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Branched chain fatty acid composition of yak milk and manure during full-lactation and half-lactation
Prostaglandins, Leukotrienes and Essential Fatty Acids 150 (2019) 16–20
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Original research article
Branched chain fatty acid composition of yak milk and manure during fulllactation and half-lactation Wancheng Suna,b, Yihao Luoa, Dong Hao Wangb, Kumar S.D. Kothapallib,c, J. Thomas Brennab,c,
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a
Animal Science Department, College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA c Dell Pediatric Research Institute and Deptartment of Pediatrics, Dell Medical School, The University of Texas at Austin, 1400 Barbara Jordan Blvd, Austin, TX 78723, USA b
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A B S T R A C T
Keywords: Branched chain fatty acids (BCFA) Yak milk Yak manure Gene expression
Background: Branched chain fatty acids (BCFA) are bioactive food compounds and are well known to be essential components of human, cow and caprine milk. In Qinghai-Tibet plateau, yaks are domesticated in large numbers and their milk in addition to meat are commercially important to millions of Tibetans and Chinese. Objective: We tested the hypotheses that concentrations of BCFA in yak milk and manure differ between lactation periods and evaluated gene expression levels of certain genes involved in the biosynthesis and elongation of fatty acids. Design: Fresh milk and manure were collected from each yak and their fatty acid compositions compared with emphasis on BCFA. Participants/setting: Yak milk and manure samples from the full lactation (October, 2015) and half lactation periods (March, 2016) were collected and BCFA levels were analyzed in detail by GC-FID and structures verified by GC-EI-MS/MS. Gene expression studies were carried out by semi-quantitative real time PCR method. Statistical analyses performed: The difference between full lactation and half lactation was tested using student's ttest. Linear regression model was modelled in Excel and its significance was tested by ANOVA. Statistical significance was determined by performing student's t-test for gene expression studies. Results: BCFA ranged from 3–6% of total fatty acids in yak milk samples. The half-lactation yak milk contained higher levels of BCFA (5.29 ± 0.53) than the full-lactation milk (4.00 ± 0.46). The total BCFA in yak manure was found to be 14.67 ± 1.21, high in anteiso-15:0 and anteiso-17:0. ELOVL1 enzyme involved in the elongation of saturated C18 to C26 acyl-CoA substrates and MCAT enzyme involved in the transfer of a malonyl group to the mitochondrial acyl carrier protein are significantly upregulated in full-lactation milk. Conclusions: BCFA in yak manure especially anteiso BCFA are positively correlated with yak milk from the same animal, indicating that these BCFA come from dietary sources. Yak milk delivers 777 mg BCFA compared to 158 mg per cup of whole U.S. dairy milk. QTP herders known to consume up to 2 kg of yak yogurt take in an estimated 3,500–5,000 mg BCFA per day. We conclude that BCFA intake for yak milk consumers is among the highest known in the world, higher when drawn from half lactating yaks.
1. Introduction Yak (Bos grunniens or Poephagus grunniens) is a versatile domestic animal that provides the basic subsistence and nutrition to the people inhabiting the Qinghai-Tibet Plateau (QTP), which comprises alpine and subalpine regions [1]. The yak is the only bovine species that has adapted to thrive in extremely harsh and high altitude conditions of QTP, at heights of 2500–6000 m above sea level [2]. The total world population of yaks is estimated at around 14.2 million, approximately
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13.3 million occur within China [3]. Just put the number in context, there are about 9.2 million milk cows in the United States [4]. Yak milk contributes to 15% of Chinese milk consumption and contains higher fat content at about 5.3 to 8.8% compared to 4.2% in bovine milk [5–7]. The high fat content is a critical energy booster for their calves’ survival on the cold QTP as well as for human consumers. Earlier reports have shown seasonal variation in the fat composition of yak milk is mainly related to the seasonal changes in the grass composition of the pasture [1,3].
