Introduction
The hormones thyroxine (T4) and triiodothyronine (T3) are synthesized exclusively by the thyroid through a series of reactions linked to the histological organization of the gland (Carvalho and Dupuy, 2017). Thyroid hormones affect growth, development, differentiation, reproduction, and several routes of intermediary metabolism. Although T3 is the biologically active hormone, T4 is considered a pro-hormone that requires conversion to T3 (Suttle, 2010). The deiodination process is catalyzed by one of the three deiodinase enzymes currently described, distributed mainly in extrathyroidal tissues (Beckett and Arthur, 2005). All deiodinases contain a residue of selenocysteine in their active site (Stadtman, 2000); therefore, an adequate metabolic balance of selenium (Se) is essential for synthesis, activation, metabolism, and secretion of thyroid hormones. An insufficient supply of Se in ruminants compromises the metabolism of thyroid hormones affecting the conversion of T4 to T3 (Contreras et al., 2002; 2005; Voudouri et al., 2003; Rowntree et al., 2004). Calves born from mothers with negative metabolic balances of Se have a compromised Se status and neonatal synthesis of thyroid hormones (Awadeh et al., 1998; Guyot et al., 2011). To correct or control clinical deficiency it is common to resort to inorganic (e.g., sodium selenite) or organic Se sources (e.g., selenized yeasts; Davis et al., 2008). In this regard, Se metabolic path depends on the source employed (Ortman and Pehrson, 1999; Juniper et al., 2008; Slavik et al., 2008), with selenite being more easily metabolized to the immediate precursors of selenocysteine in comparison with organic forms of Se. Barium selenate (BaSeO4) is an inorganic source used in the formulation of prolonged-release preparations to provide an adequate Se status for at least 7 months (Leyán et al., 2004; Judson and Badidge, 2010), generating a high and sustained concentration of Se in plasma and liver compared to other inorganic forms (Davis et al., 2008).
The hypothesis of this study was that blood concentration of thyroid hormones in newborn calves from Se-deficient mothers can be improved by supplementing the mother with barium selenate as a source of Se. Therefore, this study aimed to evaluate the blood concentration of T3 and T4 in newborn calves from mothers supplemented with barium selenate during prepartum.
Materials and methods
Ethical considerations
The experiment was developed according to the bioethics protocols for the use of animals in research of Universidad Austral de Chile (Fondecyt 119-09939).
Animals
Twelve clinically healthy black Friesian cows were used for the study, conducted in Valdivia, Chile (39º48'LS; 73º15'LO). The animals were distributed in two groups of six cows each, homogeneous in terms of age (6.5 years), number of deliveries (3.5 deliveries), body weight (594 kg), gestational age (230 days), and milk production in the last lactation (6,406 L).
Experimental design
Throughout the experimental period, animals were housed in individual stalls on concrete floor and straw bed. All cows were fed a Se-deficient diet (Se <0.05 ppm) consisting of 9.5 kg of natural pasture hay [Se = 0.02 ppm of dry matter -DM), and 1 kg of commercial concentrate (Cosetan® Biomaster IANSA, Quepe, Chile; Se = 0.12 ppm DM) in individual feeders, and water ad libitum. One group of animals was selected at random and supplemented with Se (Se-S), and the other group remained as control without supplementation (Se- D). After calving, which occurred in August (late winter), the calves received 4 L colostrum from their mothers during the first 24 hours and stayed with them for three days. Afterward, they were placed in individual pens where they continued to receive whole milk (4 L/d).
Selenium supplementation
The Se-S group cows were supplemented subcutaneously with 1 mg Se/kg using barium selenate (Deposel®, Young Animal Health Ltd., New Zealand) in a single dose of 1 mL/50 kg/ BW, administered 60 days before the expected calving date.
Sampling
Blood samples from each cow were collected by coccygeal venipuncture into heparin-coated and noncoated tubes before supplementation (day 0) and then every 15 days for 90 days. Blood samples were collected from the calves into noncoated tubes immediately after birth, before colostrum ingestion, and at 7 days post- birth. Blood plasma and serum were maintained at -20 °C until analysis.
Selenium metabolic balance
The Se balance in cows was evaluated by blood activity of glutathione peroxidase (GPx) using a commercial reagent (Ransel®, Randox), according to Contreras et al. (2002). The activity of GPx was expressed in U/g of hemoglobin (Hb).
