How Management System Affects the Concentration of Retinol and α-Tocopherol in Plasma and Milk of Payoya Lactating Goats: Possible Use as Traceability Biomarkers
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Geographical Location and Animal Management
- (a)
- Mountain grazing (MG). Goats grazed ad libitum for eight hours per day in a pasture composed of 60% Poaceae (Gramineae) and other families. The rest of the time, they were stabled and received 800 g/day of cereal-based pellet compound feed. The farm was located at 934 m of altitude and mid-mountain weather conditions.
- (b)
- Cultivated meadow (CM). Goats grazed ad libitum for eight hours per day on a pasture composed of 95% Poaceae (Avena sativa) and other families. The rest of the time, they were stabled and received 500 g/day of compound feed composed of 28.45% MG pellet compound feed + 35% sunflower seed (Helianthus annuus) + 28.3% oat seed (Avena sativa) + 8.25% pea seed (Pisum sativum). The farm was located at 298 m of altitude and valley weather conditions.
- (c)
- Total mixed ration (TMR). Goats were always stabled and fed 1.5 kg/day total mixed ration based on cereal and hay ad libitum.
- (a)
- Pasture. Three sampling times were considered: at the time of parturition (M1), at 10 days of lactation (M2) and at 30 days of lactation (M3). Samples were collected according to the procedures of Fernández et al. (1993) [24]. Briefly, samples were collected in triplicate, classified by botanical families, weighted and percentages of each family were calculated. Then, samples were stored in self-sealing transparent bags labeled and identified with the botanical family and the sampling date and thereafter placed in a freezer at −80 °C for subsequent lyophilization and analysis.
- (b)
- Pellet compound feed. Samples were obtained directly from commercial bags and placed in transparent self-closing bags labeled with the farm identifier and the sampling date. Then, the samples were stored in a freezer at −80 °C until analysis. Following commercial practices, compound feeds were supplemented with vitamins A and E using encapsulation technology.
- (c)
- Hay. Hay was provided only to TMR goats. Samples were taken directly from the hay bales on the farm, placed in self-sealing transparent bags and labeled with the sampling date. Then, they were stored in a freezer at −80 °C for subsequent lyophilization and analysis.
2.2. Sampling of Blood and Milk
- (a)
- Blood. For each sample, 10 mL was extracted from each goat in duplicate by jugular puncture and collected in VACUTAINER vacuum tubes. Li-heparin was used as an anticoagulant. The samples were labeled with the goat’s identification code, the farm and the date. The samples were cold transported to the laboratory at 4 °C in a portable icebox. The samples were centrifuged (2500 rpm, 10 min, 4 °C) in an Eppendorf 5810 R centrifuge to separate the plasma, which was stored at −20 °C in the freezer until analysis.
- (b)
- Milk. Two 30 mL samples of milk from each goat were collected aseptically in Falcon tubes and protected from light. The samples were taken in the morning before milking after eliminating the first jets. The samples were labeled with the goat’s identification code, the farm and the date. The samples were cold transported to the laboratory at 4 °C in a portable icebox. The samples were kept in a freezer at −20 °C until analysis.
2.3. Extraction of β-Carotene, Lutein, Retinol and α-Tocopherol
- (a)
- Feed. Extraction of the fibrous and concentrated feed samples was performed in duplicate according to the methodology described by Pickworth et al. [25]. Samples were homogenized with the aid of a mortar, and the solvent was slowly mixed with 5 g of sample until coloring ceased to form. The solvent used for the extractions was a mixture of hexane and ethanol (1:1, v/v). The resulting liquid was filtered and then saponified overnight at room temperature in darkness and in a nitrogen atmosphere with a potassium methanol solution of 10–15%. Subsequently, the liquid was transferred to a separatory funnel and distilled water, and 1 mL of diethyl ether was added. After the phases were separated, the aqueous phase was discarded. The organic phase was washed with demineralized water until neutralization. Subsequently, the resulting extract was evaporated with a stream of nitrogen gas, and the dry residue obtained was resuspended in 100 μL of ethyl acetate for subsequent analysis by high-performance liquid chromatography (HPLC).
