Introduction
Colostrum, the first milk produced by cows following parturition, provides essential nutrients for the calf's healthy development, including immunoglobulin G (IgG), growth factors, hormones, cytokines and numerous vitamins and minerals (McGrath et al., Reference McGrath, Fox, McSweeney and Kelly2016). The most distinctive visual feature of colostrum is its dark yellow colour, attributable to the presence of carotenoids, yellow plant pigments whose concentration is approximately five times higher in colostrum than in mature milk (Sommerburg et al., Reference Sommerburg, Meissner, Nelle, Lenhartz and Leichsenring2000). As mammals, cows cannot produce carotenoids (Álvarez et al., Reference Álvarez, Meléndez-Martínez, Vicario and Alcalde2015), and therefore must obtain carotenoids from their diet, primarily from grass in pasture-based systems. Forage contains the carotene β-carotene, and the xanthophylls (oxygenated carotenoids) lutein and zeaxanthin among others (Noziere et al., Reference Nozière, Graulet, Lucas, Martin, Grolier and Doreau2006). β-Carotene is vital for the functioning of the photosynthetic system (Liguori et al., Reference Liguori, Xu, Van Stokkum, Van Oort, Lu, Karcher, Bock and Croce2017), and serving as a precursor to hormones essential for plant architecture and oxidative stress management (Mansoor et al., Reference Mansoor, Mir, Karunathilake, Rasool, Ştefanescu, Chung and Sun2023). Once absorbed by the cow, the β-carotene pool is divided into direct incorporation into the bloodstream and conversion into vitamin A (Prom et al., Reference Prom, Engstrom and Drackley2022). Vitamin A regulates body development (Harris et al., Reference Harris, Wang, Deavila, Busboom, Maquivar, Parish, McCann, Nelson and Du2018), the immune system (Jin et al., Reference Jin, Yan, Shi, Bao, Gong, Guo and Li2014) and is essential in vision (Saari, Reference Saari2016) and reproduction (Ikeda et al., Reference Ikeda, Kitagawa, Imai and Yamada2005). β-Carotene has the ability to scavenge free radicals, thus preventing oxidative damage (Fiedor and Burda, Reference Fiedor and Burda2014). In ruminants, β-carotene has been suggested to improve rumen microbial production (Aragona et al., Reference Aragona, Rice, Engstrom and Erickson2021). Given that placental transfer of β-carotene to the pre-born calf is minimal (Prom et al., Reference Prom, Engstrom and Drackley2022), the presence of sufficient quantities of β-carotene in colostrum is critical for the calf (Zanker et al., Reference Zanker, Hammon and Blum2000). A deficiency in β-carotene in the dam results in inadequate concentrations of β-carotene in colostrum, thereby increasing the risk of infection and diarrhoea in neonatal calves (Kume and Toharmat, Reference Kume and Toharmat2001; Torsein et al., Reference Torsein, Lindberg, Sandgren, Waller, Törnquist and Svensson2011).
Colour has been suggested as an indicator for carotenoid concentrations in milk (Ugarković et al., Reference Ugarković, Rusan, Vnučec, Konjačić and Prpić2020). Rapid measurement of milk colour, as an estimation of milk carotenoids content, has been proposed to trace the feed system (Nozière et al., Reference Nozière, Graulet, Lucas, Martin, Grolier and Doreau2006) and to predict higher-quality colostrum (Prom et al., Reference Prom, Engstrom and Drackley2022), as a deeper yellow colour in colostrum correlated with higher levels of β-carotene, retinol and α-tocopherol (Prom et al., Reference Prom, Engstrom and Drackley2022). Colostrum colour can be estimated using a colostrum colour scale (Prom et al., Reference Prom, Engstrom and Drackley2022) or a portable colorimeter (Gross et al., Reference Gross, Kessler and Bruckmaier2014). In the laboratory, carotenoids in milk can be measured using high-performance liquid chromatography (HPLC) (Prom et al., Reference Prom, Engstrom and Drackley2022) or ultra-high performance liquid chromatography (UHPLC) (Chauveau-Duriot et al., Reference Chauveau-Duriot, Doreau, Noziere and Graulet2010). The latter is preferable due to its greater sensitivity and better resolution (Guillarme et al., Reference Guillarme, Nguyen, Rudaz and Veuthey2007). However, these techniques require expensive equipment, specialised personnel and are time consuming. Therefore, on-field techniques or rapid laboratory techniques are desirable for measuring colostrum and milk colour to estimate quality.
