Effect of Chromium Propionate Supplementation on Biochemical and Hormonal Parameters of Sheep During Gestation and Lactation

Abstract

Introduction

Chromium is a naturally occurring mineral and your role in mammalian organisms was first reported over 60 years ago by Schwarz and Mertz [1–3]. The authors proposed that Cr³⁺ was the active compound of a “glucose tolerance factor” (GTF) [3] shortly after showing that rats fed a “GTF” deficient diet had the glucose removal rate from bloodstream significantly delayed [2]. In the same study, by giving these rats ingredients known as “sources of GTF” (acid-hydrolyzed porcine kidney and Brewer`s yeast) and isolated concentrate GTF via stomach tube, the glucose removal rate was restored [2]. Although the GTF is now considered an artifact of its isolation process [4,5], Schwarz and Mertz were pioneers for linking Cr to insulin mode of action.

Yamamoto et al. [6]  identified and isolated a low-molecular-weight chromium-binding substance (LMWCr, now called chromodulin) present in the cytosol of liver cells. Afterward, in-vitro trials showed that chromodulin has a dose-dependent action on enhancing glucose incorporation into carbon dioxide and lipids, subsequent to the binding of insulin to the insulin receptor (IR) [7]. Several studies focus on understanding the molecular mechanisms by which Cr may alter IR signaling. The most consistent model was demonstrated by Davis and Vincent [8,9], where Cr titration, in the presence of rat adipocytic membranes fragments, apo-chromodulin (i.e., metal-free), insulin and proper substrate, presented the ability to activate the protein kinase activity (i.e., activation of IR in response to insulin binding) [8]. A range of in vivo tests presented consistent results of the direct action of Cr in increasing insulin-stimulated IR phosphorylation and downstream effects of insulin signaling in liver and muscle cells [10–13]. Moreover, as the ability of chromodulin to potentiate the intracellular responses of insulin is directly dependent on the Cr available amount, approximately four chromic ions per chromodulin are required for maximal tyrosine kinase activity. The addition of more than six Cr³⁺ per chromodulin results in the inhibition of kinase activation [5,8], which allows hypothesizing that there could be an ideal dose of Cr supplementation.

Ovine gestation lasts around 150 days and energy requirements are similar to maintenance during early and mid-gestation (first 100 days), but it increases substantially during late gestation because 70% of fetal growth occurs in this period [14]. Adequate nutrition in late pregnancy will also determine udder development and production of colostrum and milk. Furthermore, uterus expansion limits rumen space, compromising feed ingestion capacity. During lactation, ewe`s nutritional demands are highest. Negative energy balance and some loss of body condition are expected during this period as consequences of these factors, but the nutrition must be adjusted to attenuate catabolism of adipose and muscle tissue by increasing the proportion of energy sources ingredients in the diet [15,16].

Cr supplementation may have supraphysiological benefits for ruminants. As Cr potentiates insulin action and insulin improves efficiency of amino acid transportation, protein synthesis, carbohydrate and lipid utilization, Cr seems to have sparing effects on these processes [17]. Cr supplementation decreased serum triglyceride, cholesterol, and insulin concentrations in growing lambs [18–21]. In prepartum and early lactation dairy cows, Cr decreased serum non-esterified fatty-acids (NEFA) concentration [22,23]. These results suggests that Cr would not only enhance glucose utilization and, consequently, lower fat mobilization to meet high energy demands, but also affect the complete insulin action of increasing lipogenesis and reducing lipolysis [17,24]. However, some researchers reported that concentrations of insulin, triglyceride, cholesterol and NEFA were unaffected by Cr supplementation [17]. The inconsistency in results might be a consequence of differences in the amount of Cr provided in the experimental diets. In vitro experiments showed that there is a saturation of the IR activation, followed by inhibition, in response to the progressive addition of Cr ions [8]; it is likely that the same occurs in livestock performance responses to Cr supplementation.

In a previous study of our laboratory, daily supplementation of 1 mg of Cr, also as Cr propionate, had impact on body weight, serum insulin, NEFA and beta-hydroxybutyrate concentrations of ewes at late gestation fed 110% of energy requirements [25,26]. Therefore, the objective of this study was to determine the effects of 0.5 mg, 1.0 mg and 1.5 mg of Cr supplementation on some biochemical and hormonal parameters related to energy metabolism of late pregnant (final third of gestation) and lactating ewes receiving 100% of predicted energy requirements. The hypothesis is that Cr supplementation is capable to better modulate catabolism during high demand periods in a non-linear dose-dependent response.

