Effect of Chromium Methionine Supplementation on the Lactation Performance, Hepatic Respiratory Rate and Antioxidative Capacity of Early-Lactating Dairy Cows

Abstract

Backgrounds: Chromium is an important trace element that may regulate dairy cow metabolism. The objective of this study was to investigate the effect of chromium methionine (Cr-Met) supplementation on lactation performance, hepatic respiratory rate and anti-oxidative capacity in early-lactating Holstein dairy cows.

Results: Sixty-four multiparous cows were grouped into 16 blocks based on parity, days in milk and milk production, and four cows within a block then were assigned randomly to one of four dietary treatments with 0, 4, 8 or 16 g/d of Cr-Met per cow in addition to a basal diet. The experiment lasted for 14 weeks, with the first two weeks as adaptation period. Milk yield and composition were recorded weekly. Dry matter intake was measured every other week. The plasma variables were measured in weeks 4, 8 and 12 of the experiment. Supplementation of Cr-Met did not affect dry matter intake of early-lactating dairy cows. As the supplementation of Cr-Met increased, yields of milk, fat, lactose and energy corrected milk increased in a linear manner (P < 0.01). In terms of plasma variables, insulin concentration decreased in a linear manner with Cr-Met supplementation (P = 0.04). As for variables relative to hepatic respiration rate, concentrations of pyruvate and NADH in the plasma were increased in linear (P < 0.01) and quadratic manners (P < 0.01), and lactic dehydrogenase activity was linearly increased as the feeding levels of Cr-Met increased (P < 0.01). Moreover, plasma glutathione peroxidase and superoxide dismutase activity were increased in a linear manner (P < 0.01).

Conclusion: The results suggest that Cr-Met supplementation improved lactation performance of early-lactating dairy cows through enhancing antioxidant capacity and hepatic cellular respiration.

Background

During the early-lactation period, dairy cows are subject to metabolic diseases including fatty liver, ketosis, and immunosuppression [1]. One of the key issues for metabolic homeostasis disruption during this stage is that feed intake and glucose supply to the dairy cows does not meet their need for maintainace and milk production [2]. The insufficient glucose supply leads to body reserve mobilization and severe oxidative stress of early-lactating dairy cows [3, 4]. On the other hand, increased hepatic respiration can enhance its gluconeogenesis, which can be indicated by changes in concentrations of lactic dehydrogenase (LDH), NADH and pyruvate in plasma [5]. Therefore, improved respiration rate and reduced oxidative stress would be beneficial to metabolic glucose supply in early-lactating dairy cows.

Chromium (Cr) is an important trace element that may regulate dairy cow metabolism. Previous study showed that chromium supplementation can improve milk production of early-lactating cows via different mechanisms including increased blood glucose clearance, improved insulin sensitivity of liver and mammary gland [6], and reduced immune response [7]. As an important component in the super complex of cellular mitochondria, Cr may play an important role in regulating cellular ATP synthesis, respiratory rate and metabolic status [8]. In contrast, limited studies have been conducted to demonstrate the role of Cr supplementation on antioxidant capacity of ruminants. Zhang et al [9] suggested that Cr supplementation failed to improve antioxidant capacity of mid-lactating cows with heat stress. As a stressed physiological stage, the role of Cr supplementation in antioxidant capacity and respiratory status in early-lactating cows has not been evaluated yet.

On the other hand, inorganic chromium can improve the production performance of dairy cows but the absorption of tissue cells to chromium is not ideal [10]. To overcome above-mentioned issue, organic chromium, such as chromium-yeast (Cr-yeast) [10] and chromiun-propionate (Cr-Pro) [11] were developed to replace inorganic Cr in dairy cows to improve Cr absorption rate and milk yield. Nowadays, a unique chelated formation of chromium methionine (Cr-Met) is available to the feed industry, with sulphur residues on Met binding with Cr can prevent blood circulation and peripheral tissues of dairy cows from being hydroxylated.

Thus, we hypothesize that Cr-Met supplementation improves the lactation performance through enhancing the hepatic respiratory rate and antioxidant capacity of early-lactating dairy cows. Therefore, the objective of this study was to investigate the effect of Cr-Met supplementation on lactation performance, blood variables related with respiration and antioxidant capacity in early-lactating Holstein dairy cows.

