Praca oryginalna Original paper
In mammals, vitamin D is necessary for calcium-phosphorus equilibrium, cell functioning, and activa-tion of the immune funcactiva-tion (2, 6, 11). A common form of vitamin D is ergocalciferol (vitamin D2), synthesized
directly from forage through digestion. Additionally, a well-known form of this vitamin is cholecalciferol (vitamin D3), which is converted from
7-dehydrocho-lesterol by ultraviolet (UV) sunlight on the skin in some species. In both of these forms, vitamin D is converted to active metabolite type 1,25-(OH)2D3 to be a various
conversion in the liver and kidney. As the active form of vitamin D, 25-hydroxyvitamin D (25(OH)D3) is
the best biomarker of vitamin D-associated vitamin D binding protein in circulation (16, 47).
In recent years, researchers have found vitamin D levels to be notable and have conducted significant research on them. The literature has generally found that serum or plasma vitamin D concentrations above 30 ng/mL are required in mature cattle to adequately meet demands and immune function (15, 28, 29). However, in calves classified as having low (15-25 ng/mL) and high (55-65 ng of 25(OH)D3/mL) vitamin
D status (30). Other research has revealed that calves fed anionic rations supplemented with 6 mg/d of
25-(OH) vitamin D3 had higher serum vitamin D levels
compared to calves fed with other ration types up to 3 days of age (52).
Metabolic diseases in the transition period that generally originate from negative energy balance, particularly those that exist prior to dairy cows exhibit-ing health problems, are a key cause of herd trouble in dairy cows (49). Excessive increased NEFA has been presented as one of the gold indicators of deficient ad-aptation to negative energy balance (13, 35). Calcium (Ca) is required to meet the demands of lactation and foetal bone improvement in the transition period for dairy cattle (1). In particular, colostrum calcium ma-teriality is provided by mobilization of calcium from bone stores, absorption of calcium from rations, and reduction of calcium elimination by the kidneys (5). As with NEFA, increased calcium levels after parturi-tion, called hypocalcaemia, are associated with other postpartum diseases (4, 21) and immune function (24). Lean et al. (22) emphasized calcium-affected energy balance through bone function.
Various studies in humans have demonstrated that NEFA and vitamin D have a negative relationship (26, 41). Only one study found that vitamin D conversion
Interpretation of 25-OH-D
3
, NEFA, and calcium
correlations among cow and calf pairs
SONGUL ERDOGAN1, KEREM URAL1, HASAN ERDOGAN1,DENIZ ALIC URAL2, MEHMET GULTEKIN1, SERDAR PASA1
1 Department of Internal Medicine, Faculty of Veterinary, Aydın Adnan Menderes University, Aydın, Turkey 2Faculty of Veterinary, Aydın Adnan Menderes University, Aydın, Turkey
Received 02.09.2020 Accepted 10.12.2020
Erdogan S., Ural K., Erdogan H., Ural D. A., Gultekin M., Pasa S.
Interpretation of 25-OH-D3, NEFA, and calcium correlations among cow and calf pairs
Summary
In the present study, the aim was to determine alteration of NEFA, calcium and vitamin D3 levels in cow
and calf pairs at parturition as well as correlation between each parameter levels. For this purpose, a cow-side device employing the enzymatic colorimetric method was used for measurement of NEFA and calcium levels. On the other hand, serum 25(OH)D3 analysis was performed using the fluorescence immunochromatographic
method at the laboratory in the Faculty. Blood samples were taken from Vena jugularis of 15 Simmental cow and calf pairs immediately after parturition and placed in serum and heparinized tubes. In cow and calf pairs, concentration of vitamin D3 (15.6-120 and 31.8-120 ng/mL, respectively), NEFA (0.12-1.2 and 0.09-0.8
mmol/L, respectively) and calcium (1.8 ± 0.9 and 2.2 ± 0.6 mmol/L, respectively) were determined. There was no significant correlation between NEFA, Ca, and vitamin D3 in cows and calf pairs. Taking into account several
co-factors that influenced test results, which could not easily be excluded, further studies may be warranted with larger cow-calf pair populations. In conclusion, vitamin D3 concentration in calves is not affected by the
negative energy balance of dams in the parturition period. Keywords: vitamin D, NEFA, cow, calf, calcium
levels and between NEFA and vitamin D3 levels.
