Effect of Parenteral Trace Element Supplementation on the RNA-sequencing Profile of Peripheral Blood in Peripartum Dairy Cows

doi:10.21203/rs.3.rs-2953036/v1

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Effect of Parenteral Trace Element Supplementation on the RNA-sequencing Profile of Peripheral Blood in Peripartum Dairy Cows

https://doi.org/10.21203/rs.3.rs-2953036/v1

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The transition period is the most critical stage in the lactation cycle of dairy cattle. During this period, cows are subjected to high levels of oxidative stress (OS). One way of managing OS is through mineral supplementation with antioxidant micronutrients. The aim of this study was to evaluate the gene expression of transition dairy cows supplemented with the antioxidant trace elements copper (Cu), zinc (Zn), manganese (Mn) and selenium (Se). The study was carried out in a commercial Holstein dairy farm located in General Belgrano, province of Buenos Aires, Argentina. Cows (n = 200) were randomly assigned to either a supplemented or a control group. Blood samples were obtained seven days after calving and used to determine superoxide dismutase (SOD) and glutathione peroxidase activity, antioxidant capacity (AC) and thiobarbituric acid reactive substances. Additionally, RNA-sequencing analysis was performed. The OS index, SOD activity and AC differed significantly between groups, despite only two differentially expressed genes codified for second messengers (adjusted p value < 0.05). This would suggest that trace mineral supplementation of transition dairy cows would not induce changes in gene expression profiles in pathways associated with OS and immune function, since their expression is already high in response to the high OS levels and the dietary changes associated with this period. Nevertheless, considering the role of these minerals as cofactors, a higher availability in the supplemented group would increase antioxidant enzyme activity.

Holstein

Oxidative stress

Transcriptome

Transition period

Mineral supplementation

The transition from gestation to lactation is the most critical phase in the lactation cycle of dairy cattle. This phase, which includes three weeks before and three weeks after calving [1], is commonly named transition period. During this period, cows are subjected to high levels of oxidative stress (OS) [2], which is the result of an imbalance between the production of reactive oxygen species (ROS) and the antioxidant capacity (AC) of the animal metabolism to neutralize them [3]. During transition, OS may contribute to a number of health disorders, including mastitis, metritis, mammary edema and retained fetal membranes [4, 5], which can lead to decreased animal performance and poor health [6, 4]. Therefore, it is not surprising that approximately 75% of diseases that affect dairy herds occur within the first month after calving, reaching maximum values within the first 10 days [1]. The redox status of dairy cows can be assessed with the OS index (OSi), which is a combined measurement based on the ratio between antioxidants and ROS, also used to determine whether cows have experienced an oxidative challenge after calving [7].

Mineral supplementation with antioxidant micronutrients such as selenium (Se), copper (Cu), zinc (Zn) and manganese (Mn) is one of the strategies used for OS management [8]. While Se (an essential component of glutathione peroxidase [GSHpx] enzymes) [8], Cu and Zn are necessary for Cu-Zn superoxide dismutase (SOD) activity, Mn is important for Mn-SOD activity [9]. These can boost the antioxidant response of the animals by an increase of SOD, catalase and GSHpx enzyme activity that can directly catalyze ROS reduction. They can also reduce the levels of stress (lower cortisol) and enhance health status by stimulating the immune system, resulting in lower somatic cell count, increased immunoglobulin content and phagocytic activity of neutrophils and T cells. All of this ultimately improves animal performance, leading to better economic outcomes for dairy farmers [10, 11].

The complex metabolic phenotypes of the transition period can be investigated using high-throughput technologies [12]. In transition dairy cows, the adaptive changes in the gene expression profiles of different tissues and cells such as liver [13–15], rumen [16], adipose tissue, mammary gland [17] and leukocytes [18] have been identified using transcriptomics. Despite evidence suggesting that supplementation with antioxidant microminerals during transition provokes a differential antioxidant response [1], only a few studies have evaluated the effect of Cu, Zn, Mn and Se on the gene expression profiles of these cows. Therefore, the aim of this study was to evaluate the gene expression of dairy cows supplemented with the antioxidant trace elements Cu, Zn, Mn and Se during the transition period.