Materials And Methods

Animals and management

The experiment was conducted in experimental station of Institute of Dairy Science, Zhejiang University (Hangzhou, China). The study was approved by Animal Care and Use Committee of Zhejiang University. Sixty-four multiparous Holstein cows in early-stage lactation with average DIM of 50 (SD ± 4) and average BW of 726 kg (SD ± 71.3) were selected and assigned to 16 blocks based on parity and milk yield with 4 cows in a block, and then the 4 cows in a block were randomly allocated into 4 groups, and fed basal diets supplemented with different doses of Cr-Met (Availa ^® Cr 1000, which supplies 1000 mg Cr/kg from Cr-Met, Zinpro (China) Animal Nutrition Technology Co., Ltd) at the doses of 0, 4, 8, or 16 mg Cr/kg DM, respectively. All cows were housed in a tie stall barn, and fed and milked at 0630, 1400, and 1930 h every day. All cows had free access to drinking water. Feed was given in excess to allow 5% orts during the experiment. The Cr-Met was added once per day at 0630 h by scattering it on the total mixed ration for individual cows. The experiment lasted for 14 weeks, with the first 2 weeks for adaptation. During adaptation stage, Cr-Met was not supplemented to dairy cows.

Sample collection and measurements

Throughout the experimental period, DMI was recorded for 2 consecutive days (d 6 and 7 each wk) and samples of TMR and orts were collected on the same days of every other week. All the samples were analyzed for DM (105°C for 5 h), crude protein (method 988.05; AOAC, 1990) [12], crude ash (method 942.05; AOAC, 1990) [12], and acid detergent fiber (ADF) (method 973.18; AOAC, 1990). Content of neutral detergent fiber (NDF) was analyzed with method described by Van Soest et al [13] with the addition of sodium sulfite and amylase. An ANKOM²⁰⁰⁰ fiber analyzer (Ankom Technology Corp., Macedon, NY, USA) was used to extract and filter NDF and ADF, respectively. The value for NE_L in the experimental diet was estimated based on the Cornell Net Carbohydrate and Protein System (CNCPS) model using the CPM Dairy 3.0 [14]. The ingredient and nutrient composition of the experimental diet are listed in Table 1. The BW was estimated at the beginning and the end of the trail with method described by Yan et al [15].

The milk yield was recorded on d 6 and 7 of each week, and milk samples were collected on d 7 using milk-sampling devices (Waikato Milking Systems NZ Ltd., Waikato, Hamilton, New Zealand). One 50 mL aliquot of the composited milk sample was collected at each milking of the sampling day, and mixed with bronopol tablets (milk preservative, D & F Control Systems, San Ramon, CA). Milk sample was analyzed with a spectrophotometer (Foss-4000; Foss Electric A/S, Hillerod, Denmark) for milk compositions (protein, fat, lactose and milk urea).

Blood samples were collected from the coccygeal vein of each cow 3 h after the morning feeding on d 7 of wk 4, 8 and 12, respectively. The samples were deposited into lithium-heparin-containing vacuum tubes (5 mL, Becton Dickinson, Franklin Lakes, NJ), centrifuged at 3000 × g for 15 min to collect the plasma, and frozen at -20°C for subsequent analysis. Plasma samples were analyzed using an Auto Analyzer 7020 instrument (Hitachi High-technologies Corporation, Tokyo, Japan) with colorimetric commercial kits (Ningbo Medical System Biotechnology Co., Ltd.) to determine total protein, albumin, globulin, BUN, creatinine, glucose, non-esterified fatty acid (NEFA), β-hydroxybutyrate, triglyceride, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, insulin, glucagon, pyruvate, LDH and NADH, according to previously described methods [16]. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined using the ELISA method by Jiangsu MeiBiao Biological Technology Co., Ltd, Jiangsu, China [17].

Rumen fluid (50 mL) was collected using an oral stomach tube approximately 3 h after the morning feeding on d 7 of wk 4, 8 and 12, as described by Shen et al [18]. The pH of the rumen fluid was immediately measured using a portable pH meter (FE20-FiveEasy Plus™; Mettler Toledo Instruments Co. Ltd., Shanghai, China). The samples were placed on ice and kept stationary while the supernatant separated, and then, the samples were frozen at −­­20 °C for future determination of volatile fatty acids (VFAs). Two mL of rumen sample was acidified with 20 μL of 25% orthophosphate acid and then centrifuged at 20,000 × g for 10 min at 4 °C. The supernatant was then subjected to VFAs measurement using a gas chromatograph (GC-2010, Shimadzu, Kyoto, Japan) according to the methods described previously [19].