Serum T3 and T4 concentration in calves
Serum concentrations of T3 and T4 hormones was measured by electrochemiluminescence technique in an Elecsys 1010 device (Roche Diagnostics, Basel, Switzerland). The values were expressed in nmol/L. The serum T3 /T4 ratio was calculated by their quotient on serum concentration values.
Serum selenium concentration in calves
Serum selenium concentration in calves was quantified in an atomic absorption spectrophotometry device (Thermo® Series AA Solaar, Waltham, MA, USA), at 196 nm, with hydride generation in a (Thermo Scientific model VP100, Waltham, MA, USA), after acid digestion of the sample as indicated by Muñiz-Naveiro et al. (2005). The values are expressed in mg/L.
Statistical analysis
Data are expressed as means and standard deviation. The Shapiro-Wilk test was used to establish normality of the data. Significance of differences in Se-balance between supplemented and non-supplemented cows was evaluated using a variance analysis of repeated measures followed by Tukey's multiple comparisons test. Differences in concentration of thyroid hormones in the calves were assessed by Student's "t" test. The analysis was performed with the GraphPad© Prism 3 software (GraphPad Software, San Diego, CA, USA), and it was considered significant at p<0.05.
Results
Mean initial values of GPx activity in the Se-D and Se-S groups were 111 ± 52 U/g Hb and 118 ± 29 U/g Hb (p>0.05), respectively. Subsequent measurements in the Se-D group showed a reduction in blood activity of GPx up to values considered marginal (<100 U/g Hb) at day 30 and the following days (Figure 1). In contrast, in the Se-S group, an increase was observed in blood activity of GPx with respect to the initial value. The differences between groups were detected from day 45 until the end of the experiment (p<0.05).
The average T3 concentration was lower in the first hour of life in calves from the Se-S mothers compared with calves from Se-D mothers (p<0.05; Table 1). The Se-D values presented on day 7 were similar between groups (p>0.05). The T4 concentrations during the first hour of life were also similar between groups (p>0.05). On day 7, both groups presented a decrease that was significantly lower in the Se-S group (p<0.05). The T3:T4 ratio was similar between groups in the first hour of life (p>0.05) and on day 7. However, the ratio between periods was higher in the Se-D group (p<0.05; Table 1).
Discussion
The Se requirement for dairy cattle is 0.3 ppm according to the NRC (2001). The diet used in this experiment contained 0.04 ppm Se; thus, the non-supplemented cows were in negative metabolic balance for this mineral. Accordingly, blood activity of GPx in the Se-D cows remained within a low-marginal range (<100 U/g Hb; Ceballos and Wittwer, 1996). Although dietary minimum Se concentration for grazing cattle should be greater than 0.05 ppm to prevent alterations in health and productivity (Suttle, 2010), no clinical signs of Se deficiency were observed during the trial period in the present study. Supplementation with barium selenate significantly increased GPx activity of cows in the Se-S group from day 45 post-supplementation, reaching values higher than 200 U/g Hb during delivery, which are considered appropriate for lactating cows (Ceballos and Wittwer, 1996). This result agrees with reports by other researchers using different Se sources (Awadeh et al., 1998; Guyot et al., 2011). According to Gunter et al. (2013), the increase in blood Se in calves of supplemented mothers depends on the Se source.
The effect of organic and inorganic Se supplementation on different systems, including the function of the thyroid gland in cattle, has been reported by various researchers (Awadeh et al., 1998; Rowntree et al., 2004; Koenig and Beauchemin, 2009; Guyot et al., 2011; Rose et al., 2012); however, few studies refer to the effect in newborns from mothers supplemented with Se. In the present study, the average concentration of T3 was lower in the first hour of life (p<0.05) in calves from Se-S mothers (Table 1).
Figure 1
Blood activity of GPx (mean ± SD in U/g Hb) of cows maintained fed ration with low selenium, supplemented during prepartum (day 0) with barium selenate (Se-S) and non-supplemented (Se-D). Parturition period between 45 and 60 days. Asterisks (*) indicate significant differences (p<0.05) between groups.