Corresponding author. E-mail address: [email protected] (J.T. Brenna).
https://doi.org/10.1016/j.plefa.2019.09.002 Received 5 April 2019; Received in revised form 15 July 2019; Accepted 4 September 2019 0952-3278/ © 2019 Elsevier Ltd. All rights reserved.
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Branched chain fatty acids (BCFA) are primarily saturated FA (SFA) with one or more methyl branches [8]. BCFA are major components of vernix caseosa and are constituents of the term newborn gut lumen, being swallowed as vernix particulates of the amniotic fluid [8]. BCFA are bioactive components known to reduce incidence of necrotizing enterocolitis (NEC) in a neonatal rat model [9], have anti-tumor effects on T-cell non-Hodgkin's lymphoma (T-NHL) cell lines [10] and regulate post-embryonic development [11]. BCFA are widely available in the food supply, primarily from dairy and beef, seafood and are also present in human colostrum and breastmilk [8,12–15]. A thorough survey of the fatty acid profile of cow's milk from the US market identified 2% BCFA in milk fat and an estimated daily intake of 500 mg BCFA from dairy and beef [12,13]. The human breastmilk samples collected from regular dairy-consuming and low-dairy-consuming populations showed variability in BCFA content, higher BCFA concentrations found with diary consumption [15]. Recent studies have shown that ruminant foods are rich sources of BCFA, with content and profile of BCFA depending on the activity and composition of rumen microbiota [12,16]. BCFA are a major component of bacterial membranes most notably bacilli [17], as well as Bifidobacterium strains [18]. Many bacterial species have more than 50% BCFA in their membranes, including the potential pro-biotic Sporolactobacillus inulinus that have >90% BCFA [19]. BCFA constitute >90% of the fatty acids (FA) in many species of Bacilli, Lactobacilli and Brevibacterium [17,20]. The Bacillus subtilis synthesize higher proportions of anteiso-15:0 and anteiso-17:0 BCFA due to cold shock response [21]. Yaks of reproductive age and that are lactating can be divided into two types- the full-lactating yak and the half-lactating yak. The yaks in the year of calving that are lactating and suckling a calf are called “fulllactating yaks”. Yaks calved in the previous year that are not pregnant however still suckling their last calf are called Yama or “half-lactating yaks” [22,23]. The yak, even though not pregnant at the onset of winter but still suckling a calf, will continue to lactate through the following warm season and, normally, will be hand milked. The "half-lactating yak" will stop being milked at the end of her second warm season and will then go dry, irrespective of whether the yak is pregnant or not. Half-lactating yak milk yield is about half to two thirds of the yield in the year of calving. The two different lactation periods could have effects on milk composition. It is unknown whether fatty acids, especially BCFA vary in milk between these two lactation periods. Manure is a remote indicator of rumen fermentation and can be compared to milk production from the same animals. There are no studies linking BCFA composition of yak milk and manure from the same animal. Here we tested the hypotheses that concentrations of BCFA in yak milk and manure differ between lactation periods and evaluated gene expression levels of certain genes involved in the biosynthesis and elongation of fatty acids.
Fig. 1. BCFA methyl esters distribution in yak milk and manure (mean ± SD). The iso-13:0, anteiso-13:0 and iso-17:0 are under detection limits in yak milk.