Animals and Experimental Design

The study was carried out in a commercial dairy farm of Holstein cows (herd size, 600) located in General Belgrano, Buenos Aires, Argentina (35°46′00″S 58°30′00″O). The feed intake of dairy cows complied with the Nutrient Requirements of Dairy Cattle recommendations [19]. Two hundred Holstein cows meeting the following inclusion criteria were selected: number of calvings (2 to 5), no clinical alterations, not receiving parenteral supplementation with minerals 90 days before calving and not receiving pharmacological treatment 60 days before calving. Cows were randomly separated into two groups: supplemented (SG, n = 100) and control (CG, n = 100). Animals from SG received 5 ml of a mineral suspension containing Cu (10 mg/mL as edetate; Sigma-Aldrich®, Missouri, US), Zn (60 mg/mL as edetate; Sigma-Aldrich®), Se (5 mg/mL as sodium selenite; Sigma-Aldrich®) and Mn (10 mg/mL as edetate; Surfactan®) 20 days before calving (day -20) and on the day of calving (day 0). At the same time, CG cows received 5 ml of sterile saline solution.

Seven days after calving, blood samples were obtained from the jugular vein of randomly selected animals (n= 25 from each group) to determine SOD and GSHpx activity, AC and thiobarbituric acid reactive substances (TBARS). Blood samples were collected in Vacutainer® EDTA tubes for hematocrit and GSHpx determination and in tubes without anticoagulant for colorimetric, SOD activity, AC and TBARS determinations.

Six randomly selected blood samples from each group were extracted, preserved in RNAlater (Thermo Fisher Scientific, USA) and stored at -80 °C until processing by RNA-sequencing analysis. The study design is presented in Figure 1.

Biochemical Determinations

Hematocrit and GSHpx Activity

After blood collection, samples were divided into two aliquots, one for hematocrit determination and the other for erythrocyte hemolysis with high-performance liquid chromatography (HPLC)-grade water (1:4). Samples were frozen at -70 °C until complete sample collection (n = 50). After centrifugation to separate plasma from red blood cells, plasma was discarded and red blood cells were used to measure GSHpx activity by ultraviolet visible (UV-vis) spectrophotometry with the Glutathione Peroxidase Assay Kit (Cayman Chemical Company, MI, USA) (340 nm wavelength), whose rate of decrease in A₃₄₀ is directly proportional to GSHpx activity.

SOD Activity, AC and TBARS

After centrifugation, samples were aliquoted into two tubes, one for SOD activity and AC assessment and the other for TBARS determination. SOD activity was determined by UV-vis spectrophotometry using the Superoxide Dismutase Assay Kit® (Cayman Chemical). One SOD unit was the necessary amount of enzyme to produce a 50% reduction of the superoxide radical. Absorbance was measured at 440-460 nm. The determination of AC was done by UV spectrophotometry with the Total Antioxidant Assay Kit (Cayman Chemical) at 750 nm. TBARS were determined with the Total Antioxidant Assay Kit (Cayman Chemical) and measured at 530-540 nm.

RNA-seq and Differential Gene Expression (DGE) Analysis

The blood samples preserved in RNAlater were delivered to the Novogene Sequencing Facility (Sacramento, CA, USA) for RNA extraction with assay and hemoglobin depletion. RNA quality was verified with an Agilent bioanalyzer (Agilent, CA, USA). After quality control procedures, the samples were sequenced in a NovaSeq 6000 sequencing platform (Illumina Inc, CA, USA) and the paired-end 150bp (PE150) strategy. Raw data were filtered to remove low-quality and adapter sequences. Then, the nf-core/RNAseq project pipeline [20] was used to perform RNA-seq analysis using the ARS-UCD1.2 version as the reference bovine genome. In brief, this workflow includes the following analyses: quality control of reads with FastQC [21], STAR software for alignment to reference genome [22] and featureCounts software for counting reads [23]. Readings not having in average 10 total reads in SG and CG were filtered to delete non-expressed genes and transcripts with low expression levels. To identify differentially expressed genes (DEG), the DESeq2 software in R was applied [24].