Table 1
Serum concentrations (mean ± SD in nmol/L) of T3, T4, and T3:T4 ratio in calves at 1 hour (h) and 7 days after birth from cows fed a low-selenium diet supplemented with barium selenate (Se-S) and non- supplemented (Se-D) during prepartum.
| 1 h | T3 | 7-day | 1 h | T4 | 7-day | T3:T4 ratio 1 h | 7-day | |
|---|---|---|---|---|---|---|---|---|
| Se-S | 5.92 ± 0.6 | 4.64 ± 0.94 | 181 ± 51 | 101 ± 21≠ | 0.0347 | 0.0464 | ||
| Se-D | 8.64 ± 2.46* | 4.23 ± 1.16≠ | 188 ± 31 | 88 ± 31 | 0.0472 | 0.0488≠ |
Although no studies conducted under similar conditions were found in the literature, other researchers have reported values of thyroid hormones in the newborn by applying experimental models different from those in this study (Davicco et al., 1982; Stojić et al., 2002; Kirovski et al., 2008). These differences can be attributed not only to the hormone determination methodologies but also to the potential influence of the environment on the animals subjected to experimentation. In this regard, Stanko et al. (1991) indicated that calves born during the winter period have higher concentrations of T3 and T4 than those born in other seasons because cold stimulates synthesis and secretion of these hormones in the newborn. Colostrum intake is another factor related to the concentrations of thyroid hormones in the neonate (Grongnet et al., 1985). In the present study, the calves received colostrum ad libitum from their mothers, which presented different metabolic Se status (Figure 1); however, some studies have indicated that iodine transferred transplacentally from the mother to the fetus would be more relevant than iodine transferred by colostrum (Davicco et al., 1982; Guyot et al., 2011).
The decrease in hormone concentration after birth is consistent with other reports (Awadeh et al., 1998; Takahashi et al., 2001; Stojić et al., 2002; Guyot et al., 2011; Rose et al., 2012). In this sense, the high initial T3 concentration in the newborn is related to the synthesis of thermogenin, a protein necessary for heat generation in the brown adipose tissue (Carstens, 1994). When analyzing Se between groups, T3 concentration was lower in the calves from Se-supplemented mothers compared with the non-supplemented. This unexpected effect could be attributed to the administration of barium selenite; a low-soluble salt formulated for subcutaneous deposition of Se to be released over a one year period. The uncontrolled release can reach values that, in the conditions of this test, could be considered supra-nutritional, leading to lower conversion of T4 to T3 in newborn calves. With a similar Se source, this paradoxical effect has been observed in heifers supplemented with barium selenate. The erythrocyte GPx activity exceeded by far metabolically adequate values, decreasing the cellular immune response (Leyán et al., 2006). This hypothetical effect associated with Se formulation is supported by studies conducted with different Se sources. In this regard, Awadeh et al. (1998) reported that T3 concentration in calves from mothers supplemented with 60 ppm Se, in the form of organic Se, was higher than in calves from mothers supplemented with the same amount of inorganic Se, with the highest T3:T4 ratio in calves from mothers who received salts with 120 ppm Se. However, in other trials, no effect on the concentration of thyroid hormones in newborns was observed by supplementing prepartum cows with selenized yeast or selenite (Rowentree et al., 2004; Koenig and Beauchemin, 2009).
There was also no effect on thyroid hormone concentrations of newborns when using intra- ruminal boluses formulated on the basis of Se, iodine and cobalt, and supplied two months before delivery (Rose et al., 2012). The effect of different Se formulations (selenite and selenium yeast) on thyroid hormones concentration in adult animals has also been studied and, although they increase GPx activity and blood Se concentration, they do not cause differences in T3, T4 concentration and T3:T4 ratio with respect to non-supplemented animals (Gunter et al., 2013). These results were attributed to a compensatory response of non-supplemented animals, in which the expression of the enzyme iodothyronine deiodinase type I in the thyroid would be increased.
The studies conducted on Se effects on the thyroid gland function have used organic Se, selenite salts and elemental selenium with varied results. To the best of our knowledge, this is the first study using slow-release barium selenate in cows during prepartum to assess its effect on the concentration of thyroid hormones in the calve, which results do not allow for establishing criteria for practical use. The results of this study raise questions about the adequate balance of Se in the prepartum cow and the consequences of supranutritional contributions that could cause negative metabolic responses in the newborn. Accordingly, further studies are required on this subject.