2.2. Fatty acid extraction and analysis Fatty acid methyl esters (FAME) were prepared using a modified one-step digestion and methylation method [24]. FAME mixtures were analyzed quantitatively by Hewlett Packard 5890 series II gas chromatograph-flame ionization detector (GC-FID) using an equal weight mixture for response factor calibration. Detection limits are estimated to be about 0.02% w/w. FAME structural identity was verified by gas chromatography covalent adduct chemical ionization tandem mass spectrometry (GC-CACI-MS/MS) and/or gas chromatography electron ionization tandem mass spectrum (GC-EI-MS/MS) as described earlier [13,25]. The section of a typical chromatogram demonstrating resolution of BCFA from adjacent peaks is presented in Supplementary Materials (Fig. 1). 2.3. RNA isolation and cDNA synthesis Total RNA was isolated from lyophilized yak milk powder samples using the E.Z.N.A. Total RNA kit (Omega Bio–Tek, GA). The quantity and quality of RNA was analyzed by 260/280 nm ratios using a microspectrophotometer (NanoDrop 2000, Thermo Scientific). The total RNA was reverse-transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, NY) according to the manufacturer's instructions. The resulting cDNA was used as template for semi-quantitative real time PCR reactions. 2.4. Semiquantitative RT-PCR We designed gene specific PCR primers using Bos mutus (wild yak) mRNA sequences and the PrimerQuest software from Integrated DNA Technologies (IDT; Coralville, IA). Primer pairs were ordered from IDT. The primer sequences and standardized annealing temperatures of each primer pair are presented in supplementary Table 1 (Table S1). Semiquantitative RT-PCR amplification reactions were run on an Eppendorf gradient thermal cycler (Eppendorf, NY) using EmeraldAmp GT PCR Master Mix (Clontech, CA). PCR products were separated on 2% agarose gel stained with ethidium bromide and bands visualized under UV light. The intensities of the expressed amplicons were quantified densitometrically by ImageJ software (National Institutes of Health, USA). Expression levels of each transcript were normalized to expression values of control Beta-Actin gene.
2. Materials and methods The study was approved by the institutional review board of Qinghai University on animal subjects research.
2.1. Collection of yak milk and manure The milk and manure samples were all collected from the same farm of yak herds located on the plateau altitude of 3200 m in Qilian county of Qinghai Province in China. Fresh milk samples were collected from free range ten full-lactating yaks in October 2015 and eight half-lactating yaks in March 2016. The milk samples were directly milked from the yak and stored in vials covered with ice packs. All the milk samples were lyophilized to yak milk powder and kept in −20 °C for further processing in the laboratory. Manure from the same animals were also collected, labeled and kept in −20 °C until laboratory analysis.
2.5. Statistical analysis Data was analyzed using Microsoft Excel (2010) software. All data in figures are presented as mean ± SD. The difference between full lactation and half lactation was tested using student's t-test. Linear regression model was modelled in Excel and its significance was tested by 17
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Table 1 Fatty acid composition of yak milk from full lactation (n = 10) and half lactation (n = 8) (%, w/w; mean ± SD).