Statistical analysis

The biochemical determinations in both groups were compared using the Student´s t-test. Statistical significance was set at p < 0.05. DEG were identified using the Benjamini and Hochberg method implemented in the DESeq2 software in R [24]. Transcripts with an adjusted p value < 0.05 were considered to have DGE, as identified by the Ensembl database.

Effects of Mineral Supplementation on Phenotypic Traits

The biochemical determinations showed that one week after birth, OSi was significantly higher in CG than SG, while AC and SOD activity were higher in SG than CG. No differences in GSHpx activity and TBARS were found between groups (Table 1).

Table 1. Biochemichal determinations of oxidative stress index, antioxidant capacity (AC), Cu/Zn-SOD activity (SOD), GSHpx activity (GSHpx) and T-BARs in the control and supplemented groups.

	Control group	Supplemented group	P value
OSi (arbitrary units)	1.29 ± 0.174	0.811 ± 0.032	0.028
AC (mM)	0.643 ± 0.089	0.938 ± 0.044	0.0083
Cu/Zn SOD activity (U/ml)	0.372 ± 0.176	0.506 ± 0.097	0.026
GSHpx activity (U/ml PCV)	80.376 ± 6.836	85.441 ± 8.227	0.322
TBARS* (µM MDA)	0.771± 0.051	0.721 ± 0.032	0.223

Results are presented as means ± standard deviations. Statistical significance was set at p < 0.05.

*One week later, both groups were clearly different (0.743 ± 0.019 vs 0.887 ± 0.067; p= 0.033).

OSi, oxidative stress index; AC, antioxidant capacity; Cu/Zn-SOD, copper/zinc superoxide dismutase; GSHpx, glutathione peroxidase; TBARS, thiobarbituric acid reactive substances.

DGE Analysis

The bioanalyzer measures evidenced that samples from SG and CG exhibited an RNA integrity number (RIN) greater than 7.5 (average 8.40). RNAseq analyses of blood samples resulted in 116,969,995 million reads. Of these, 96.3% (% of global alignment) were successfully mapped to the ARS-UCD1.2. Low expression transcripts were filtered (< 10 reads in total), with 116,947,762 transcripts remaining for the following analysis.

RNA-seq reads of the 12 blood samples from SG and CG (n = 6 each) detected a total of 27,429 genes in at least one of the samples. After filtering, 14,288 genes remained.

Principal component analysis (PCA) based on the expression profiles showed that the two first components accounted for 82.1% of the total variance (PC1 = 70% and PC2 = 12.1%; Figure 2), without discriminating between SG and CG. The DGE analysis resulted in only two DEG (ENSBTAG00000034185 and ENSBTAG00000053198; adjusted p value < 0.05) that encoded for guanosine triphosphate (GTP) protein activity.

In this study, supplementation with trace minerals improved the antioxidant response of transition dairy cows. Cows supplemented with Cu, Se and Zn had lower OSi, higher SOD activity and AC, and similar GSHpx activity. This is in accordance with prior studies in which trace minerals were found to improve animal AC by reducing OS [10, 11], probably due to the effect of Se, Cu, Mn and Zn on the activity of different antioxidant enzymes [8, 9]. The main focus of our work was to detect changes in the gene expression profiles of dairy cows parenterally supplemented with trace minerals during the transition period, mainly in pathways related to antioxidant response mechanisms. In this sense, we found no differences in gene expression, with the exception of GTP protein activity genes, whose expression was found to be higher in supplemented cows. These genes have a wide role as secondary messengers and are important regulators of gene expression and physiological changes such as proliferation, differentiation, migration, survival, apoptosis and depolarization [25].