In addition to the quality of the feed (Nozière et al., Reference Nozière, Graulet, Lucas, Martin, Grolier and Doreau2006) factors such as breed (Stergiadis et al., Reference Stergiadis, Leifert, Seal, Eyre, Larsen, Slots, Nielsen and Butler2015), health status (Andrei et al., Reference Andrei, Matei, Rugină, Bogdan and Ștefănuț2016) and stage of lactation (Jadhav et al., Reference Jadhav, Kulkarni and Chavan2008) also influence the deposition of carotenoids in milk. However, despite ongoing efforts to identify optimal micro-nutrient formulations aimed at meeting the physiological needs of cows (Kurćubić et al., Reference Kurćubić, Ilić, Vukašinović and Đoković2010), preventing metabolic diseases postpartum (Mion et al., Reference Mion, Van Winters, King, Spricigo, Ogilvie, Guan, DeVries, McBride, LeBlanc, Steele and Ribeiro2022), preparing animals for lactation (Erickson and Kalscheur, Reference Erickson and Kalscheur2020) and improving milk quality (Osorio et al., Reference Osorio, Trevisi, Li, Drackley, Socha and Loor2016), there remains a lack of data on how micro-nutrient supplementation affects carotenoid absorption and status in cows, and subsequently in their milk.
Selenium (Se) and choline are essential micro-nutrients, known for their roles in preventing postpartum metabolic diseases (Kommisrud et al., Reference Kommisrud, Østerås and Vatn2005; Delesalle et al., Reference Delesalle, de Bruijn, Wilmink, Vandendriessche, Mol, Boshuizen, Plancke and Grinwis2017), improving milk production (Sales et al., Reference Sales, Homolka and Koukolova2010), increasing IgG content in colostrum (Zenobi et al., Reference Zenobi, Gardinal, Zuniga, Dias, Nelson, Driver, Barton, Santos and Staples2018) and mitigating oxidative stress in mammals (Zakeri et al., Reference Zakeri, Asbaghi, Naeini, Afsharfar, Mirzadeh and Kasra Naserizadeh2021). Moreover, Se and choline have been reported to influence lipid metabolism in animals (Yao and Vance, Reference Yao and Vance1990; Goselink et al., Reference Goselink, Van Baal, Widjaja, Dekker, Zom, De Veth and Van Vuuren2013; Nido et al., Reference Nido, Shituleni, Mengistu, Liu, Khan, Gan, Kumbhar and Huang2016). It has been reported that vitamin E, which has an absorption mechanism similar to that of carotenoids, increases in the meat of broiler chicken (Skrivan et al., Reference Skrivan, Dlouha, Mašata and Ševčíková2008) and in bovine milk (Pinotti et al., Reference Pinotti, Baldi, Politis, Rebucci, Sangalli and Dell'Orto2003) following supplementation with Se and choline, respectively. This suggests that carotenoid levels in milk may also be influenced by Se and choline supplementation. Therefore, the primary objective of this study was to assess the impact of prepartum supplementation of dairy cows with inorganic Se, organic Se or rumen-protected choline (RPC) on the carotenoid content of colostrum and transition milk (TM) using UHPLC-DAD. Additionally, we aimed to develop a method for the rapid estimation of carotenoid levels in colostrum and TM through colour measurement.
Materials and methods
This study was carried out at the Teagasc, Animal & Grassland Research and Innovation Centre, Moorepark, Fermoy, Co. Cork, Ireland between December 2020 and May 2021. Ethical approval was granted by the Teagasc Animal Ethics Committee (TAEC) (TAEC263/2020). Experiments were undertaken in accordance with the Cruelty to Animals Act (Ireland 1876, as amended by European Communities regulations 2002 and 2005) and the European Community Directive 86/609/EC.
Experimental design
The experimental design and prepartum supplementation for this study was previously reported by McDermott et al. (Reference McDermott, Kennedy, Drouin, Brennan, O'Callaghan, Egan and Hogan2024). Briefly, 57 (12 primiparous and 45 multiparous) Holstein-Friesian (HF) (n = 36) and HF × Jersey (JEX) (n = 21) cows were enrolled and balanced based on parity 2.4 (±0.8), expected calving date 8th February 2021 (±12.7 d), prepartum body weight (BW) 595 (±57.4) kg, prepartum body condition score (BCS) 3.23 (±0.041) and Economic Breeding Index (EBI; Berry et al., Reference Berry, Shalloo, Cromie, Veerkamp, Dillon, Amer, Kearney, Evans and Wickham2007) €173 (±39.5).