C10:0 C12:0 iso-14:0 C14:0 iso-15:0 Anteiso-15:0 C15:0 iso-16:0 C16:0 C16:1 Anteiso-17:0 C17:0 C17:1 C18:0 C18:1 C18:2 C19:1 C18:3 C20:0 CLA C20:1 C20:2 C22:0 C22:1 C22:4n-6 C24:0 C22:5n-3 Σ BCFA Σ PUFA
Full lactation
Half lactation
p value
1.13 ± 0.28 1.29 ± 0.23 0.32 ± 0.06 7.68 ± 1.02 0.92 ± 0.19 1.09 ± 0.15 1.83 ± 0.18 0.60 ± 0.07 33.26 ± 1.67 2.93 ± 0.50 1.07 ± 0.07 1.44 ± 0.08 0.63 ± 0.05 11.84 ± 2.90 28.70 ± 2.10 1.58 ± 0.25 0.33 ± 0.04 0.89 ± 0.28 0.69 ± 0.13 0.49 ± 0.12 0.46 ± 0.09 0.21 ± 0.04 0.39 ± 0.07 0.10 ± 0.02 0.19 ± 0.04 0.15 ± 0.04 0.29 ± 0.06 4.00 ± 0.46 3.17 ± 0.59
0.95 ± 0.23 1.19 ± 0.15 0.44 ± 0.06 7.09 ± 0.41 0.99 ± 0.08 1.45 ± 0.17 2.35 ± 0.27 1.05 ± 0.16 33.13 ± 3.26 2.54 ± 0.97 1.36 ± 0.13 1.88 ± 0.20 0.90 ± 0.11 11.32 ± 2.70 27.23 ± 2.76 1.93 ± 0.18 0.48 ± 0.05 0.48 ± 0.09 0.76 ± 0.09 0.32 ± 0.10 0.41 ± 0.04 0.24 ± 0.04 0.17 ± 0.06 0.39 ± 0.04 0.94 ± 0.37 0.13 ± 0.04 0.18 ± 0.03 5.29 ± 0.53 3.77 ± 0.39
0.1422 0.2535 0.0003 0.1233 0.3145 0.0001 0.0001 0.0000 0.9108 0.2557 0.0000 0.0000 0.0000 0.6872 0.1892 0.0030 0.0000 0.0006 0.1892 0.0034 0.2037 0.0787 0.0000 0.0000 0.0000 0.3360 0.0001 0.0000 0.0175
ANOVA. Statistical significance was determined by performing student's t-test for gene expression studies. 3. Results 3.1. BCFA in yak milk samples Total BCFA ranged from 3–6% of total FA in yak milk with an overall mean of 4.57 ± 0.82 regardless of lactation periods. BCFA identified in yak milkfat are iso-14:0, iso-15:0, anteiso-15:0, iso-16:0, and anteiso-17:0. The anteiso BCFA constituted about half of the BCFA pool. PUFA are 3.43 ± 0.59 of the total fatty acids. To determine the effect of lactation period on BCFA content, Table 1 summarizes the fatty acid profile of full-lactation and half-lactation yak milk expressed as mean ± SD, respectively. BCFA from half-lactation milk are significantly higher (5.29 ± 0.53) than full-lactation milk (4.00 ± 0.46, p < 0.001). PUFA levels are also significantly higher in half-lactation milk (3.77 ± 0.39) than full-lactation milk (3.17 ± 0.59, p = 0.02). In comparison, conjugated linoleic acid (CLA) is lower in half-lactation milk (0.32 ± 0.10) than full-lactation milk (0.49 ± 0.12, P < 0.0034). Table S2 & S3 present the fatty acid profile of ten yak milk's collected in October, 2015 (full lactation) and eight in March subsequent year (half lactation). Levels of BCFA and PUFA are summarized at the bottom of the two tables.
Fig. 2. Manure and milk BCFA correlation reveal a rumen origin of milk BCFA in yak. (a) anteiso-15:0 (r2 = 0.63, p < 0.001); (b) anteiso-17:0 (r2 = 0.63, p < 0.001); (c) total BCFA (r2 = 0.12, p = 0.17).
17:0 are the top two BCFA by weight. A full fatty acid profile of yak manure collected in October, 2015 and in March, 2016 are presented in supplementary Table S4 and Table S5. Fig. 2 shows the correlation between manure and milk BCFA. The two major BCFA, anteiso-15:0 and anteiso-17:0 are explored separately for this purpose. One outlier, animal No.4 in October was excluded because manure sample was high in both anteiso-15:0 and anteiso-17:0, presumably due to post-fermentation or contamination. Both anteiso15:0 and anteiso-17:0 followed a positive linear regression between manure BCFA and milk BCFA (r2 = 0.6, p < 0.001). Total manure and milk BCFA show a weak correlation (Fig. 3c, r2 = 0.1).
