Effects of Intramuscular Injections of Vitamins AD3E and C in Combination on Fertility, Immunity, and Proteomic and Transcriptomic Analyses of Dairy Cows during Early Gestation
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cows and Experimental Design
- (1)
- C: the control treatment for which cows were injected with normal saline;
- (2)
- VAD3E: cows were administered a single intramuscular injection (I/M) of vitamin AD3E (Phenix, Brussels, Belgium; vitamin A 300,000 IU, vitamin D3 100,000 IU, vitamin E acetate 50 mg; 1 mL per 50 kg live weight use) and progesterone release device (CIDR) was removed at night on day 7 (cows received the CIDR on day 0);
- (3)
- VAD3EC: cows received vitamins AD3E and C in combination (a total dose of 1500 mg of vitamin C, ascorbic acid, Q.P., Reasol; 500 mg) at night on day 7;
- (4)
- VC: cows were injected with a single dose of vitamin C on the same day as the other treatment.
2.2. Estrus Synchronization
2.3. Blood and Serum Sample Collection
2.4. Serum Assay
2.5. Proteomic Analysis Using LC-MS/MS Technique
2.6. Gene Expression Analysis
2.7. Statistical Analysis
3. Results
3.1. Conception Rate and Pregnancy Rate
3.2. Hematological Parameters
3.3. Biochemical Parameters and Hormone Contents
3.4. Immune and Antioxidation Activity
3.5. Functional Classification of Identified Serum Proteins
3.6. LC-MS/MS Identification
3.7. Differentially Abundant Protein in All Treatments during Induction Times
3.8. Quantitative Real-Time Reverse Transcription PCR (qPCR)
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Gene 1 | Sequence (5′-3′) | Accession No. | Ref. |
---|---|---|---|
IL-10 | F: TTCTGCCCTGCGAAAACAA | NM_174088 | [16] |
R: TCTCTTGGAGCTCACTGAAGACTCT | |||
TNFα | F: TGACGGGCTTTACCTCATCT | AF_348421 | [16] |
R: TGATGGCAGACAGGATGTTG | |||
ISG15 | F: GGTATCCGAGCTGAAGCAGTT | NM_174366 | [16] |
R: ACCTCCCTGCTGTCAAGGT | |||
SOD1 | F: TGTTGCCATCGTGGATATTGTAG | NM_174615 | [17] |
R: CCCAAGTCATCTGGTTTTTCATG | |||
CAT | F: GCTCCAAATTACTACCCCAATAGC | NM_001035386 | [17] |
R: GCACTGTTGAAGCGCTGTACA | |||
Beta-actin | F: GCGTGGCTACAGCTTCACC | AY141970 | [17] |
R: TTGATGTCACGGACGATTTC |
Items | C | VAD3E | VAD3EC | VC | p-Value |
---|---|---|---|---|---|
Estrus detection AI% | 100.0 | 90.00 | 100.00 | 100.00 | 0.380 |
Detections/all cows | 10/10 | 9/10 | 10/10 | 10/10 | |
Conception Rate AI% | 10.0 | 30.0 | 20.0 | 40.0 | 0.446 |
Detections/all cows | 1/10 | 3/10 | 2/10 | 4/10 | |
Pregnancy Rate% | 10.0 | 30.0 | 20.0 | 40.0 | 0.446 |
Detections/all cows | 1/10 | 3/10 | 2/10 | 4/10 |
Items | C | VAD3E | VAD3EC | VC | p-Value |
---|---|---|---|---|---|