Animals were assigned to one of three treatment groups:
1. Supplemental sodium (Na) selenite at 50 mg/kg to provide 0.5 mg Se/kg dry matter (DM)/d (INORG),
2. Supplemental Na selenite at 30 mg/kg along with Se-yeast (Selsaf®) at 20 mg/kg to provide 0.5 mg Se/kg DM/d (ORG),
3. Supplemental Na selenite at 50 mg/kg with the addition of 20 g/kg DM/d of rumen-protected choline (RPC). The choline was rumen-protected with hydrogenated, high melting point rape oil providing choline of high bioavailability (Cargill Animal Nutrition, Naas, Co. Kildare). Each treatment group consisted of an equal number of first parity (primiparous) (n = 4), second parity (n = 4) and ≥third (n = 11) animals. The mean length of prepartum supplementation was 49 ± 12.9 d over a range of 45 ± 69 d.
A standard dry cow mineral blend, with the addition of one of the three micro-nutrients (Agritech, Nenagh, Co. Tipperary, Ireland) was top dressed twice daily to the silage. Animals were group penned by treatment, all within the same house and offered 13–14 kg/DM of grass silage with ad lib access to water until day of calving.
Forage sample collection
Silage samples were collected weekly from each treatment group throughout the duration of the experiment, starting from the initiation of mineral supplementation (21/12/2020) until all cows had calved (17/03/2021). These samples were dried at 40°C for 48 h to determine the DM content (25.4 ± 5.51% DM).
Milk sample collection
Colostrum and TM samples were collected as previously described by McDermott et al. (Reference McDermott, Kennedy, Drouin, Brennan, O'Callaghan, Egan and Hogan2024). In brief, colostrum samples were collected from each cow, in the calving pen immediately following parturition using a portable milking unit (InterPuls, Wiltshire, United Kingdom) or in the milking parlour (DairyMaster, Causeway, Co. Kerry, Ireland) if calving occurred within one hour of the scheduled milking time (07:30 h or 14:30 h). Transition milk samples one to five (TM1 to TM5) were collected in the milking parlour at scheduled milking times (as above) using an individual milking unit. Cognisance was taken of colostrum collection time in order to ensure a minimum of six ± one hour time periods between colostrum and TM1 sample collection. Each milk sample (approximately 500 ml) was aliquoted immediately into 15 ml centrifuge tubes and stored at −20°C until analysis. All analyses were carried out upon collection of the entire sample set.
Chemicals and reagents
Tetrahydrofuran (THF) (Fisher Scientific, Belgium, UK), butylated hydroxytoluene (BHT) (Fluka, Switzerland), n-hexane (VWR chemicals, Gliwice, Poland), sodium chloride (NaCl) (Sigma-Aldrich, St. Louis, USA), acetone (VWR chemicals, France), methanol (VWR chemicals, France) and methyl tert-butyl ether (MTBE) (Fisher Scientific, Belgium, UK).
Carotenoid extraction
From the total milk samples collected (n = 142), seven samples were excluded from analysis due to poor solubility of the fat fraction (n = 5) or due to the presence of blood in the milk sample (n = 2). Prior to extraction, milk samples were thawed overnight at 8°C in the dark, to prevent light oxidation and heated to 35°C in a water bath (Clifton Range, NE2D Unstirred Digital Water Bath) for 10 min. To completely homogenise the fat fraction, each sample was vortexed for 30 s (Vortex-T Genie 2, Scientific Industries). Carotenoid extraction was performed as described by Domingos et al. (Reference Domingos, Xavier, Mercadante, Petenate, Jorge and Viotto2014) with modifications. 2 g of each milk sample was transferred to a 50 ml polypropylene tube containing 5 ml THF with the addition of 0.1% BHT to prevent the oxidation of the carotenoids during the extraction process. The remaining proportion of each milk sample was kept in a water bath at 35°C for colour analysis on the same day. Samples re-suspended in THF were extracted three times by vortexing for 30 s and centrifuged at 3500 × g for 5 min at 20°C (3–18 K centrifuge, Sigma, UK). After extraction, no colour remained in the milk residue. 15 ml of n-hexane and 5 ml of saturated NaCl solution were added to the collected organic phase. After vigorous manual shaking for 15 s, the upper organic phase was collected, and the extraction was repeated with an additional 10 ml of n-hexane. The two extracts were combined and dried in a vacuum centrifuge (miVac Centrifugal Concentrator, Genevac™, Ireland) at <30°C, re-suspended in 5 ml acetone and centrifuged again for 5 min. The supernatant was dried in the vacuum centrifuge and re-dissolved in HPLC mobile phase (1:1 methanol:MTBE, v/v) for ultra-high performance liquid chromatography – diode array detection (UHPLC-DAD) analysis.