3.2. Manure BCFA and its correlation with milk BCFA For each yak milk sample, we also analyzed a sample of the same yak's manure. Fig. 1 shows the BCFA concentrations of yak manure beside yak milk. The total BCFA in yak manure was found to be 14.67 ± 1.21, w/w. BCFA are much higher in manure samples, than the milk samples. BCFA identified in yak manure are iso-13:0, anteiso13:0, iso-14:0, iso-15:0, anteiso-15:0, iso-16:0, iso-17:0 and anteiso-17:0. The iso-13:0, anteiso-13:0 and iso-17:0 present in yak manure are under detection limits in yak milk. In yak manure, anteiso-15:0 and anteiso-
3.3. Expression levels of genes involved in the biosynthesis and elongation of fatty acids Semi-quantitative RT-PCR was performed to test expression levels of certain genes involved in the biosynthesis and elongation of fatty acids (Fig. 3). ELOVL1 enzyme involved in the elongation of saturated C18 to 18
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milk can be accounted for by the grass composition of the pasture, which in turn having influence on the gut microbiota. We found certain differences in BCFA profile in yak manure compared to yak milk. The shorter chain iso-13:0 and anteiso-13:0 and longer chain iso-17:0 are detected only in yak manure. As the fatty acid are metabolized in the rumen before their absorption in ruminants [34], the presence of iso-13:0, anteiso-13:0 and iso-17:0 in yak manure shows that these BCFA are not metabolized in the rumen or they might be of lower gut microbiome origin. The anteiso BCFA are positively correlated with yak milk from the same animal, indicating that these BCFA come from dietary sources. ELOVL1 belongs to a highly conserved microsomal enzyme family that are involved in fatty acid biosynthesis. ELOVL1 elongates straightchain SFA of chain lengths C18:0 to C26:0, with the highest activity toward C20:0 and C22:0 FA [35]. ELOVL1 was found to be ubiquitously expressed in human and mouse tissues [35,36]. ELOVL1 is 7-fold upregulated in full lactation milk compared to half lactation and is well correlated with significant increase in C22:0 levels in full lactation milk samples (Table 1). The protein encoded by MCAT gene is found exclusively in the mitochondrion and the malonyl-CoA-dependent system is capable of synthesizing straight-chain SFA C14-C18 and 20:5n-3 [37,38]. MCAT is 2.8 fold upregulated in full lactation milk compared to half lactation. In the present study we were not able to detect 20:5n3, however, it's elongation product 22:5n-3 is significantly increased in full lactation milk, whereas, straight-chain SFA of chain lengths C14C18 are slightly increased in full lactation milk but are not statistically significant (Table 1). Earlier we have estimated the contribution of BCFA to the nutrition of Americans, using measured and estimated cow's milk intake [13]. Similarly, here we estimate the contribution of BCFA from yak milk to the nutrition of QTP population. One cup (244 g = 8 oz) of yak milk (6% of fat) contains about 14.6 g fat. If we take 4.57% BCFA into the calculations, one cup of yak milk would provide 669 mg BCFA. This number is more than 4 times as cow's whole milk, which yields 158 mg BCFA per cup [13]. There is no data on how much and how frequent QTP habitants consume yak milk. If for example the QTP habitants consume three cups of yak milk, as recommended for dairy consumption in 2015–2020 Dietary Guidelines for Americans, yak milk would contribute 2007 mg of BCFA per capita per day, compared with an average of 500 mg BCFA per capita per day for the Americans. If we add intakes from yak meat and butter, these estimates will rise.
Fig. 3. Expression levels of genes involved in the biosynthesis and elongation of fatty acids from full lactation and half lactation yak milk samples. Gene expression levels are measured by semi-quantitative real-time PCR and normalized to the expression levels of house keeping β-actin. Values represent the means ± SD. *P < 0.05.
