Survey of different responses of non-pregnant fat-tailed ewes to the intravenous glucose tolerance test during the feed restriction period

Summary
Introduction: Blood glucose concentration varies according to the physiological stages and daily milk yield. In ruminants, the gluconeogenesis process supplies most of the glucose requirements, which plays a prominent role in the metabolism and maintaining energy homeostasis. Due to the pivotal role of glucose and its metabolic processes in ruminants, particularly in milk production and optimizing reproductive efficiency, comprehensive studies have been conducted on dairy cows. The glucose tolerance test (ivGTT) is one of the techniques to assess glucose metabolism and the capacity of various tissues to react over insulin secretion (Kaneko et al., 2008). Although ivGTT studies have achieved significant success in glucose and insulin metabolism, the effect of some factors, such as feed restriction, physiological status, and dose of glucose infusion, need to be sufficiently clarified. The published results demonstrated that feed restriction at 65% energy content of the diet increases the plasma glucose concentration in non-pregnant ewes (Zarrin et al., 2021b). This study evaluated the effect of intravenous glucose infusion on blood metabolite and insulin concentration in non-pregnant fat-tailed ewes during feed restriction.

Materials and Methods: Ten multiparous non-pregnant fat-tailed Turki-Qashqai ewes (3-4 years old and 49.2 ± 3.60 kg body weight), after two weeks of adaptation, were randomly allocated into two treatment groups: the control group (Control; n =5) and the feed restriction group (Restriction; n =5). The Control ewes had free access to feed throughout the experiment (weeks 1 to 5). The restricted ewes received a diet equivalent to 100, 50, 65, 80, and 100% of the energy content of the diet at weeks 1, 2, 3, 4, and 5 relative to the start of the experiment (Ding et al., 2016). Intravenous glucose infusion (1 ml/kg BW) was administered in the third week of the experiment. Blood samples were taken 10 and 5 minutes before and 2, 5, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120, and 180 minutes after the infusion. The area under the curve (AUC) was calculated for each parameter during the ivGTT (0-180 min) using the trapezoidal roll (sum of the rectangular and triangular areas under the curve). The Revised Quantitative Insulin Sensitivity Check Index (RQUICKI) was calculated using the proposed equation (Holtenius and Holtenius, 2007). For the calculation of the Glucose clearance rate (the rate at which the concentration of glucose is removed from the blood, k (%/min)) ) and the half-life of glucose (T1/2; the time required to reduce the concentration of glucose by half), were calculated by Kaneko et al.(2008). Data was analyzed by the MIXED procedure of SAS. Treatments (Control and Restriction), time points (0-180 min), and the interactions (treatment × time) were considered as fixed effects. The animal breed was considered as random effect. Measured data were considered as dependent variables. The data obtained for ivGTT parameters were evaluated using the SAS GLM Procedure. The results from reference samples were included in the model as covariates for individual differences between ewes. Data are presented as Means ± SEM and differences were considered significant if P ≤ 0.05.

Results and discussion: Glucose infusion increased glucose concentration in both groups (P<0.01). No significant changes were observed for glucose turnover, half-life, and the area under the glucose curve between the groups. Insulin concentration increased in both groups a few minutes after glucose infusion, but the increase was slighter in Restriction (P<0.05). Free fatty acids declined due to intravenous glucose infusion in Control (P<0.05). Glucose infusion did not affect beta-hydroxybutyrate concentration. The current study was designed and conducted to study the glucose clearance pattern during feed restriction regardless of the many potentially confounding effects of the pregnancy presented in other studies. The animals were not matted during the experiment to avoid the influences of the particular endocrine and metabolic changes during pregnancy and the transition period. Researchers indicated that glucose and insulin increased in response to dextrose administration in pregnant and non-pregnant sheep (Lunesu et al., 2020; Morgante et al., 2012). An instantaneous increase in insulin concentration after the glucose infusion is expected because of its glucose-regulatory role (Zarrin et al., 2015; Francis and Bickerstaffe, 1996). In agreement with the current study, increases in insulin levels were reported in Ghezel ewes from zero to 1st hour after dextrose administration (Chalmeh et al., 2020). Previous study documented the high level of insulin concentration in response to to intravenous glucose infusion (Wolfe et al., 1986). Glucose infusion, increases glucose, insulin, cortisol, and prolactin concentration, while the it decreased the concentration of free fatty acid, beta-hydroxybutyrate, and insulin like growth factor-1 during pre- and post-parturition in sheep (Chalmeh et al., 2020). These researchers suggested that induced hyperglycemia through intravenous infusion causes metabolic and hormonal changed in fat-tailed sheep that may due to providing energy sources and glucose as a metabolic regulator (Chalmeh et al., 2020). Although feed restriction-induced insulin resistance (Petterson et al., 1993), the low insulin concentration during the ivGTT in the restriction group compared to Control showed a different glucose homeostasis in the fat-tailed sheep. The low insulin concentration in Restriction compared to Control might be a comeback to the decreased energy during the whole experiment in this group (Zarrin et al., 2021b, 2021c). Circulatory levels of beta-hydroxybutyrate and free fatty acids significantly decreased following intravenous hypertonic dextrose administration in Ghezel ewes (Chalmeh et al., 2020). Low insulin concentration and reduced sensitivity of the tissues around parturition increase lipid mobilization and induce further rise in plasma free fatty acid concentrations (Hayirli, 2006). The rapid drop in free fatty acid concentrations after the glucose infusion demonstrated the low lipid mobilization (Chalmeh et al., 2020). Previous studies have authenticated the negative correlation between free fatty acid concentrations, area under the curve of insulin, and insulin concentration at its peak (Bossaert et al., 2008). Adipose tissue and its derivatives are crucial in determining and modulating insulin sensitivity during glucose metabolism in dairy cows (De Koster and Opsomer, 2013). Direct manipulative studies have also demonstrated that providing excess free fatty acid by abomasal fat infusion produced peripheral insulin resistance in nonlactating cows (Pires et al., 2007; Pires and Grummer, 2007). The current results indicate the ability of fat-tailed sheep to preserve blood glucose levels in periods of feed restriction, which is accomplished through hormonal mechanisms, especially hormones involved in energy metabolism, such as insulin. The reduction of insulin in Restriction compared to Control, even when a rich source of glucose is provided through intravenous infusion, might be due to increased insulin sensitivity in the restricted group ewes compared to Control. Therefore, the finding results proved the physiological ability of fat-tailed sheep to spare glucose concentration through hormones and metabolites involved in energy metabolism. This ability is important for these breeds in harsh environmental conditions or husbandry systems.