Carotenoid quantification
Carotenoids were separated and quantified using UHPLC (Nexera SCL-40, Shimadzu, Japan) with a reversed phase sub-2 micron C30 column (Sunshell C30, 2.6 μm, 3.0 × 150, ChromaNik, Japan). A gradient programme was applied with mobile phases A (methanol) and B (MTBE). The flow rate was 0.3 ml/min and the column temperature maintained at 20°C. The gradient started at 0% mobile phase B, increased to 5% during the first 8 min, 20% over the next 7 min and to 50% over the next 5 min. From 20 to 23 min, the concentration of mobile phase B was maintained at 50% and then returned to 0% within 6 min. Sample analysis was monitored at 444 nm for lutein and 451 nm for β-carotene. The identification of β-carotene, cis-β-carotene and lutein was confirmed by the comparison of the retention time and UV-visible spectrum of sample peaks at 451 nm with an authentic standard (Carotenature GmbH, Münsingen, Switzerland). Cis-β-carotene peaks were confirmed by the presence of the cis peak and spectral shifts to blue when compared with the all-trans β-carotene authentic standard. All-trans lutein and all-trans zeaxanthin were identified by retention time and spectrum comparison at 444 nm with a commercial product (UltraLutein™) used as standard.
Quantification was performed by reference to calibration curves for lutein and for β-carotene, with at least five calibration standards, analysed in duplicate, using a UV-Vis spectrophotometer (UV-1900i, Shimadzu, Kyoto, Japan). The molar extinction coefficients used were 149 600 and 136 400 l mol−1 cm−1 for lutein and β-carotene in methanol, respectively. These calibrants were analysed in duplicate, whereas the lowest concentration calibrants were injected five times in order to establish the lower limit of quantification (LLOQ), provided the relative standard deviation (RSD) was lower than 5%. The upper limit of quantification (ULOQ) was allocated as the highest concentration calibrant, with no requirements for RSD. The equation of the calibration line obtained for lutein was y = 0.02018x + 0.47636 (where y is amount of carotenoid standard as ng and x is the peak area), with LLOQ and ULOQ 4.6 ± 0.01 and 62.76 ± 0.08 ng/ml, respectively. For β-carotene the equation was y = 0.02067x + 0.70897, with LLOQ and ULOQ 2.93 ± 0.01 and 72.08 ± 0.04 ng/ml, respectively. The correlation coefficients were 0.9997 and 0.9993 for each calibration line, respectively.
Colour analysis
Colour of the homogenised milk samples was assessed by measuring transmittance spectra using a Shimadzu UV-1900i spectrophotometer. Readings were taken from 300 to 800 nm in 0.5-nm steps using a 1 mm path cuvette (Macro cell 100-1-40, Hellma, Mason Technology, Germany). The spectrophotometer was calibrated using distilled water. Colour analysis was performed concurrently with the extraction process described above, with the milk samples maintained in a 35°C water bath. The cuvette was loaded and transmittance was recorded to calculate the colour index by measuring the upper area of the spectrum between 450 and 530 nm, with transmittance at 530 nm set to zero (Nozière et al., Reference Nozière, Graulet, Lucas, Martin, Grolier and Doreau2006). Colour evaluation followed the CIE system; b* represents the position on the blue-yellow axis, a* represents the position on the green-red axis and L* represents the position of the sample on the dark-light axis.
Statistical analysis
All statistical analyses were conducted using SAS (version 9.4; SAS Institute Inc., Cary, NC). Data were checked for normality using the Shapiro–Wilk test (PROC UNIVARIATE). As all P-values from tests for normality were >0.15, this ensured that all data were normally distributed. The effects of prepartum supplementation on β-carotene concentration (μg/g), colour index and colour parameters (a*, b* and L*) of colostrum and TM1 – TM5 were analysed using linear mixed models (PROC MIXED); P-values < 0.05 were considered statistically significant.