C26 acyl-CoA substrates and MCAT enzyme involved in the transfer of a malonyl group to the mitochondrial acyl carrier protein are significantly upregulated in full-lactation milk (Fig. 3). No significant changes in the expression levels of FASN, which catalyzes the conversion of Acetyl-CoA to palmitate, FADS2 and FADS1, fatty acid desaturases that introduce double bonds into fatty acyl chains and ELOVL6, enzyme involved in the elongation of saturated C12 to C16 acyl-CoA substrates (Fig. 3). 4. Discussion The BCFA content in foods is low-1–3% of total FA, compared to other saturates, and it is not routinely reported in literature [26]. One more obstacle is their quantification complicated by predominant straight-chain SFA and monounsaturated FA (MUFA) peaks in gas chromatography analyses [27]. In this study total BCFA ranged from 3 to 6% in yak milk and over 14% in yak manure. Our data show yak milk has higher 3 to 6% BCFA content when compared to 1.6 to 3.1% in cow, moose, goat and camel milk's [28,29]. Earlier reports of BCFA in dairy products contained both iso- and anteiso-type with chain length from 14 to 17 carbons, with very little iso-18:0 [12,26]. The odd chain anteiso15:0 and anteiso-17:0 comprised half of the total BCFA concentrations in cow milk–based dairy products [12,26]. BCFA in yak milk consisted of both iso- and anteiso-type with chain length from 14 to 17 carbons, iso-18:0 was under detection limits. In addition, the odd chain anteiso15:0 and anteiso-17:0 comprised more than half of the total BCFA in yak milk, similar in lines with human, goat and cow milk [13,30]. In goat milk, BCFA content was shown to depend on the forage concentration ratio and the forage level [29]. In QTP region the grass growing season is very short, lasting from May to October [31]. In QTP, the month of March is considered the beginning of the spring season with the pasture consisting of Achnatherum splendens and Carex qinghaiensis species. The Achnatherum splendens grow only during the months of March to May, there is no A. splendens pasture in October. The C. qinghaiensis grows in all seasons, however, its herbage height is 12 cm in March and April, significantly higher than in other months. In A. splendens pasture the dry matter, organic matter and acid-detergent fiber content was found to be higher than all other pastures [31]. It has also been shown that changes in diet composition induce changes in rumen bacterial populations [29,32]. In ruminants, the gut microbiota was shown to be influenced by several host and environmental factors, such as diet, age, the general host health, geographical location, season, and feeding regimen [33]. The higher BCFA content in half-lactation
5. Conclusions We report for the first time the profile and amount of BCFA in the yak milk and manure. Milk BCFA had chain lengths of 14–17 carbons, whereas, manure BCFA had chain lengths of 13–17 carbons which includes both iso- and anteiso-BCFA. The anteiso-BCFA are found to be more than half of the total BCFA in yak milk. The half-lactation yak milk contained higher levels of BCFA than the full-lactation milk. ELOVL1 and MCAT are significantly upregulated in full-lactation milk. BCFA in yak manure positively correlated with yak milk from the same animal, indicating that these BCFA come from dietary sources. CRediT authorship contribution statement Wancheng Sun: Conceptualization, Data curation, Formal analysis, Resources, Validation, Writing - original draft, Writing - review & editing. Yihao Luo: Data curation, Formal analysis, Validation, Writing - review & editing. Dong Hao Wang: Validation, Writing - original draft, Writing - review & editing. Kumar S.D. Kothapalli: Conceptualization, Resources, Validation, Writing - original draft, Writing - review & editing. J. Thomas Brenna: Conceptualization, Resources, Validation, Writing - original draft, Writing - review & editing. 19