Material and methods
Animal selection and sampling. A total of 15 clinically
healthy multiparous Simmental cow and calf pairs (0 days at parturition) were randomly enrolled from a Simmental herd located in Umurlu district of Aydın, Turkey. The herd had 150 cows placed in free-stall housing fed with total mix rations according to their lactation period and milk yield. Cows nearing parturition were transferred to individual pens for several postpartum days. Calves were separated from dams soon after parturition (Fig. 1).
Blood samples were drawn from the V. jugularis of both dams and their calves and inserted into serum (4 ml) and heparinized (4 ml) tubes for assays. Newborn calf blood was withdrawn after the first (30 minutes) colostrum con-sumption.
Biochemical analysis. To determine serum NEFA and
Ca levels, a Vet Photometer 700 DP (Diaglobal, Germany) was used for the cows, as it is able to provide results quickly (in the range 0.02-4.00 mmol/L) by use of an enzymatic colorimetric method. Briefly, 1 mL of blood taken was immediately transferred to eppendorf tubes to separate serum and plasma using a mini-centrifuge device. After the separation process, the NEFA levels for the cows were tested as follows: 1) 1000 µL of R1 reagent was added to
and sample tubes, and they were mixed. 2) after the first step, 50 µL of standard and plasma of each sample were added into only standard and sample tubes, and they were mixed again. 3) after the tubes were mixed, samples and standard cuvettes were immediately measured against the blank cuvette with the device at a wavelength of 520 nm.
The procedure to determine Ca levels was as follows: 1) 1500 µL of colour reagent and buffer solution were pipet-ted into each sample cuvette, including blank, standard, and sample cuvettes, respectively; 2) after adding 50 µL of hepa-rinized plasma into sample cuvettes and 50 µL of standard solution into standard cuvettes, excluding the blank cuvette, all cuvettes were mixed well; 3) immediately after step 2, the device was turned on, and calcium analysis was selected; 4) first the blank cuvette was inserted into the device, fol-lowed by the standard cuvette. Following this, all samples were measured at a wavelength of 520 nm.
Separated serum samples, on the other hand, were shipped to the laboratory, where cold chain and serum 25(OH)D3 analysis were performed using the
immuno-chromatographic fluorescence method with a Savant POCT analyser (Beijing Savant Biotechnology Co., Ltd., China). Tests were performed based on the manufacturer’s prin-ciple of serum 25(OH)D3: 1) test kits were left at room
temperature, at 18-28°C, and ID card information was loaded and limited information from 10 vitamin D kits
was automatically instructed by the device; 2) 30 µL of serum and 25(OH)D3 diluent solution were mixed in the
eppendorf tube; 3) for each sample, 60 µL of the solution mixed in the eppendorf tube was pipetted onto the sample septum of one of the vitamin D test kits that had waited for 15 minutes at room temperature; 4) the vitamin D test kits were then inserted into the device, and the results were read by the device (Fig. 2).
Statistical analysis. The mean and standard deviation
of the results obtained from mothers and offspring were determined and tabulated. Normality tests of the data within the groups were performed, and an independent t test was used to determine the differences between the groups. Pearson’s correlation was used to determine the relation-ship between vitamin D3 concentrations and NEFA and Ca
values of mothers and offspring. The program SPSS 22 was used in all analyses, and p < 0.05 was accepted as statisti-cally significant.
Results and discussion
In cattle and their calves, the serum concentration of vitamin 25(OH)D3 (39.9 ± 29.1 and 49.1 ± 27.8 ng/
mL, respectively), NEFA (0.6 ± 0.9 and 0.42 ± 0.25 mmol/L, respectively), and calcium (1.8 ± 0.9 and 2.2 ± 0.6 mmol/L, respectively) were determined. A nonsignificant positive correlation exists between NEFA and calcium concentrations of dams’ vitamin D levels; in the offspring, a negative correlation exists between 25(OH)D3 vitamin and NEFA concentrations,
and a statistically insignificant correlation exists be-tween NEFA and calcium levels.