The model included milking postpartum as the repeated measure with animal as the random effect and the fixed effects included treatment (n = 1 to 3), each milking postpartum (time) (n = 6 (Colostrum, TM1… TM5)), parity (n = 1 to 3) and breed (n = 1 (HF) to 2 (JEX)). Non-significant interactions (P > 0.05) were eliminated from the model, with the final model consisting of the main effects. Predicted Transmitting Ability figures (PTAs), obtained from the EBI, were used as covariates to improve the accuracy of the models. Colostrum yield (kg) and duration of mineral supplementation were also included as covariates. Spearman correlation coefficients were calculated using PROC CORR, correlations which were considered ‘strong’ for values ≥0.60, ‘moderate’ between 0.30 and 0.59 and ‘weak’ for ≤0.29.
Results
Carotenoid profile
β-Carotene was the main carotenoid present in all milk samples along with minor amounts of β-carotene cis-isomers, observed at a ratio of 88:12. Lutein was present in trace amounts. However, as the lutein peak area consistently remained below LLOQ throughout the analysis of each milk sample, this carotenoid was not quantifiable. Zeaxanthin was detected in a small number of samples, with peak areas smaller than those of lutein. Overall, the carotenoid profiles of colostrum and TM samples were similar across the three prepartum supplementation groups (Fig. S1, Supplementary material).
Colostrum
Prepartum supplementation with INORG, ORG or RPC did not have a significant effect on colostrum β-carotene concentrations (P = 0.104), colour index values or colour parameters a*, b* or L* (Table 1). Colostrum β-carotene concentrations were higher in JEX (+0.13 μg/g) compared to HF, although this difference was not statistically significant (Table 2). The differences observed in β-carotene concentrations between the two breeds was also evident in the colour index values, with a tendency (P = 0.072) for JEX animals to produce colostrum with a higher colour index (+2.59) compared to HF animals. This difference was also reflected in the b* parameter as JEX animals produced colostrum with a higher b* parameter than HF (P = 0.030). Breed did not have a significant effect on the other colour parameters a* and L* (P = 0.950 and P = 0.973, respectively). Parity did not have a significant effect on β-carotene concentrations, colour index a* or L* parameters in colostrum (Table 3). First parity animals produced colostrum with significantly lower b* parameter compared to higher parity animals (−0.67 and −0.51 for second and third or greater parity, respectively). Second and third or greater parity animals produced colostrum with similar b* parameters (4.75 and 4.59, respectively).
Table 1. β-Carotene (μg/g) and colour parameters of bovine colostrum from pasture-based dairy cows supplemented with inorganic selenium (INORG), organic selenium (ORG) and rumen-protected choline (RPC) during the prepartum period
Evaluating the impact of prepartum supplementation with INORG, ORG or RPC on β-carotene concentrations and colour parameters in bovine colostrum using a one-way ANOVA. Values are represented as mean ± standard error (se). Groups were separated based on supplement received during the prepartum period where INORG, inorganic selenium; ORG, organic selenium and RPC, rumen-protected choline.
1 P-value refers to the impact of treatment.
Table 2. Impact of breed on the β-carotene (μg/g) concentrations and colour measurements of bovine colostrum from pasture-based dairy cows
Evaluating the impact of breed on β-carotene concentrations and colour parameters in bovine colostrum using a one-way ANOVA. Values are represented as mean ± standard error (se). Groups were separated based on breed where HF, Holstein-Friesian and JEX, HF × Jersey.
1 P-value refers to the impact of breed.
a, bMean ± se marked with different letter differ significantly (P < 0.05).
Table 3. Impact of parity on the β-carotene (μg/g) concentrations and colour measurements of bovine colostrum from pasture-based dairy cows
Evaluating the impact of parity on β-carotene concentrations and colour parameters in bovine colostrum using a one-way ANOVA. Values are represented as mean ± standard error (se). Groups were separated based on parity where 1, first parity (primiparous) animals; 2, second parity animals and ≥3, multiparous animals (≥third parity).
1 P-value refers to the impact of parity.
a, bMean ± se marked with different letter differ significantly (P < 0.05).