Material And Methods

The experiment was conducted at the Fernando Costa Campus of the University of São Paulo, Pirassununga, Brazil. All procedures were approved by the Institutional Ethics Committee of Animal Use (protocol n^o CEUA 2148190220).

Animals and design

Thirty-one ewes, single birth, were selected from an initial flock of sixty-seven Santa Inês and Dorper crossbreed, 63 ± 10 kg body weight before mating, 3 ± 1 years old. At the 100^th day of gestation, ewes were randomly assigned into four treatments, according to the dose of Cr to be supplemented daily: without supplementation (CR0, n = 8), supplementation of 0.5 mg (CR0.5, n = 8), 1 mg (CR1.0, n = 8) and 1.5 mg (CR1.5, n = 7) of chromium per ewe. The Cr doses described were administered orally, previously incorporated into 100 g of a corn meal for each animal, 30 minutes before morning feeding (KemTrace Chromium, 0.4% Cr; Kemin Agrifoods South America). Cr was provided from the 100^th day of gestation to the 60^th day after lambing (110 ± 5 days). 

After complete ingestion of the Cr meal, all ewes had access to the same diet (Table 1) composed of corn silage, finely ground corn, soybean meal, minerals and vitamin premix. The total diet was formulated according to the nutritional requirements for late pregnancy and lactation [14]. To ensure consumption ad libitum, the amount of feed offered was adjusted daily according to the remainder from the previous day.

Samples collection and measurements

Blood samples were collected on day 135 of pregnancy and days 7, 30 and 60 after lambing, obtained via jugular vein 1 h after morning feeding. Serum and fluoride tubes were centrifugated (3,500 rpm, 15 min) for separation of serum and plasma, stored in microtubes at -20^oC until analysis. Blood concentrations of urea (ref. 104-4), creatinine (ref.  96), aspartate aminotransferase (AST; ref. 109), gamma glutamyl transferase (GGT; ref. 1058), total proteins (ref. 99), albumin (ref. 19), calcium (ref. 90-2/60), phosphorus (ref. 12-200), triglycerides (ref. 87-2/100), total cholesterol (76-2/100) and glucose (ref. 133-1) were determined with commercially available assay kits from Labtest Diagnóstica S.A. (Lagoa Santa, Minas Gerais, Brazil; reference number as indicated) and analyzed by Mindray BS-120 equipment (Mindray Bio-Medical Electronics Co., Ltd., China). Globulin was calculated subtracting mathematically the level of albumin from total proteins. Blood concentrations of insulin (ref. 2425-300), triiodothyronine (T3; ref. 125-300), thyroxine (T4; ref. 225-300) and cortisol (ref. 3625-300) hormones were determined with ELISA assay kits from Monobind Inc. (California, USA) and analyzed by Multiskan MS 352 reader (LabSystems Inc., USA). Blood non-sterified fatty acids (NEFA; ref. FA115) and beta-hydroxybutyrate (BHB; ref. RB1007) concentrations were determined with assay kits from Randox Laboratories Ltd. International (Kearneysville, West Virginia, USA), analyzed by Randox RX Daytona equipment. Ewes had their weigh and body condition score (BCS) [25] (in a scale of 1, emaciated, to 5, obese, in 0.25 intervals) measured at the first day of Cr supplementation and right after every blood sample collection. Estimated daily milk production was measured at 30 days of lactation by using the weigh-suckle-weight method [28].

Statistical analysis

The statistical design was completely randomized. Using the statistical package SAS 9.4 (SAS Institute Inc., USA), data were analyzed applying Proc Mixed and Tukey’s test. Significant differences were accepted when P value ≤ 0.05 and tendencies, when 0.05 < P ≤ 0.1. It was used regression analysis to evaluate the doses of Cr. The model included Cr supplementation (i, fixed effect), ewe (j, random effect) and random error (e) as shown:

Yij = µ+ Ti + eij 

Where: Yij is the dependent variable, (i = treatment; j = repetition), μ is the overall mean, Ti is the fixed effect of the treatment (i = 1-4), and eij is the residual error. 

Gestation and lactation data were analyzed separately. Lactation parameters obtained in the three days of collection (days 7, 30 and 60 after lambing) were analyzed over time.

Results

The body weight, BCS did not different between treatment during late gestation (Table 2). Serum phosphorus concentration was higher in the CR1.0 (6.92 mg/dL) compared to CR0 (5.44 md/dL, P < 0.05) in late gestation (Table 2). The CR0.5 (0.33 mmol/L) tended to reduce plasma NEFA level compared to the other treatments (P < 0.1). Triglycerides, cholesterol, AST, BHB, glucose, insulin, urea, creatinine, total protein, albumin, globulin, calcium, T4, T3 and corrisol concentration were not significantly different between treatments in gestation (P > 0.1).