RBC (×106 cells/mm3) | 6.44 ± 0.34 | 4.96 ± 0.55 | 5.78 ± 0.49 | 5.22 ± 0.40 | 0.138 |
Ht (%) | 30.20 ± 1.53 | 23.20 ± 2.76 | 28.40 ± 3.37 | 24.40 ± 2.99 | 0.270 |
Hgb (g/dL) | 9.52 ± 0.41 | 7.46 ± 0.81 | 8.48 ± 0.88 | 7.94 ± 0.87 | 0.299 |
MCV (fL) | 43.38 ± 1.27 | 47.92 ± 2.85 | 49.04 ± 1.82 | 46.82 ± 2.68 | 0.910 |
MCHC (g/dL) | 31.28 ± 0.33 | 31.26 ± 0.33 | 28.74 ± 0.87 | 32.20 ± 1.48 | 0.075 |
WBC (cells/mm3) | 16,640 ± 7124.79 b | 16,620 ± 5956.37 b | 46,580 ± 7362.50 a | 8460 ± 1148.74 b | 0.002 |
Neu (%) | 51.80 ± 12.46 a | 24.80 ± 8.35 b | 14.80 ± 3.11 b | 19.40 ± 4.83 b | 0.015 |
Eos (%) | 2.80 ± 0.86 | 1.40 ± 0.24 | 1.40 ± 0.40 | 2.00 ± 0.55 | 0.280 |
Lin (%) | 43.80 ± 13.07 b | 72.40 ± 8.29 a | 81.40 ± 1.40 a | 77.40 ± 2.23 a | 0.015 |
Mon (%) | 1.80 ± 0.20 | 1.80 ± 0.37 | 2.20 ± 0.49 | 1.60 ± 0.40 | 0.730 |
PLT (cells/mm3) | 201,600 ± 29,512.03 b | 208,400 ± 8518.22 b | 231,200 ± 35,319.12 b | 376,400 ± 36,912.87 a | 0.002 |
Items | C | VAD3E | VAD3EC | VC | p-Value |
---|---|---|---|---|---|
BUN, mg/dL | 4.60 ± 0.51 c | 28.60 ± 1.12 a | 15.00 ± 1.52 b | 8.00 ± 2.07 c | ≤0.01 |
GLU, mg/dL | 4.50 ± 0.22 | 4.60 ± 1.29 | 2.00 ± 0.32 | 2.96 ± 0.50 | 0.057 |
ALB, g/dL | 2.48 ± 0.12 b | 3.14 ± 0.18 a | 3.32 ± 0.17 a | 3.16 ± 0.06 a | 0.003 |
TG, mg/dL | 8.20 ± 0.20 | 9.20 ± 0.37 | 10.60 ± 1.36 | 8.40 ± 1.25 | 0.303 |
CHO, mg/dL | 82.40 ± 2.98 c | 168.40 ± 3.97 a | 122.60 ± 8.73 b | 126.40 ± 7.34 b | ≤0.01 |
Ca, mg/dL | 8.36 ± 0.27 | 8.36 ± 0.42 | 8.56 ± 0.25 | 9.24 ± 0.09 | 0.132 |
P, mg/dL | 9.54 ± 0.74 | 8.30 ± 0.18 | 9.40 ± 0.33 | 8.94 ± 0.88 | 0.486 |
P4, ng/mL | 0.80 ± 0.25 | 0.82 ± 0.34 | 1.02 ± 0.10 | 1.09 ± 0.39 | 0.856 |
E, pg/mL | 21.20 ± 10.80 | 11.60 ± 1.03 | 40.16 ± 16.83 | 11.60 ± 1.03 | 0.168 |
Cortisol, µg/dL | 1.19 ± 0.46 | 0.43 ± 0.24 | 0.71 ± 0.55 | 0.33 ± 0.18 | 0.190 |
Items | C | VAD3E | VAD3EC | VC | p-Value |
---|---|---|---|---|---|
ACH50, U/mL | 294.11 ± 6.24 c | 394.95 ± 20.04 a | 365.05 ± 22.94 ab | 342.14 ± 10.04 bc | 0.004 |
Total Ig, mg/mL | 1.34 ± 0.12 b | 2.61 ± 0.13 a | 2.53 ± 0.20 a | 2.59 ± 0.14 a | ≤0.01 |
LZY, U/mL | 335.10 ± 13.26 b | 499.00 ± 33.60 a | 478.83 ± 30.80 a | 328.53 ± 24.05 b | <0.01 |
SOD, inhibition rate, % | 85.22 ± 0.73 c | 89.26 ± 1.72 b | 94.48 ± 0.72 a | 91.42 ± 0.49 ab | ≤0.01 |
CAT, mU/mL | 0.20 ± 0.00 b | 0.01 ± 0.00 d | 0.23 ± 0.01 a | 0.07 ± 0.01 c | ≤0.01 |
GPx, mU/mL | 0.09 ± 0.01 c | 0.09 ± 0.00 bc | 0.10 ± 0.00 ab | 0.11 ± 0.01 a | 0.003 |
GR, U/mL | 0.16 ± 0.01 c | 0.23 ± 0.02 a | 0.26 ± 0.01 a | 0.20 ± 0.01 b | ≤0.01 |
Log2 Abundance of Protein 1 | |||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Day 0 | Day 7 | Day 14 | |||||||||||||||