Transition milk
No significant interactions were observed between treatment and each milking postpartum for β-carotene concentrations (P = 0.161), colour index (P = 0.076), b* (P = 0.628), a* (P = 0.491) or L* (P = 0.256; Table 4). Therefore, the results for each milking are presented as the average of the three prepartum supplementation groups in Table 4 and separately for each treatment group in Fig. 1. The highest concentrations of β-carotene were found in colostrum and decreased significantly to TM3. Concentrations of β-carotene were similar in TM3 and TM4 followed by a further decrease observed from TM4 to TM5. Concentrations of β-carotene decreased by 81.8% from colostrum to TM5 (Fig. 1A). Colour index was highest in colostrum and decreased significantly to TM2 after which values plateaued, until a further decrease occurred from TM4 to TM5. Colour index decreased by 52.5% from colostrum to TM5 (Fig. 1B). Each milking postpartum had no significant impact on the a* parameter (P = 0.539). The highest b* parameter was observed in colostrum and decreased significantly, a decrease of 50.4% from colostrum to TM5 (Fig. 1C). In contrast, L* increased by 37.7% from colostrum to TM5 (Fig. 1D).
Table 4. β-Carotene (μg/g) concentrations and colour measurements of bovine colostrum and transition milk one to five collected at each consecutive milking following parturition
Evaluating the impact of each milking postpartum on β-carotene concentrations and colour parameters as milk transitions from colostrum to transition milk using a repeated measures ANOVA. Prepartum supplementation with inorganic Se (INORG), organic Se (ORG) and rumen-protected choline (RPC) did not have a significant effect on the composition of colostrum or transition milk, with no significant interaction between treatment × time (each milking). Values are given as averages between the three prepartum supplementation treatments groups.
1 P-values refers to the impact of treatment (INORG, ORG or RPC), time (each milking postpartum; colostrum – transition milk five (TM5)) and the interaction between treatment and time.
a, eMean ± se marked with different letter denote significance between each milking time period (P < 0.05).
Figure 1. Changes in β-carotene concentrations (μg/g), colour index and colour parameters b* and L* as milk transitioned from colostrum (colos) to transition milk one to five (TM1…TM5) (each milking postpartum). Groups were separated based on supplement received during the prepartum period where INORG, inorganic selenium; ORG, organic selenium and RPC, rumen-protected choline. Values are represented as mean ± standard error (se). The reader is referred to Table 4; for the individual P-values for the impact each milking postpartum had on β-carotene concentrations, colour index, b* and L* as milk transitioned from colostrum to TM5. Colour parameter a* is not included in Fig. 1 as a* was not significantly affected by each milking postpartum.
The reduction in β-carotene concentration from colostrum to TM5 was significantly correlated with concomitant reductions in colour index (R 2 = 0.560; P < 0.001; Fig. 2A) and in b* parameter (R 2 = 0.671; P < 0.001; Fig. 2B). Conversely, β-carotene concentration was negatively correlated with the a* parameter (R 2 = 0.062; P = 0.003; Fig. 3A) and L* parameter (R 2 = 0.297; P < 0.001; Fig. 3B).
Figure 2. Coefficient of determination observed between β-carotene concentrations (μg/g) in milk and colour. Panel A: Correlation observed between β-carotene concentrations (μg/g) in milk and colour index (R 2 = 0.560; P < 0.001). Panel B: Correlation observed between β-carotene concentrations (μg/g) in milk and colour parameter B (b*) (R 2 = 0.671; P < 0.001).
Figure 3. Panel A: Correlation observed between β-carotene concentrations (μg/g) in milk and colour parameter A (a*) (R 2 = 0.062; P = 0.003). Panel B: Correlation observed between β-carotene concentrations (μg/g) in milk and colour parameter L (L*) (R 2 = 0.297; P < 0.001).