Statistical analysis

The effect of Cr-Met on DMI, lactation performance, and plasma variables of dairy cows were analyzed using the MIXED procedure in SAS software version 2000 (SAS Institute Inc., Cary, NC), with covariance type AR (1) for repeated measures analysis. A randomized block design with repeated measures was used for the analysis, with wk, treatment, interaction of treatment × wk, and block as the main effects and cow within the diet as a random effect. The linear and quadratic effects of treatment on the variables were evaluated with orthogonal polynomials accounting for unequal spacing of Cr-Met supplement levels. The results were listed as least squares means and were separated using the PDIFF option when the fixed effects were significant. P < 0.05 was defined as statistical significance, and 0.05 ≤ P < 0.10 was considered as tendency of significance.

Results

Feed intake and lactation performance

The results of DMI and lactation performance of lactating dairy cows fed with different doses of Cr-Met are shown in Table 2. Throughout the whole experimental period, DMI was not affected by Cr-Met addition (P > 0.05). Milk yield, ECM, and feed efficiency of lactating dairy cows increased linearly with the increase of Cr-Met supplementation, with the greatest value at the dose of 16 g/d (P < 0.01, Figure 1). As the supplemented Cr-Met increased, yields of fat (P < 0.01), protein (P = 0.01), and lactose (P = 0.01) increased in a linear manner. However, concentrations of milk components, inlcuding fat, protein, lactose, and MUN, were not changed by dietary supplementation of Cr-Met (P > 0.05).

Plasma variables related with oxidative stress and liver function

The effect of Cr-Met supplementation on plasma parameters related with liver function in early-lactating cows fed different doses of Cr-Met were presented in Table 3. In terms of antioxidant capacity related index, activities of SOD and GSH-Px were increased in a linear manner (P < 0.01, Table 3). In wk 8 and 12, concentrations of SOD (Figure 2A) and GSH-Px (Figure 2B) were higher in cows supplemented with 16 g/d Cr-Met, compared with the control, respectively (P < 0.01). In terms of variables relative to liver respiration, concentrations of pyruvate, NADH and LDH in the plasma were increased in both linear and quadratic manners, respectively (P < 0.01, Table 3). Similarly, in wk 8 and 12, concentrations of pyruvate (Figure 2C), LDH (Figure 2D), and NADH (Figure 2E) in the plasma of Cr-Met-fed cows were greater than that of the control, respectively (P < 0.05). In terms of liver health status index, total bilirubin, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase in plasma were not changed by supplementation of Cr-Met, respectively (P > 0.05, Table 3).

Plasma variables related protein and energy substrates and antioxidant capacity

The effect of Cr-Met supplementation on plasma index in concerned with protein and energy substrate and glucose metabolism of early-lactating cows were presented in Table 4. As supplemented Cr-Met increased, NEFA increased quadratically (P = 0.09, Table 4) and BHB decreased quadratically (P = 0.10, Table 4) in plasma of dairy cows. The overall glucose concentrations in the plasma across cows in different groups were not different (P > 0.10). However, in wk 8 and wk12, glucose concentration was lower in cows supplemented with 16 g/d Cr-Met, than cows without Cr-Met, respectively (P <0.01, Figure 2H). As added Cr-Met increased, plasma insulin concentration was reduced linearly (P = 0.04, Table 4). Additionally, insulin (wk 12, Figure 2F, P < 0.05) and insulin:glucagon (wk 12, Figure 2G, P < 0.05) were lower in cows consuming Cr-Met, compared with the control, respectively. However, glucagon and insulin:glucagon were not changed by Cr-Met supplementation (P > 0.05, Table 4). The Cr-Met supplementation did not affect concentrations of metabolites related with protein metabolism, including total protein, albumin, globulin, A/G, and BUN, respectively (P > 0.10).

Rumen fermentation parameters

The rumen fermentation parameters of the lactating cows are listed in Table 5. The Cr-Met supplementation did not affect concentrations of VFAs, including acetate, propionate, butyrate, isobutyrate, valerate, isovalerate and ratio of acetate to propionate, respectively (P > 0.10). In addition, there was no difference in the rumen pH and total VFA (P > 0.05).