High-throughput technologies have been used to characterize the different phenotypes of transition dairy cows, which have been shown to undergo adaptive changes in the gene expression of different tissues. For instance, Minuti et al. found major changes after calving, which were mainly related to immunity, inflammation and endocrine aspects [18]. Other groups have reported gene expression changes related to epidermal growth factors of the rumen [16].

Omics technologies have also been used to study the effect of nutrient availability on the gene expression profiles of transition dairy cows. Loor et al. reported that prepartum energy overfeeding of cows resulted in transcriptional changes, predisposing cows to fatty liver [14]. Similarly, underfed cows also suffered changes in the gene expression profiles of the liver [15]. In cows receiving a daily oral bolus of a trace mineral complex (minerals linked to organic compounds) containing Cu, Zn, Mn and Co, DGE was related to inflammation and OS response in peripheral leukocytes and endometrial biopsies [26] and hoof transcriptomics [27] when compared with cows receiving inorganic supplementation.

As reported by different authors [13, 16–18], transition dairy cows naturally undergo changes in the expression of genes related to OS and immune function, concomitant with changes in their physiological state, metabolic requirements and the feed provided. The current results suggest that supplementation of transition dairy cows with trace minerals would not induce changes in the gene expression profiles of these pathways, since they already have an elevated expression in response to the high levels of OS and the metabolic and dietary changes associated with this period. However, a higher availability of these minerals in SG would increase antioxidant enzyme activity, as they are necessary cofactors [28]. Most of the antioxidant defenses are dependent on antioxidant enzymes using their specific substrates to reduce ROS. Enzymes like Cu/Zn or Mn-SOD and GSHpx constitute the first line of defense against free radicals of oxygen. Despite different strategies haven been used to prevent OS, including enzymes and small molecules acting as scavengers, the improvement of enzyme activity would be the most promising antioxidant therapeutic strategy [29]. The ideas discussed above would be supported by the low number of DEG currently found in the study groups and the significant differences in OSi.

In conclusion, supplementation of transition dairy cows with antioxidant micronutrients would be essential, considering that their gene expression profiles naturally undergo changes in response to the conditions of the transition period. However, changes in gene expression alone would not be enough for this response to be effective, and the provision of micronutrients like Cu, Zn, Mn and Se would be fundamental to ensure higher levels of activity in enzymes such as GSHpx and SOD, as shown by the lower levels of OSi detected in supplemented animals.

Data Availability

The transcriptomic data used in this study are available from GEO https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE231491.

Acknowledgements

The authors are grateful to the cattle owners who participated in the study and donated samples. Thanks are also due to Adriana Di Maggio for correcting and editing the manuscript.

Funding

This study was funded by the National Scientific and Technical Research Council of Argentina (Grant PUE-2016 N° 22920160100004CO) and the Research Incentive Program of the National University of La Plata (Grant V247).

Contributions

Conceptualization, F.C., G.G., and S.J.P.; data curation F.C., L.H.O., and M.E.F.; investigation, F.C., M.E.F., L.H.O., A.G.L., G.G., and S.J.P.; supervision, G.G. and S.J.P.; writing—original draft, G.G., and S.J. P.; writing—review and editing, F.C., M.E.F., L.H.O., A.G.L., G.G., and S.J.P.

Competing Interests

The authors declare no competing interests.

Ethics Approval

All animal procedures were performed by a veterinarian and reviewed and approved by the Institutional Committee on Care and Use of Experimental Animals from the School of Veterinary Sciences of the National University of La Plata (Buenos Aires, Argentina; Protocol 123-4-22T).

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No competing interests reported.

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Effect of Parenteral Trace Element Supplementation on the RNA-sequencing Profile of Peripheral Blood in Peripartum Dairy Cows

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