Discussion
Previous research has shown that the cow's diet can impact on the carotenoid profile of bovine milk (Kalac, Reference Kalac2012). In the present study, prepartum supplementation with Se or RPC did not impact colostrum carotenoid composition. However, our results contribute to previous research regarding the impact of prepartum supplementation on carotenoid deposition in colostrum and TM (Antone et al., Reference Antone, Zagorska, Sterna, Jemeljanovs, Berzins and Ikauniece2015; Alhussien et al., Reference Alhussien, Tiwari, Panda, Pandey, Lathwal and Dang2021; Erasmus et al., Reference Erasmus, Machpesh, Coertze and Du Toit2021). The range of β-carotene concentrations within colostrum of the current study was 0.57–2.65 μg/g, which coincided with previous research carried out by Kehoe et al. (Reference Kehoe, Jayarao and Heinrichs2007) who reported β-carotene colostrum concentrations in the range from 0.1–3.4 μg/g (with an average value of 0.68 μg/g). Similarly, Aragona et al. (Reference Aragona, Rice, Engstrom and Erickson2021) observed colostrum β-carotene concentrations with an average of 1.66 μg/g from their control animals, which received no supplementation.
Colostrum colour is influenced directly by the concentration of β-carotene (Faulkner et al., Reference Faulkner, O'Callaghan, McAuliffe, Hennessy, Stanton, O'Sullivan, Kerry and Kilcawley2018). Calderón et al. (Reference Calderón, Chauveau-Duriot, Martin, Graulet, Doreau and Nozière2007) reported that β-carotene accounted for 65% of the variation in the colour index in colostrum and milk over the first 12 weeks postpartum. This correlation was non-linear with the colour index plateauing at β-carotene concentrations above 2 μg/ml, suggesting that the colour index could not reflect further increases in β-carotene content in colostrum. Our results differ from those of Calderon et al. (Reference Calderón, Chauveau-Duriot, Martin, Graulet, Doreau and Nozière2007), as the β-carotene concentrations in the milk samples analysed in the current study were below 2.5 μg/ml, and the correlation with β-carotene and the colour index was linear. In this correlation, β-carotene accounted for 56% of the variation in the colour index. The L* parameter increased significantly with each milking postpartum, reflecting a reduction in β-carotene content in the milk (Chudy et al., Reference Chudy, Bilska and Kowalski2020). The presence of blood components, the varying degree of transfer of carotenoids from blood to milk and individual cow metabolism may contribute or influence to the variation in colostrum colour from cows consuming the same diet (Madsen et al., Reference Madsen, Rasmussen, Nielsen, Wiking and Larsen2004; Nozière et al., Reference Nozière, Graulet, Lucas, Martin, Grolier and Doreau2006; Calderón et al., Reference Calderón, Chauveau-Duriot, Martin, Graulet, Doreau and Nozière2007).
Ugarković et al. (Reference Ugarković, Rusan, Vnučec, Konjačić and Prpić2020) and Prom et al. (Reference Prom, Engstrom and Drackley2022) measured colostrum β-carotene concentrations and the b* parameter and reported moderate, positive correlations (r = 0.54 and r = 0.57 in each study, respectively). The stronger correlation between β-carotene concentrations and the b* parameter observed in the current study may be attributed to the use of transmission spectroscopy. Although reflectance spectroscopy, as used by and Ugarković et al. (Reference Ugarković, Rusan, Vnučec, Konjačić and Prpić2020) and Prom et al. (Reference Prom, Engstrom and Drackley2022) is reliable, transmittance may be more precise to determine colostrum colour, as it avoids the influence of sample structure and angle of measurement (Gardner, Reference Gardner2018). To the best of the authors' knowledge, this study is the first to measure colostrum and TM colour using transmittance.
Previous studies reporting correlations with various colour parameters and β-carotene concentrations have used whole milk samples rather than colostrum (Agabriel et al., Reference Agabriel, Cornu, Journal, Sibra, Grolier and Martin2007). From the correlations presented in the current study, the colour difference between colostrum and TM for the evaluated colour parameters is evident. As the L* represents the dark to light axis, the increase in L* as colostrum returns to mature milk is reflected in the decrease in β-carotene concentrations (Chudy et al., Reference Chudy, Bilska and Kowalski2020). Of all colour parameters assessed in the current study, the b* obtained the highest correlation with β-carotene, which indicates that this colour parameter may be used as an indicator of β-carotene concentrations in colostrum and milk.