Discussion

In the current study, Cr-Met had no effects on DMI in dairy cows. This result is inconsistent with previous study [20], who reported that Cr-Met improved DMI in periparturient dairy cows. In our study, multiparous lactating cows with 50 DIM were used to evaluate the effect of Cr on lactation performance. Thus, differences in DMI of dairy cows could be attributed to their different physiological stages between our study and observation in Hayirli et al [20]. Several studies showed that postpartum DMI was greater when Cr-yeast [10] or Cr-Pro [1, 21] were introduced. Previous studies have shown an increase in DMI in response to Cr-Met supplementation [21, 22, 23]. Most of these studies began supplementation prepartum and the study timeline was in the transition period, whereas the current study began at 50 DIM limiting the ability to demonstrate differences in DMI. Sadri et al [24] found that supplemental Cr-Met increased DMI prepartum and tended to increase postpartum DMI of cows fed barley-based diets, but not cows fed a corn-based diet. Additionally, Bryan et al [25] showed no response in grazing cows, indicating that when DMI is physically limiting, cows also may not show a production response to Cr-Met.

In the current study, Cr-Met supplementation improved yields of milk and lactose in early-lactating cows, which is in agreement with previous studies [15.1 mg Cr/d in Cr-Met for early lactation in Hayirli et al [20] and Kafilzadeh et al [26], but not consistent with Yasui et al [27], who found that milk yield was not changed by Cr-Pro addition in early-lactating cows, suggesting that the effect of Cr supplementation on lactation performance of early-lactating dairy cows depended on chelated ligands of Cr. The Met can effectively eliminate free radicals in the mammalian cells, normalize hematocrit, and suppress abnormal activities of enzymes [28]. When Met is chelated with Cr³⁺ by forming a complex, Cr³⁺ can be absorbed by mammary gland more efficiently [29]. Thus, the positive effect of Cr-Met supplementation on milk yield in early-lactating may be attributed to relatively higher absorption rate of Cr-Met in the mammary gland. In addition, overall milk composition (fat, true protein, MUN) was not affected by Cr-Met supplementation in our study, which is in agreement with previous studies [2, 20, 22, 25]. In the current study, as Cr-Met doses increased, more lactose was synthesized in the mammary gland of lactating cows when they consumed similar DMI. It is because lactose production plays a predominant role in milk yield of dairy cows by maintaining osmotic pressure [30]. Thus, increased milk yield induced by Cr-Met in early-lactating dairy cows is attributed to greater lactose production in cows consumed Cr-Met.

In the current study, limited change in rumen propionate concentration indicated that Cr-Met supplementation did not improve available precursors for hepatic gluconeogenesis [2]. Activities of LDH, NADH and pyruvate in the plasma were important index of hepatic respiration [5]. The increased pyruvate, LDH and NADH activities in the plasma of dairy cows with increasing Cr-Met supplementation indicated that hepatic cellular respiration was improved by Cr-Met supplementation. The increased hepatic respiratory rate would lead to a greater level of hepatic gluconeogenesis in dairy cows [31]. Moreover, greater hepatic gluconeogenesis is associated with decreased insulin concentrations in the plasma [32]. In the later period of the experiment, changes of milk yield of cows fed Cr-Met is accompanied with changes of pyruvate, LDH, NADH and insulin in plasma. Thus, the improved milk lactose production induced by Cr-Met supplementation is partly attributed to the increased hepatic respiratory rate and gluconeogenesis of dairy cows. Future research should be conducted to determine how Cr-Met regulates hepatic respiratory rate and gluconeogenesis.

Glucose utilization efficiency in mammary gland plays an important role in lactose synthesis. The limited changes in NEFA and BHB in plasma indicated that cows with different treatments had similar status of body reserve mobilization. In the current study, the lower glucose concentrations in the plasma (wk 4, 8, and 12) of cows fed Cr-Met may suggest that more glucose was absorbed by these cows [20]. Other research has shown Cr-Met supplementation to decrease serum glucose [33]. The decrease of insulin concentration in cows fed Cr-Met may be due to the cumulative effect of Cr supplementation. Nikkhah et al [23] used cows 38 DIM and showed that cows fed supplemental CrMet had decreased blood insulin, NEFA and insulin:glucagon ratio. Moreover, lactose synthesis activity of Golgi could be depressed by oxidative stress in early-lactating cows, which finally led to reduced lactose synthesis [1, 34]. On the other hand, improved anti-oxidative capacity of dairy cows would be beneficial for mammary gland health status and milk synthesis efficiency [35]. In the current study, the linearly increased SOD and GSH-Px activities in the plasma indicate the improved antioxidant capacity of the animal. The improved activities of SOD and GSH-Px in the plasma of cows fed Cr-Met-fed (wk 8 and 12) seem to be consistent with the greater milk yield of cows fed Cr-Met through experimental wk 4-12. Thus, the increased lactose synthesis in animals fed Cr-Met can be partly attributed to their improved anti-oxidative capacity. Thus, the reduced mammary oxidative stress could be another cause for the improved milk lactose yield in cows with Cr-Met.

