Cow performance was not affected by Se-biofortified hay
Previous studies on peripartum ewes lambs indicated a positive effect of Se supplementation during pregnancy on mammary gland growth, development, and vascularity [34, 35] that can increase milk production. In our study, only a tendency for increased milk production during the first 120 days of lactation in primiparous Holstein cows was detected and not in Jersey cows, but, except for lactose which increased in Holstein but decreased in Jersey cows receiving the Se-biofortified hay, none of the milk components were affected by the treatment. Our findings are similar to prior studies where Se supplementation did not affect milk yield and composition [36, 37], but, as in our study, the same treatment increased the lactose yield in Holstein dairy cows [37]. Different than those prior studies and our study, in another study it was observed a significant increase in fat % in lactating multiparous cows supplemented with Se-yeast compared to cows supplemented with inorganic Se [20].
In prior studies, Se supplementation increased the proportion of polyunsaturated fatty acids (PUFA) in the milk of cows [38] and humans [39]. It is thought that the effect of Se on PUFA is due to its role via GPx enzyme. The GPx can prevent the oxidation by free radicals of PUFA present in milk [40]. Contrary to the above experiments, we did not detect any important effect on milk PUFA by feeding Se-biofortified hay. This appears to be consistent with our data indicating that feeding Se-biofortified hay does not affect milk GPx activity [24].
Selenium biofortified hay improves albumin production by the liver
In our experiment, supplementation of dairy cows with Se-biofortified hay improved plasma albumin concentration early post-partum. Similar results were detected previously in Jersey cows supplemented with Se-yeast during the last eight weeks of gestation [14] and in Holstein cows supplemented with selenomethionine from 21 days prepartum to 21 days post-partum [41] or inorganic Se + vitamin E [42].
The reason for the consistent higher concentration of circulating albumin when organic Se is provided to cattle is unclear. Albumin is a negative acute-phase protein, and larger increase in circulating albumin is consequence of lower inflammation and better liver function [11]. In our experiment, we did not observe any effect of the treatment on inflammation; thus, the larger plasma albumin in Se-treated cows cannot be the consequence of lower inflammation. The reason for the greater albumin observed in our experiment by feeding Se-biofortified hay is likely due to an increase in liver synthesis of albumin or decreased turnover. Unfortunately, we did not measure synthesis of albumin, but preliminary data indicate not effect of feeding Se-biofortified hay on the transcription of albumin gene in the liver [43].
Urea concentration in plasma is the result of the balance between urea input (i.e., produced by the liver) and output (i.e., rumen utilization, elimination by the kidneys, and passive loss via feces, sweat, and milk) [44]. Hence, increased plasma urea can be caused by increased urea production, decreased urea elimination, or a combination of the two. We can exclude the increase of urea by dietary protein intake considering that there was no difference in DMI and dietary crude protein between groups [24]. We can also eliminate any kidney problem, considering that larger urea to creatinine ratio in cows supplemented with Se-biofortified hay was detected. The higher urea to creatinine ratio however suggests a larger reabsorption of urea by the kidney in Sel vs. Ctr cows. Urea in plasma can also increase as a consequence of increased muscle proteolysis [45]. This does not appear to be the case in our study due to the lack of any difference in BW between treatment groups. Thus, it is possible that the larger urea in the blood was the consequence of a combined improved capacity of the liver to synthesize urea and larger re-absorption of urea by the kidney. Data produced in vitro [46] and in vivo [47] demonstrated a positive effect of organic Se supplementation on rumen fermentation in dairy cows. Thus, it is possible that the larger concentration of plasma urea observed in our study was also due to greater production of microbial proteins. However, this did not translate in more milk protein synthesis. In the in vivo study, Wei and collaborators detected a linear reduction of ammonia in the rumen by an increased dose of organic Se supplemented. This would indicate a larger amount of ammonia utilization by the microbes that would decrease the urea production by the liver. Thus, the observed effect on urea level by Se-biofortified hay remains still unclear.
To further complicate the interpretation of the urea data, an increase in blood urea concentration in Se supplemented animals was also observed in rats supplemented with inorganic Se [48] and in one year old Angus steers fed organic Se [49] but not in multiparous dairy cows [41, 50]. Thus, it is possible that the observed increased circulating urea by feeding Se-biofortified hay might only be associated with the young age of the cows used.
Overall, the higher albumin and urea concentration in plasma appears to partly support a liver in better condition in cows treated with Se-biofortified hay compared to cows fed a normal hay. It is also possible that the effects observed were due to a better rumen fermentation or larger kidney re-absorption of urea. Finally, the effect can be also associated with the use of young animals.
Selenium biofortified hay does not affect the oxidative status and the immune system but improves the antioxidant function of albumin
One of the main antioxidant enzymes in the blood of cows is the Se-dependent GPx that acts to remove free radicals normally produced by cells [51]. The activity of GPx was increased significantly in erythrocytes and plasma of the cows supplemented with Se-biofortified hay compared to control cows in our experiment [24], indicating an enhanced antioxidant response. However, none of the measured parameters associated with the oxidative status reported in the present manuscript were affected by the treatment with the exception of the AOPP, that was larger in cows few Se-biofortified hay compared to control animals.