McDermott et al. (Reference McDermott, Visentin, McParland, Berry, Fenelon and De Marchi2016) collected milk samples from various dairy breed animals from five to 375 d in milk (DIM). Their findings indicated that JEX cows produce colostrum with a darker colour compared to other breeds with JEX cows, having a greater b* parameter value of 10.03 compared to HF cows with an average b* value of 7.48. Consistent with results of the current study, the darker colour of colostrum from JEX cows is attributable to producing milk with a greater fat and carotenoid content (Ludwiczak et al., Reference Ludwiczak, Składanowska-Baryza, Kuczyńska and Stanisz2020), while also producing lower milk yields than HF cows, resulting in colostrum with higher milk solids (Alessio et al., Reference Alessio, Neto, Velho, Pereira, Miquelluti, Knob and da Silva2016). As expected, parity did not have a significant effect on β-carotene concentrations as previous research has indicated that parity contributes a restricted degree on milk carotenoid variation (Nozière et al., Reference Nozière, Graulet, Lucas, Martin, Grolier and Doreau2006).
Given the changes in composition and biological functions observed by O'Callaghan et al. (Reference O'Callaghan, O'Donovan, Murphy, Sugrue, Mannion, McCarthy, Timlin, Kilcawley, Hickey and Tobin2020) from milk obtained between days zero to day five postpartum, it is accurate to identify colostrum as milking zero and TM defined as milking's two to six after calving (Godden, Reference Godden2008). Similar to colostrum, TM contains elevated bioactive components and nutrient levels although at reduced concentrations than colostrum (van Soest et al., Reference Van Soest, Nielsen, Moeser, Abuelo and VandeHaar2022). The concentrations of β-carotene in mature milk has been reported at 0.07 μg/g (Strusińska et al., Reference Strusinska, Antoszkiewicz and Kaliniewicz2010) which is significantly lower than the β-carotene concentrations observed in TM5 of the current study (0.31 μg/g). Bovines have the ability to accumulate carotenoids, (mainly β-carotene) at high concentrations. Álvarez et al. (Reference Álvarez, Meléndez-Martínez, Vicario and Alcalde2015) reported that calves at 14 months of age stored carotenoids within plasma, adipose tissue and liver. Although the elevated concentrations of IgG in colostrum play a pivotal role in providing immunity to the calf (Lombard et al., Reference Lombard, Urie, Garry, Godden, Quigley, Earleywine, McGuirk, Moore, Branan, Chamorro and Smith2020), the elevated concentrations of β-carotene found within colostrum, and TM1 to TM3 in particular, are critical as colostrum acts as a main source of antioxidants including β-carotene, which represent the non-enzymatic link of the adverse outcome pathway for immune-mediated responses in the neonate (Zanker et al., Reference Zanker, Hammon and Blum2000). This elevated concentrations of β-carotene in TM may be attributable to the inclusion of JEX animals in the current study as Strusińska et al. (Reference Strusinska, Antoszkiewicz and Kaliniewicz2010) solely enrolled HF animals. As neonatal calves are born with low levels of tissue and blood β-carotene (Ghaffari et al., Reference Ghaffari, Bernhöft, Etheve, Immig, Hölker, Sauerwein and Schweigert2019), providing TM over the first few days of life may provide the neonate with additional support for immune function and improved vitamin status. Given the prevalence of calf scour within the first two weeks of birth (Ma et al., Reference Ma, Wo, Li, Chang, Wei, Zhao and Sun2020) the elevated concentrations of β-carotene in TM may generate stronger intestinal epithelial immunity against Escherichia coli (Puppel et al., Reference Puppel, Gołębiewski, Grodkowski, Slósarz, Kunowska-Slósarz, Solarczyk, Łukasiewicz, Balcerak and Przysucha2019) in preventing calf scour.
In conclusion, athough prepartum supplementation with INORG, ORG or RPC to pasture-based dairy cows did not significantly increase β-carotene concentrations in colostrum or transition milk, this paper further characterises the changes occurring in milk as it evolves from colostrum to mature milk. Breed had a significant effect on the b* parameter of colostrum as Jersey cows produced a higher b* value when compared to colostrum from Holstein-Friesian cows. Primiparous animals produced colostrum with the lowest b* value when compared to multiparous animals. This study is the first to measure colostrum and transition milk colour using transmittance. The strong correlation observed between β-carotene concentrations and b* parameter suggests that this colour parameter may be the most suitable for estimating β-carotene concentrations in colostrum and transition milk.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0022029924000463
Acknowledgements
This research has emanated from research conducted with the financial support of Teagasc, the Irish Agriculture and Food Development Authority and Science Foundation Ireland (SFI) and the Department of Agriculture, Food and Marine (DAFM) on behalf of the Government of Ireland under Grant Number [16/RC/3835] – VistaMilk.
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