In lactation, serum creatinine level was lower in the CR0.5 (0.89 mg/dL) compared to CR1.5 (0.98 mg/dL, P < 0.05, Table 3). Serum BHB tended to be lower in the CR0.5 (0.68 mmol/L) compared to CR1.0 (0.85 mmol/L, P < 0.1). The CR0.5 (0.43 µUI/mL) tended to have lower insulin concentration compared to CR0 (1.23 µUI/mL P < 0.1) and had lower insulin:glucose ratio also compared to CR0 (P < 0.05). Serum phosphorus level was significantly affected by Cr supplementation, where the CR1.5 (8.13 mg/dL) had higher concentration compared to CR0 (6.40 mg/dL), with CR0.5 (6.75mg/dL) and CR1.0 (7.71 mg/dL) levels in the middle (P < 0.001). All others blood parameter means were not significantly different between treatments (P > 0.1).

Discussion

Ovine blood parameters concentrations of specific physiological states, such as late gestation and lactation, are not as available on literature as there is for other farm animals. Although many of the substances analyzed in the present study are somehow associated with insulin activity, but were not affected by Cr supplementation, the data obtained by this experiment is scientific and clinically relevant for comparations.

During late gestation and early lactation, ruminants have a large and expected drop in lipogenesis and increase in lipolysis, which can lead to accumulation of triglycerides in the liver and abnormal production of ketone bodies [29,30]. In the present study, blood parameters indicate no compromise of liver functions [31]. However, there were responses to Cr supplementation that suggests an interference in nutrient partition at elevated metabolic demands.

Blood NEFA level have a direct relation with fat mobilization, and, therefore, with negative energy balance. Reduction in serum NEFA concentrations is one of the most relevant effects of Cr supplementation in ruminants during prepartum and early lactation periods [17,22,29,30,32]. An improvement on glucose uptake by insulin-dependent organs, modulated by more Cr available, decreases the reliance on body reserves and reduces fat mobilization and circulating NEFA [29]. Moreover, whereas increased supply of Cr improves insulin sensitivity in adipose tissue, it may also increase lipogenesis and, consequently, decrease net fatty-acid release from the cell [32]. Dairy cows receiving Cr methionine had lower serum NEFA concentration in prepartum period than cows not supplemented, with similar insulin concentrations [22]. In the present study, at late gestation, only the dose of 0.5 mg of Cr lowered NEFA concentration. This result agrees with the founds of lactating cows fed Cr at 0.05 and 0.10 g/kg BW^0.75 where only the lower dose decreased NEFA levels, compared to control [29].

Kafilzadeh et. al [22], supplementing dairy cows Cr methionine during gestation, partially attributed the lower NEFA levels in prepartum to the reduced blood cortisol levels, as insulin did not change in comparison to the control group. In the present study, cortisol level was not affected by Cr supplementation neither in late gestation, nor in lactation. However, 0.5 mg of Cr lowered blood insulin level at lactation with no change in glucose level, reenforcing that there is a non-linear dose-dependent improvement in insulin sensitivity.

Increase in insulin sensitivity is one of the most documented responses to Cr supplementation [17]. Cr is the active compound of chromodulin, a cofactor for insulin action, which has an intrinsic role in insulin sensitivity and cellular signaling pathway. The increment of available Cr upregulates intracellular mRNA levels of the glucose transporter type 4, increasing membrane associated GLUT4, boosting the rate of glucose uptake [33].

Unchanged glucose levels during lactation with lower secretion of insulin by CR0.5 in comparison with CR0 suggest that the lower dose of Cr supplemented was capable of enhance insulin efficiency. Glucose homeostasis is strictly controlled and do not oscillate in ruminants as much as in monogastric animals [34]. Thus, to sustain the same basal glucose concentration of the non-supplemented treatment, CR0.5 ewes required a smaller amount of insulin. Non-linear insulin responsiveness to increased Cr supply were observed in post-partum dairy cows receiving 0.03, 0.06 and 0.12 mg of Cr/kg of metabolic BW [30].

In accordance with blood insulin level at lactation, BHB level was also reduced by 0.5 mg of Cr supplementation, suggesting that the referred dose enabled a more effective utilization of energy provided by the diet. The lack of benefits on energy partition with higher Cr doses compared to CR0 might suggest an alleviated Cr mediation of improved insulin sensitivity and signaling. 