- (b)
- Plasma and milk. The compounds were extracted from plasma samples using the protocol described by Lyan et al. [26] and for the milk samples, the protocol described by Nozière et al. [11] was used. Both extractions were performed in duplicate for each sample and shared the initial stages of the procedures. For each extraction, 2 mL of sample, 1 mL of distilled water, 2 mL of ethanol and 2 mL of hexane were mixed in 50-mL Falcon tubes. The organic phase was separated with Pasteur pipettes after centrifugation (2500 rpm, 10 min, 4 °C). In the aqueous phase, hexane extraction was repeated two more times. On the one hand, the extracts obtained from plasma were evaporated to dryness with a stream of nitrogen gas. The dry residue was resuspended in 50 μL of ethyl acetate for subsequent analysis by HPLC. On the other hand, the extracts obtained from milk were saponified with 15% ethanolic potassium hydroxide overnight at room temperature in darkness and in a nitrogen atmosphere to hydrolyze the triacylglycerols integrated into the milk fat [27]. The next day, the samples were washed by adding distilled water to remove excess potash and subsequently evaporated to dryness using a stream of nitrogen gas. The resulting dry residue was resuspended in 50 μL of ethyl acetate for HPLC analysis.
2.4. Chromatographic Conditions
2.5. Statistical Analysis
3. Results and Discussion
3.1. Feed (Pasture, Hay and Pellet Compound Feed): Effect of Management System
3.2. Goat Plasma and Milk: Effects of Management System and Sampling
3.2.1. Plasma
3.2.2. Milk
3.2.3. Relationship between Fluids (Plasma/Milk)
3.3. Discriminant Analysis
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Mountain Grazing (MG) | Cultivated Meadow (CM) | Total Mixed Ration (TMR) | |
---|---|---|---|
Pasture (% of each botanical family) | ad libitum | ad libitum | none |
60% Poaceae 11% Fagaceae 8% Geraniaceae 6% Malvaceae 6% Fabaceae 5% Asteraceae 4% Others | 95% Poaceae 3% Asteraceae 1% Rubiaceae 1% Malvaceae | ||
Hay | none | none | ad libitum |
33% Avena sativa 33% Vicia sativa 33% Hordeum vulgare var hexastichon | |||
Compound feed | 800 g/day | 500 g/day | 1.5 kg/day |
Graminae: maize, oats, barley, wheat bran. Legumes: broad beans, field beans, sweet lupins, Oilseeds: genetically modified soybean, sunflower seeds. | 28.45% MG concentrate, 35% sunflower seed, 28.3% oat seed, 8.25% pea seed. | Graminae: genetically modified maize, barley, triticale, wheat bran, oatmeal, beet pulp, genetically modified soy peel. Legumes: broad beans, field beans, sweet lupins, fava bean, kidney beans Oilseeds: sunflower seed, genetically modified soybean. |
β-Carotene | α-Tocopherol | Lutein | ||
---|---|---|---|---|
Mountain grazing (MG) | Pasture | 164.319 b ± 2.011 | 147.839 b ± 2.915 | 54.887 b ± 1.428 |
Compound feed * | 2.150 y ± 0.006 | 17.893 y ± 0.020 | 0.195 x ± 0.001 | |
Cultivated meadow (CM) | Pasture | 495.334 c ± 0.046 | 37.047 a ± 0.001 | 139.327 c ± 0.001 |