In ruminants, numerous dynamics may regulate vita-min D3 synthesis. Factors such as exposure to sunlight,
season, and latitude could directly influence vitamin D3 production independent of these animals’ hair coat
(19, 20). Furthermore, supplementation of vitamin D3
increases the serum levels in winter but has no posi-tive effects in summer if the cows are on pasture (18). Cattle obtain vitamin D through skin exposure to UV sunlight and through ingestion. Livestock rations are commonly supplemented with vitamin D to regulate calcium balance (31), but this traditional aspect of pre-ventive medicine for a cow’s transition period involves the risk of many types of diseases related to metabolic and immunity dysfunction in early lactation (3, 45, 46). In recent years, a significant body of research has emphasized the effects of vitamin D on the regulation of immunity and metabolic homeostasis in transition dairy cows and neonatal calves (7, 8, 33, 36). Generally, serum or plasma vitamin D concentrations above 30 ng/ mL are required for cattle to adequately meet demands and immune function (15, 28, 29). Holcombe et al. (14) argue that depletion of the level of vitamin D (82.6 ± 1.7 ng/mL) in the first week postpartum, diversely. It might be classified low (15-25 ng/mL) and high (55-65 ng of 25(OH) D3/mL) in calves (30). Vieira-Neto et al. (51) state that administering vitamin D3 (at a dose of
300 µg) at parturition increases ionized calcium, total phosphorus, and vitamin D3 levels for the first week
after calving and promotes innate immune status in early lactation. Their study also found that vitamin D3
administration did not influence NEFA concentrations during the first 2 weeks after calving. Weiss et al. (52) found that using rations with a negative cation-anion difference and vitamin D3 in dams had no effect on
their calves’ vitamin D status during the first 3 days. However, vitamin D concentrations in milk doubled in cows fed supplemental anions and vitamin D3 to
control on 28 days in lactation. Similarly, the present study found no significant correlation between vitamin D and NEFA levels or between vitamin D and calcium
to 1 mmol/L in the first week of parturition increase the risk of transition period disorders (35). In calves after parturition, before colostrum intake increases lipolysis as a result of elevated sympathomedullary activity for thermoregulation, NEFA concentrations may be high and may remain elevated if calves are not supplied with colostrum (10). Concentrations of NEFA and β-hydroxybutyrate (BHBA) are a practical biomarker of calves’ fat mobilization and energy bal-ance (12). Energy balbal-ance alterations could affect the metabolic and endocrine pathways to health protection and the ability to manage diseases (9). Collard et al. (5) state that higher concentrations of NEFA regress immune status and simplify disease development. In newborn calves (1-2 weeks of age) exposed to 24-hour transport, NEFA concentrations were found to be 0.46 mmol/L by Radostits et al. (38). We found no correlation between NEFA concentrations in calves and in their dams; NEFA concentrations were found to be 0.6 ± 0.9 in dams and 0.42 ± 0.25 mmol/L in calves. These concentrations are within the normal ranges and within the ranges indicated in the above literature; this outcome can be explained by the fact that the present study was performed with healthy animals and with only one measurement – a limitation to this study.
Parathormone plays a major role in hypocalcaemia through various physiological processes in monogas-tric mammals and its relationship to vitamin D3. Briefly,
parathormone causes an increase in 1,25(OH)2D in
kidneys, which plays a key role in the effect of vitamin D receptors (VDR) on osteoblasts. Vitamin D recep-tors induce osteoblasts to produce nuclear factor KB ligand (RANKL), which activates the located osteo-clasts principal to bone absorption (48). In addition, vitamin D elevates the expression of calbindin-D9k. Calbindin-D9k has an affinity to bind and transfers the calcium to the basolateral membranes of cells by diffusion through the cytosol (23). However, the aforementioned mechanisms are not specific to rumi-nants. Independently, rumen epithelium has an ability to provide active and passive transport for calcium (43). In the postpartum period, one reason for reduced dry matter intake and negative energy metabolism is subclinical hypocalcaemia (25). It is well known that vitamin D3 and parathormone are directly related to
hypocalcaemia in cows. However, using vitamin D3 on
calving did not affect dry matter intake, milk yields, or
tokines (e.g., IL-17A, INF-γ; 28). Reinhardt et al. (39) state that vitamin D application (200 µg, İM) before vaccination increases immunoglobulin levels in milk and blood serum in dairy cattle. Especially in calves and cows in the transition period, an adequate immune state plays an important role for infectious agents. However, the relationship between vitamin D and NEFA and calcium concentrations should be investigated in further studies, which should consider the factors and biomarkers related to tight homeostatic regulation in transition periods.
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Corresponding author: Songul Erdogan, DVM, PhD; Department of Internal Medicine, Faculty of Veterinary, Adnan Menderes University, Isikli, Aydın, 09017, Turkey; e-mail: songultp.09@gmail.com