 The relatively low concentrations of blood BHB between treatments and the absence of BCS change over time during trial indicates moderate or even low negative energy balance. There was no evidence of triglycerides accumulation on liver, for its synthesis functions were not compromised, as seen on blood protein concentrations. However, blood creatinine levels during lactation may suggest that 0.5 mg of Cr increase the effectiveness of aminoacidic turnover and protein synthesis in comparison to 1.5 mg of Cr, although there were no differences of indicators of protein metabolism between CR0.5 and CR0.

There was a notable interaction between Cr and serum levels of phosphorus (P) in the present study. As seen during lactation, serum P concentration increased linearly according to the amount of Cr provided, with CR1.5 treatment exceeding the reference range [35]. Calcium (Ca), on the other hand, was unaffected. Mineral intake at higher concentrations may have antagonist functions on the retention of other minerals. Sankaramaniel et al. [36], administering Cr intraperitoneally in rats, found that Cr inhibited both bone mineralization and resorption, while the concentrations of Ca and P in bone remained unaltered. Furthermore, Cr treatment had a 1.5-fold increase in serum P level, with serum Ca unchanged. Authors suggested that there may be increased absorption and/or decreased excretion of P, as there is less chance for the mobilization of P from the bone [36]. However, in a previous study [26], the femur density of lambs born from ewes that received 1 mg of Cr during gestation was negatively affected by the supplement [26]. Like other tissues, the bone has the ability to accumulate Cr when supplemented. It is expected that accumulated Cr might alter the metabolic activities of these sites, leading to altered bone turnover [36,37]. Further studies are needed to stablish the exact mechanism of Cr action on the bone and whereas it is mediated by insulin or not.

Summarizing, the correlation of the results of the presented study with the previous results of literature shows indications that Cr has the capacity to modulate insulin sensitivity in ruminants and interfere positively on the management of energy metabolism during ovine gestation and lactation. However, limitations of benefits of Cr supplementation were found regarding higher doses, implying why there are controversial conclusions towards the literature. It was also concluded that Cr supplementation doses used interfered progressively in serum phosphorus concentration. Moreover, the connection between Cr and mineral metabolism is a subject to be more investigated and better comprehended.

Declarations

Ethics approval

Animal investigations were approved by the Institutional Ethics Committee of Animal Use (protocol n^o CEUA 2148190220).

Declaration of interest 

None.

Financial support statement 

This work was supported by FAPESP - São Paulo Research Foundation, process number 2020/06433-0, and CAPES for the scholarship.

Author contributions 

Sarita Bonagurio Gallo, Flávia Mallaco Moreira and Daniela Lázara de Almeida contributed to the study’s conception and design. Material preparation, data collection and analysis were performed by all authors. The first draft of the manuscript was written by Sarita Bonagurio Gallo, Flávia Mallaco Moreira and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”

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Tables

Table 1 Ingredients and chemical composition of basal diet.

^a Mineral and vitamins: Ca, 140 g; P, 65 g; Mg, 10 g; S, 12 g; Na, 130 g; Co = 80 mg, Fe = 1000 mg, I = 60 mg, Mn = 3.000 mg, Se = 10 mg, Zn = 5.000 mg, F = 650 mg, vitamin A = 50.000 IU, vitamin E = 312 IU.

Table 2 Biochemical and hormonal parameters of ewes at late gestation supplemented with chromium propionate.

Numbers within each row followed by different letters in columns denote differences between treatments, where ^a-b P < 0.05 and ^x-y 0.05 < P ≤ 0.1

CR0 – 0.0 mg of chromium supplementation per ewe, CR0.5 – 0.5 mg of chromium supplementation per ewe, CR1.0 – 1.0 mg of chromium supplementation per ewe, CR1.5 – 1.5 mg of chromium supplementation per ewe, SEM - standard error of means.

Table 3 Biochemical and hormonal parameters of ewes at lactation supplemented with chromium propionate.

Numbers within each row followed by different letters in columns denote differences between treatments, where ^a-c P < 0.05 and ^x-y 0.05 < P ≤ 0.1

Empty fields refer to parameters measured only at day 7 of lactation.

CR0 – 0.0 mg of chromium supplementation per ewe, CR0.5 – 0.5 mg of chromium supplementation per ewe, CR1.0 – 1.0 mg of chromium supplementation per ewe, CR1.5 – 1.5 mg of chromium supplementation per ewe, SEM - standard error of means.