The AOPP is a marker of protein oxidation that was first described in the plasma of uremic human patients [52], and it has been recognized as a marker of inflammation in several diseases in humans [53]. Higher concentration of AOPP in plasma was also observed in dairy cows experiencing endometritis [54]. AOPP are products of proteins exposed to free radicals, and they are formed by the reaction between plasma proteins and chlorinated oxidants mediated by the neutrophil enzyme MPO [55, 56]. AOPP can also trigger the oxidative burst and the synthesis of inflammatory cytokines in neutrophils and monocytes [57]. Increased chemotaxis and respiratory burst of neutrophils was detected in sheep supplemented with Se nanoparticles compared to sodium selenite [58]. Unfortunately, we did not measure respiratory burst capacity in neutrophils in our experiment, but MPO activity tended to be larger in cows supplemented with Se-biofortified hay. Despite being both overall larger in Sel vs. Ctr, the negative correlation between AOPP and MPO and AOPP to albumin ration and MPO do not support a role of MPO in determining the level of AOPP in our study. In humans, a positive correlation between levels of AOPP and MPO was observed in plasma but only in patients with kidney failure and not in healthy patients [59]. Our cows, including the ones fed with Se biofortified hay groups, were all healthy with no kidney problems as supported by the normal level of creatinine; thus, the lack of correlation between AOPP and MPO in healthy animals appears to be confirmed.
The above data do not explain the larger concentration of plasma AOPP detected in animals treated with Se-biofortified hay compared to control cows in our study. Interestingly, the negative correlation of AOPP with inflammatory parameters and indexes of poor liver activity/health and the positive correlation with parameters related to low inflammation and/or good liver function suggest higher AOPP in plasma as a positive outcome.
Serum albumin is one of the major antioxidant proteins in the blood [60]. Interestingly, the results of albumin oxidation are AOPP [61]. The AOPP produced from oxidized albumin bind to high-density lipoprotein scavenger receptor and are primarily eliminated by the liver and the spleen [62]. Thus, the larger concentration of AOPP can partly be explained by the greater amount of circulating albumin in cows fed Se-biofortified hay. This is also supported by the positive correlation between plasma levels of albumin and AOPP. In albumin, methionine (0.8% of all amino acids) and cysteine (5.8%) account for up to 80% of its total antioxidant activity [63]. The thiol group Cys34 in albumin that acts as a scavenger for reactive oxygen and nitrogen species accounts for 80% of the reduced thiols in human plasma.
Our data appear to suggest, contrary to prior data, that larger concentration in plasma of AOPP is associated with lower inflammation and/or better oxidative status. Due to this apparent contradiction with the classical interpretation of higher oxidation by a larger level of AOPP, the role of AOPP in dairy cows deserve further investigations.
The increase in GPx activity but lack of effect of Se-biofortified hay on other parameters of the oxidative status in our study is very similar to a recent study reported in pregnant heifers [64]. In that study, heifers were supplemented with hydroxy-selenomethionine during the prepartum, and the authors observed in blood a significantly larger GPx activity but no effect on the lipid peroxidation marker malondialdehyde. However, our results differ substantially with another recent study performed in multiparous cows [16]. In that study, supplementation of organic Se prepartum improved most of the antioxidant parameters measured, most of them assessed also in our study. Thus, it is very likely that the lack of effects observed in our study was due to the use of pregnant heifers instead of using multiparous cows.
Selenium-biofortified hay affects Ca and Mg metabolism
The effect on the concentration of serum Ca by feeding Se-biofortified hay can partly be driven by the known relationship between albumin and Ca since Ca ions (approx. 50% of the total serum Ca) is bound in blood to albumin [65]. However, the free Ca appears to be positively associated with Se-biofortified hay supplementation prepartum in Jersey cows. This is of importance, considering that this is the active form of Ca, that when decreased induces the release of parathyroid hormone via the Ca2+ sensing receptor, controlling the Ca homeostasis [66]. Ca homeostasis prepartum is of paramount importance in dairy cows, especially Jersey cows, which are known to be more at risk of this post-partum hypocalcemia. Probably even more important for the prevention of hypocalcemia is the level of Mg in plasma prepartum. As a cofactor in parathyroid hormone action, Mg is required for the efficient absorption and resorption of Ca; hence, cows with low blood magnesium around calving are more prone to get milk fever [67]. Our data indicated a higher Ca prepartum in Jersey cows and a lower Mg during the first 2 DIM, suggesting a higher risk of milk fever. However, none of our cows had milk fever. Thus, the effect of Se-biofortified on Ca and Mg is of interest, but the consequences remain unclear.
Selenium biofortified hay improves red blood cells
The hematocrit was positively affected by feeding Se-biofortified hay in our study. This was not caused by dehydration as supported by the lack of change in plasma concentration of creatinine. While there are no previous data concerning cows, Se supplementation increase hematocrit in mice [68] and fattening lamb [69]. Selenium has a role in regulating red cell homeostasis by mitigating oxidative stress-dependent modulation of genes that affect the differentiation of erythroid progenitors [68]. Furthermore, oxidative stress is known to decrease the function and the half-life of red blood cells [70]. Se supplementation can prevents erythrocyte lysis and formation of methemoglobin by improved GPx activity, as observed in vitro in the rat [71]. However, the lack of correlation between the activity of GPx in whole blood previously measured [24] with hematocrit does not support the higher GPx activity in blood and plasma of cows supplemented with Se-biofortified hay improved erythropoiesis and/or protected red blood cells.