and B). The results indicated that fluctuations in the values of all parameters over time were similar in both groups. The highest value for ROS and OSi and the lowest value for SAC occurred at calving.
Effect of the health of the udder (healthy or mastitic) on ROS (CarrU), SAC (μmol HClO/ml) and OSi (ROS/SAC) in the blood of dairy cows at dry-off, calving and 30d post-partum. Group A – healthy animals; Group B – mastitic animals. Values are presented as LSM ± SEM
Parameter | Group A | Group B |
---|---|---|
Dry-off | ||
ROS | 51.87a, 1 ± 0.85 | 54.85a, 1 ± 1.87 |
SAC | 447.31a, 1 ± 2.52 | 431.64a, 1 ± 5.52 |
OSi | 0.117a, 1 ± 0.002 | 0.127a, 1 ± 0.005 |
Calving | ||
ROS | 65.89a, 2 ± 0.86 | 75.04b, 2 ± 1.87 |
SAC | 347.38a, 2 ± 2.54 | 312.38b, 2 ± 5.52 |
OSi | 0.191a, 2 ± 0.002 | 0.240b, 2 ± 0.005 |
30 d post-partum | ||
ROS | 53.46a, 1 ± 0.86 | 57.07a, 1 ± 1.87 |
SAC | 451.42a, 1 ± 2.54 | 440.36a, 1 ± 5.52 |
OSi | 0.118a, 1 ± 0.002 | 0.130a, 1 ± 0.005 |
a,b Different superscripts within the same row indicate significant difference (P < 0.001) between the two groups. 1,2 Different superscripts within the same column for the same parameter indicate significant difference (P < 0.001) between sampling points
The values of
The graphical representation of the ROC analysis curve using each of ROS, SAC, OSi, and
Diagnostic performance (receiver operating characteristic curve coordinates) of ROS, SAC, OSi and
Biomarker /Sampling point | Area (AUC) Under Curve | Cut-off (Threshold) | Sensitivity | Specificity | P-value |
---|---|---|---|---|---|
Dry-off | |||||
ROS | 0.586a ± 0.060 | >50.0 | 89.5 | 42.9 | ns (0.16) |
SAC | 0.674ab ± 0.067 | ≤450.4 | 89.5 | 45.1 | ** |
OSi | 0.663b ± 0.057 | >0.113 | 89.5 | 48.4 | ** |
0.730b ± 0.048 | ≤ 5.2 | 100 | 49.5 | *** | |
Calving | |||||
ROS | 0.869a ± 0.046 | >70.2 | 94.7 | 71.1 | *** |
SAC | 0.949b ± 0.027 | ≤320.4 | 89.5 | 88.9 | *** |
OSi | 0.952b ± 0.026 | >0.224 | 89.5 | 88.9 | *** |
0.805a ± 0.045 | ≤1.9 | 94.7 | 69.2 | *** | |
30 d post-partum | |||||
ROS | 0.614ab ± 0.064 | >46.7 | 100 | 26.7 | ns (0.075) |
SAC | 0.592a ± 0.063 | ≤467.3 | 100 | 26.7 | ns (0.14) |
OSi | 0.636ab ± 0.064 | >0.106 | 94.7 | 32.2 | * |
0.710b ± 0.053 | ≤4.5 | 68.4 | 75.8 | *** |
a,b Different superscripts within the same column for each sampling point (dry-off, calving, and 30 d post-partum) indicate significant difference (ns -not significant; *P < 0.05; **P < 0.01; ***P < 0.001)
The first finding emerging from the present study is that there is a strong association between the health of the udder and all three oxidative stress biomarkers determined (ROS, SAC, OSi) at calving but not at dry-off or 30 d post-partum. The association at calving does not imply a cause-effect relationship, but it is tempting to speculate that the increased oxidation load at calving might make cows more prone to develop mastitis during the subsequent lactation. Higher oxidation loads during the perinatal and early lactation period in dairy cows result in dysfunctional immune response activity (2, 14).
This suppression of immune response impairs how the organism counteracts inflammation, and thus, cows are more prone to mastitis (5, 11, 14). Our results are not in agreement with a previous study by our group, which reported that there were no differences between ROM and thiol group (SH) levels, and ferric-reducing ability in the blood serum of healthy cows and these levels and ability in the serum of cows with mastitis at dry-off or at calving (12). There is no explanation as to why these differences between the two studies occurred, except the possibility of the set of parameters determined in this study being more sensitive.
The second finding from the study published here is that the determined levels of the oxidative stress biomarkers and blood
The third finding brought to light by this study is the lower
In conclusion, a higher oxidation load was detected in group B (cows that developed mastitis) compared to group A (healthy cows) during the periparturient period. All three oxidative stress biomarkers exhibited perfect or excellent ability at calving for predicting which cows would develop mastitis in the subsequent lactation. The values of