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Declaration of Competing Interest
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All authors declare no competing interests. Acknowledgements Funding by Department of Science and Technology of Qinghai (2017-ZJ-711, 2018-ZJ-728). Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.plefa.2019.09.002. References [1] Y. Ma, S. He, H. Li, Yak Milk, in: Y.W. Park, G.F.W. Haenlein (Eds.), Milk and Dairy Products in Human Nutrition: Production, Composition and Health, John Wiley & Sons, Oxford, 2013. [2] H.N. Liu, F.Z. Ren, L. Jiang, Z.L. Ma, H.J. Qiao, S.S. Zeng, B.Z. Gan, H.Y. Guo, Fatty acid profile of yak milk from the Qinghai-Tibetan Plateau in different seasons and for different parities, J. Dairy Sci 94 (2011) 1724–1731. [3] L. Ding, Y. Wang, M. Kreuzer, X. Guo, J. Mi, Y. Gou, Y. Zhang, J. Zhou, H. Wang, R. Long, Seasonal variations in the fatty acid profile of milk from yaks grazing on the Qinghai-Tibetan Plateau, J. Dairy Res 80 (2013) 410–417. [4] USDA, Milk Cows: Invertory by Year, US. National Agricultural Statistics Service2017. [5] H. Li, Y. Ma, Q. Li, J. Wang, J. Cheng, J. Xue, J. Shi, The chemical composition and nitrogen distribution of Chinese yak (Maiwa) milk, Int. J. Mol. Sci. 12 (2011) 4885–4895. [6] X. Guo, R. Long, M. Kreuzer, L. Ding, Z. Shang, Y. Zhang, Y. Yang, G. Cui, Importance of functional ingredients in yak milk-derived food on health of Tibetan nomads living under high-altitude stress: a review, Crit. Rev. Food Sci. Nutr. 54 (2014) 292–302. [7] J. Luo, Z. Huang, H. Liu, Y. Zhang, F. Ren, Yak milk fat globules from the QinghaiTibetan Plateau: membrane lipid composition and morphological properties, Food Chem 245 (2018) 731–737. [8] R.R. Ran-Ressler, S. Devapatla, P. Lawrence, J.T. Brenna, Branched chain fatty acids are constituents of the normal healthy newborn gastrointestinal tract, Pediatr. Res 64 (2008) 605–609. [9] R.R. Ran-Ressler, L. Khailova, K.M. Arganbright, C.K. Adkins-Rieck, Z.E. Jouni, O. Koren, R.E. Ley, J.T. Brenna, B. Dvorak, Branched chain fatty acids reduce the incidence of necrotizing enterocolitis and alter gastrointestinal microbial ecology in a neonatal rat model, PLoS One 6 (2011) e29032. [10] Q. Cai, H. Huang, D. Qian, K. Chen, J. Luo, Y. Tian, T. Lin, T. Lin, 13-methyltetradecanoic acid exhibits anti-tumor activity on T-cell lymphomas in vitro and in vivo by down-regulating p-AKT and activating caspase-3, PLoS One 8 (2013) e65308. [11] M. Kniazeva, T. Euler, M. Han, A branched-chain fatty acid is involved in postembryonic growth control in parallel to the insulin receptor pathway and its biosynthesis is feedback-regulated in C. elegans, Genes Dev 22 (2008) 2102–2110. [12] R.R. Ran-Ressler, S. Bae, P. Lawrence, D.H. Wang, J.T. Brenna, Branched-chain fatty acid content of foods and estimated intake in the USA, Br. J. Nutr. 112 (2014) 565–572. [13] R.R. Ran-Ressler, D. Sim, A.M. O'Donnell-Megaro, D.E. Bauman, D.M. Barbano, J.T. Brenna, Branched chain fatty acid content of United States retail cow's milk and implications for dietary intake, Lipids 46 (2011) 569–576. [14] D.H. Wang, J.R. Jackson, C. Twining, L.G. Rudstam, E. Zollweg-Horan, C. Kraft, P. Lawrence, K. Kothapalli, Z. Wang, J.T. Brenna, Saturated branched chain, normal odd-carbon-numbered, and n-3 (Omega-3) polyunsaturated fatty acids in freshwater fish in the Northeastern United States, J. Agric. Food Chem. 64 (2016) 7512–7519. [15] K.A. Dingess, C.J. Valentine, N.J. Ollberding, B.S. Davidson, J.G. Woo, S. Summer, Y.M. Peng, M.L. Guerrero, G.M. Ruiz-Palacios, R.R. Ran-Ressler, Branched-chain fatty acid composition of human milk and the impact of maternal diet: the Global
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Prostaglandins, Leukotrienes and Essential Fatty Acids journal homepage: www.elsevier.com/locate/plefa
Original research article
Branched chain fatty acid composition of yak milk and manure during fulllactation and half-lactation Wancheng Suna,b, Yihao Luoa, Dong Hao Wangb, Kumar S.D. Kothapallib,c, J. Thomas Brennab,c,