Compound feed * | 1.120 x ± 0.06 | 19.547 z ± 0.233 | n.d. | |
Total mixed ration (TMR) | Hay | 2.063 a ± 0.041 | n.d. | 1.000 a ± 0.003 |
Compound feed * | 37.640 z ± 0.017 | 0.250 x ± 0.006 | 14.022 y ± 0.001 | |
Fibrous feeds (pasture and hay) p-value | ˂0.001 | ˂0.001 | ˂0.001 | |
Compound feed p-value | ˂0.001 | ˂0.001 | ˂0.001 | |
(*) Vitamin supplementation added to the compound feed | ||||
Vitamin E (all-rac-alpha-tocopherol acetate) | 30 UI/kg | 30 UI/kg | 30 UI/kg | |
Vitamin A (retinyl acetate) | 10.000 UI/kg | 10.000 UI/kg | 10.000 UI/kg |
Retinol | α-TocopheroL | |||||||
---|---|---|---|---|---|---|---|---|
M1 | M2 | M3 | p-Value | M1 | M2 | M3 | p-Value | |
MG | 1.89 ± 0.52 ax | 9.00 ± 1.63 ay | 11.80 ± 1.58 ay | ˂0.001 | n.d. | n.d. | 64.26 ± 14.56 b | |
CM | 3.68 ± 1.52 a | 6.83 ± 2.26 a | 12.06 ± 0.60 a | 0.086 | 6.36 ± 2.81 x | 9.10 ± 2.95 x | 44.65 ± 5.75 by | ˂0.001 |
TMR | 18.95 ± 3.36 b | 20.17 ± 4.51 b | 25.92 ± 3.61 b | 0.390 | 2.06 ± 0.38 x | 3.62 ± 0.85 xy | 5.43 ± 0.90 ay | 0.001 |
p-value | ˂0.001 | 0.007 | ˂0.001 | 0.158 | 0.112 | ˂0.001 |
Retinol | α-Tocopherol | |||||||
---|---|---|---|---|---|---|---|---|
M1 | M2 | M3 | p-value | M1 | M2 | M3 | p-value | |
MG | 6.62 ± 1.46 | 5.59 ± 0.34 b | 3.84 ± 0.51 | 0.092 | n.d. | n.d. | n.d. | |
CM | 9.51 ± 2.84 y | 2.46 ± 0.54 ax | 3.86 ± 0.62 xy | 0.019 | n.d. | n.d. | n.d. | |
TMR | 10.69 ± 1.23 y | 8.26 ± 0.79 cxy | 4.04 ± 0.65 x | ˂0.001 | 3.15 ± 0.19 z | 2.10 ± 0.40 y | 0.90 ± 0.28 x | ˂0.001 |
p-value | 0.389 | ˂0.001 | 0.965 |
Assigned/Real Data | MG | CM | TMR |
---|---|---|---|
MG | 83.3 | 16.7 | 0 |
CM | 25 | 75 | 0 |
TMR | 18.2 | 0 | 81.8 |
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Roncero-Díaz, M.; Panea, B.; Argüello, A.; Alcalde, M.J. How Management System Affects the Concentration of Retinol and α-Tocopherol in Plasma and Milk of Payoya Lactating Goats: Possible Use as Traceability Biomarkers. Animals 2021, 11, 2326. https://doi.org/10.3390/ani11082326
Roncero-Díaz M, Panea B, Argüello A, Alcalde MJ. How Management System Affects the Concentration of Retinol and α-Tocopherol in Plasma and Milk of Payoya Lactating Goats: Possible Use as Traceability Biomarkers. Animals. 2021; 11(8):2326. https://doi.org/10.3390/ani11082326
Chicago/Turabian StyleRoncero-Díaz, Mercedes, Begoña Panea, Anastasio Argüello, and María J. Alcalde. 2021. "How Management System Affects the Concentration of Retinol and α-Tocopherol in Plasma and Milk of Payoya Lactating Goats: Possible Use as Traceability Biomarkers" Animals 11, no. 8: 2326. https://doi.org/10.3390/ani11082326
APA StyleRoncero-Díaz, M., Panea, B., Argüello, A., & Alcalde, M. J. (2021). How Management System Affects the Concentration of Retinol and α-Tocopherol in Plasma and Milk of Payoya Lactating Goats: Possible Use as Traceability Biomarkers. Animals, 11(8), 2326. https://doi.org/10.3390/ani11082326