Conclusion

Under the current experimental conditions, Cr-Met supplementation improved milk yield in a linear manner, which is attributed to the linear increase in milk lactose production. The improved lactose yield could be attributed to the improved hepatic respiratory and greater anti-oxidative capacity of early-lactating dairy cows. The specific role and mechanisms of Cr-Met on liver and mammary gland functionality should be further investigated. Our study indicate that Cr-Met supplementation is beneficial to early-lactating dairy cows.

Abbreviations

ADF: Acid detergent fiber; ALP: Alkaline phosphatase; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; BUN: Blood urea nitrogen; BW: Body weight; CP: Crude protein; DM: Dry matter; DMI: Dry matter intake; ECM: Energy-corrected milk yield; LDH: Lactic dehydrogenase; GSH-Px: Glutathione peroxidase; MUN: Milk urea nitrogen; N: Nitrogen; NDF: Neutral detergent fiber; NEFA: Non-esterified fatty acids; SOD: Superoxide dismutase; VFA: Volatile fatty acids

Declarations

Acknowledgements

The authors appreciate the staff of the Hangjiang Dairy Farm (Hangzhou, China) for their assistance in milking and caring for the animals. The members of the Institute of Dairy Science, Zhejiang University (Hangzhou, China) are acknowledged for their assistance with sampling and analysis of the samples of feeds and blood.

Funding

This research was financially supported by the grants from China-USA Intergovernmental Collaborative Project in S & T Innovation under the National Key R & D Program (No. 2018YFE0111700), China Agriculture Research System (CARS-36), and Zinpro Corporation (Eden Prairie, MN).

Availability of data and materials

All data generated or analyzed during this study are presented in the main

manuscript.

Authors’ contributions

ZZW performed the experiments, analyzed the data and wrote the manuscript.

DMW and JXL contributed in designing the study and revising the manuscript.

WCP were involved in the animal experiment. All authors read and approved

the final manuscript.

Ethics approval

All experimental procedures involving animals were approved by the Animal

Care and Use Committee of Zhejiang University (Hangzhou, China) and were

followed the university’s guidelines for animal research.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Tables

^aPremix, formulated to provide (per kilogram of DM): 150 g of Salt, 220,000-400,000 IU of vitamin A, 50,000-100,000 IU of vitamin D₃, ≥2250 IU of vitamin E, 1000-3800 mg of Zn, 8-25 mg of Se, 12.5-100 mg of I, 600 mg of Fe, 2.5-50 mg of Co, 800-3000 mg of Mn and 200-700 mg of Cu, water ≤ 100 g.

^bPremix, formulated to provide (per kilogram of DM): 0.82% of baking soda, 0.41% of calcium hydrogen phosphate, 0.59% of calcium carbonate, 1.23% of fatty acid calcium, 0.41% of salt, 0.07% of mold adsorbent, 0.07% of active yeast.

^cAll value were estimated using the CPM dairy nutritional model (Tedeschi et al., 2008).

^aECM energy corrected milk, calculated with the equation: ECM = 0.3246 × milk yield + 13.86 × milk fat yield + 7.04 × milk protein yield (Orth, 1992); Feed efficiency = kg of ECM / kg of DM intake; N conversion = milk protein yield /CP intake.

^bL linear, Q quadratic, W week effect, T treatment, T × W the interaction between treatment and week.

^cMilk / DMI = kg of Milk yield / kg of DMI.

^aPlasma variables were measured in the week 4, 8 and 12 of experiment.

^bTBIL total bilirubin; ALP alkaline phosphatase; AST aspartate aminotransferase; ALT alanine aminotransferase; LDH lactic dehydrogenase; SOD superoxide dismutase; GSH-Px glutathione peroxidase.

^cL linear, Q quadratic, W week, T treatment, T × W the interaction between treatment and week.

^aPlasma parameters were measured in weeks 4, 8 and 12 of experiment.

^bBUN blood urine nitrogen; NEFA non-esterified fatty acid.

^cL linear, Q quadratic, W week, T treatment, T × W the interaction between treatment and week.

^aVolatile fatty acids were measured in the week 4, 8 and 12 of experiment.

^bL linear, Q quadratic, W week, T treatment, T × W interaction between treatment and week.