Uniprot Accession Number | Protein Name | Unique Peptide Sequence | ID Scores | MH+(Da) | C | VAD3E | VAD3EC | VC | C | VAD3E | VAD3EC | VC | C | VAD3E | VAD3EC | VC | |
1. | A0A3Q1M894 | Lymphocyte-specific protein 1 | LEQYTQAVEIAGR | 7.23 | 1477.08 | 0 | 0 | 0 | 0 | 0 | 0 | 17.51 | 0 | 0 | 17.43 | 0 | 0 |
2. | A0A3Q1MVU8 | Cytokine synthesis inhibitory factor (interleukin-10) | CHRFLPCENK | 9.90 | 1359.95 | 15.81 | 15.43 | 15.79 | 14.74 | 16.20 | 14.08 | 14.90 | 15.30 | 14.78 | 17.09 | 14.97 | 13.20 |
3. | E1BLB2 | TNF-alpha-induced protein 1 | DVIGDEICCWSFYGQGRK | 2.61 | 2190.44 | 15.67 | 14.40 | 16.02 | 17.99 | 15.37 | 14.21 | 13.39 | 14.81 | 14.55 | 14.76 | 13.90 | 14.52 |
4. | F6Q4D3 | ISG15 protein conjugation | KAEVAAEATR | 14.75 | 1045.19 | 16.60 | 15.79 | 16.06 | 15.29 | 16.18 | 15.48 | 15.16 | 16.57 | 16.87 | 16.58 | 15.34 | 15.71 |
5. | E1BHL1 | Superoxide dismutase (Mn), mitochondrial (EC 1.15.1.1) | AGGGAAAVVSLR | 5.20 | 1029.35 | 16.47 | 16.50 | 16.36 | 16.41 | 16.59 | 16.24 | 16.28 | 16.30 | 15.41 | 16.05 | 15.54 | 16.04 |
6. | A0A3Q1LKF1 | Catalase (EC 1.11.1.6) | GIPDGHRHMNGYGSHTFK | 12.03 | 2009.90 | 14.91 | 16.18 | 15.46 | 16.14 | 15.120 | 13.40 | 15.39 | 14.87 | 15.57 | 20.47 | 14.83 | 15.68 |
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Likittrakulwong, W.; Poolprasert, P.; Hanthongkul, W.; Roytrakul, S. Effects of Intramuscular Injections of Vitamins AD3E and C in Combination on Fertility, Immunity, and Proteomic and Transcriptomic Analyses of Dairy Cows during Early Gestation. BioTech 2022, 11, 20. https://doi.org/10.3390/biotech11020020
Likittrakulwong W, Poolprasert P, Hanthongkul W, Roytrakul S. Effects of Intramuscular Injections of Vitamins AD3E and C in Combination on Fertility, Immunity, and Proteomic and Transcriptomic Analyses of Dairy Cows during Early Gestation. BioTech. 2022; 11(2):20. https://doi.org/10.3390/biotech11020020
Chicago/Turabian StyleLikittrakulwong, Wirot, Pisit Poolprasert, Worawatt Hanthongkul, and Sittiruk Roytrakul. 2022. "Effects of Intramuscular Injections of Vitamins AD3E and C in Combination on Fertility, Immunity, and Proteomic and Transcriptomic Analyses of Dairy Cows during Early Gestation" BioTech 11, no. 2: 20. https://doi.org/10.3390/biotech11020020
APA StyleLikittrakulwong, W., Poolprasert, P., Hanthongkul, W., & Roytrakul, S. (2022). Effects of Intramuscular Injections of Vitamins AD3E and C in Combination on Fertility, Immunity, and Proteomic and Transcriptomic Analyses of Dairy Cows during Early Gestation. BioTech, 11(2), 20. https://doi.org/10.3390/biotech11020020