Abstract
Simple SummaryGlycerol is widely used as a feed supplement in ruminant nutrition. However, its administration at moderate and high doses results in an increase in plasma osmolality and in an alteration of red blood cell (RBC) indices. The present study aimed at further elucidating the effect of glycerol on RBCs’ functionality by evaluating the effect of different glycerol concentrations on RBC homeostasis under in vitro conditions. Obtained results showed that glycerol permeates the RBC membrane and leads to hemolysis when glycerol concentration exceeds 200 mg/dL. However, RBCs’ antioxidant defenses appear to protect cell membranes without causing an increase of oxidative stress markers. Moreover, no alteration in RBCs’ intracellular Ca2+ ion concentrations and metabolic activity were found. In conclusion, glycerol-based nutritional treatment should be designed in sheep to avoid exceeding 200 mg/dL glycerol circulating concentration to prevent RBC osmotic stress.The use of high doses of glycerol as a livestock feed supplement is followed by a rapid increase in plasma concentrations and consequently in plasma osmolality. Moreover, glycerol is a highly diffusible molecule that can readily permeate the red blood cell (RBC) membrane following a concentration gradient. A rise in glycerol plasma concentrations can thus alter RBC homeostasis. The present study aimed at investigating both glycerol osmotic effects on sheep RBCs and their oxidative response under in vitro conditions. Sheep blood samples were suspended in media supplemented with increasing glycerol concentrations (0, 25, 50, 100, 150, 200, 250, 300, 350, 400 mg/dL), which reflected those found in vivo in previous studies, and incubated at 37 °C for 4h. Thereafter, osmolality and hemolysis were determined in spent media, while cell extracts were used to assay intracellular concentration of glycerol, ATP, Ca2+ ions, oxidative stress markers and reactive oxygen species (ROS).The study confirmed that glycerol intracellular concentrations are directly related with its concentration in the incubation media, as well as hemolysis (p < 0.001) which increased significantly at glycerol concentrations higher form 200 mg/dL. ROS intracellular level increased at all glycerol concentration tested (p < 0.01) and total thiols decreased at the highest concentrations. However, RBCs proved to be able to cope by activating their antioxidant defense system. Superoxide dismutase activity indeed increased at the highest glycerol concentrations (p < 0.001), while total antioxidant capacity and malonyldialdehyde, a typical product of lipid peroxidation by ROS, did not show significant changes. Moreover, no alterations in intracellular Ca2+ ions and ATP concentrations were found. In conclusion, glycerol-induced hemolysis can be related to the induced osmotic stress. In sheep, nutritional treatments should be designed to avoid reaching glycerol circulating concentrations higher than 200 mg/dL.
Highlights
Glycerol is widely used as a feed supplement in small ruminant nutrition, both as replacer of corn grain in the diet [1,2,3,4,5] and to increase productive performance [6,7,8,9,10,11,12]
The osmocloanlicteynotrfadtiilount.ioTnhemseedoisamsoulpaplilteymveanluteeds awreithininthcereraasningge ogflytcheorsoel fcoounncdenintrav-ivoe[x1p6e]citnedew, me’esdpilaasomsma oalfatelirtyorianlcaredamseindisptrroaptioorntioofngallylycewroitlhas nutriti glycerol concepnltermateionnt. These osmolality values are in the range of those found in vivo [16] in ewe’s plasma after oral administration of glycerol as nutritional supTpalbelme 2e.nOt.smolality values of media supplemented with increasing glycerol concentrations
red blood cell (RBC) proved to be able to cope with the increased reactive oxygen species (ROS) production: MDA did not increase and the antioxidant defenses did not decrease following the glycerol treatment
Summary
Glycerol is widely used as a feed supplement in small ruminant nutrition, both as replacer of corn grain in the diet [1,2,3,4,5] and to increase productive performance [6,7,8,9,10,11,12]. In previous studies [15,16], we reported that oral administration of glycerol-based formulation in ewes causes a significant increase in circulating glycerol concentration (from 14.19 to 289.8 mg/dL) and in plasma osmolality (from 0.316 to 0.378 osmol/kg) These changes were associated with significant alteration in red blood cell (RBC) indices with a dose-related effect, which included shape alterations (increase in RBCs volume and distribution width) and a dilution in the amount of hemoglobin per volume unit [16]. These alterations were found 2 h after the oral administration of glucogenic formulations when glycerol concentrations in the bloodstream were higher than ≈ 200 mg/dL. Similar effects were described in in vitro human glycerolized RBCs and were associated with an impaired functionality and reduced cell deformability, which in turn can affect the cells’ capability to enter and transit the capillaries and to exchange gases, nutrients, and cell waste [17,18]
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