T
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a
Animal Science Department, College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA c Dell Pediatric Research Institute and Deptartment of Pediatrics, Dell Medical School, The University of Texas at Austin, 1400 Barbara Jordan Blvd, Austin, TX 78723, USA b
A R T I C LE I N FO
A B S T R A C T
Keywords: Branched chain fatty acids (BCFA) Yak milk Yak manure Gene expression
Background: Branched chain fatty acids (BCFA) are bioactive food compounds and are well known to be essential components of human, cow and caprine milk. In Qinghai-Tibet plateau, yaks are domesticated in large numbers and their milk in addition to meat are commercially important to millions of Tibetans and Chinese. Objective: We tested the hypotheses that concentrations of BCFA in yak milk and manure differ between lactation periods and evaluated gene expression levels of certain genes involved in the biosynthesis and elongation of fatty acids. Design: Fresh milk and manure were collected from each yak and their fatty acid compositions compared with emphasis on BCFA. Participants/setting: Yak milk and manure samples from the full lactation (October, 2015) and half lactation periods (March, 2016) were collected and BCFA levels were analyzed in detail by GC-FID and structures verified by GC-EI-MS/MS. Gene expression studies were carried out by semi-quantitative real time PCR method. Statistical analyses performed: The difference between full lactation and half lactation was tested using student's ttest. Linear regression model was modelled in Excel and its significance was tested by ANOVA. Statistical significance was determined by performing student's t-test for gene expression studies. Results: BCFA ranged from 3–6% of total fatty acids in yak milk samples. The half-lactation yak milk contained higher levels of BCFA (5.29 ± 0.53) than the full-lactation milk (4.00 ± 0.46). The total BCFA in yak manure was found to be 14.67 ± 1.21, high in anteiso-15:0 and anteiso-17:0. ELOVL1 enzyme involved in the elongation of saturated C18 to C26 acyl-CoA substrates and MCAT enzyme involved in the transfer of a malonyl group to the mitochondrial acyl carrier protein are significantly upregulated in full-lactation milk. Conclusions: BCFA in yak manure especially anteiso BCFA are positively correlated with yak milk from the same animal, indicating that these BCFA come from dietary sources. Yak milk delivers 777 mg BCFA compared to 158 mg per cup of whole U.S. dairy milk. QTP herders known to consume up to 2 kg of yak yogurt take in an estimated 3,500–5,000 mg BCFA per day. We conclude that BCFA intake for yak milk consumers is among the highest known in the world, higher when drawn from half lactating yaks.
1. Introduction Yak (Bos grunniens or Poephagus grunniens) is a versatile domestic animal that provides the basic subsistence and nutrition to the people inhabiting the Qinghai-Tibet Plateau (QTP), which comprises alpine and subalpine regions [1]. The yak is the only bovine species that has adapted to thrive in extremely harsh and high altitude conditions of QTP, at heights of 2500–6000 m above sea level [2]. The total world population of yaks is estimated at around 14.2 million, approximately
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13.3 million occur within China [3]. Just put the number in context, there are about 9.2 million milk cows in the United States [4]. Yak milk contributes to 15% of Chinese milk consumption and contains higher fat content at about 5.3 to 8.8% compared to 4.2% in bovine milk [5–7]. The high fat content is a critical energy booster for their calves’ survival on the cold QTP as well as for human consumers. Earlier reports have shown seasonal variation in the fat composition of yak milk is mainly related to the seasonal changes in the grass composition of the pasture [1,3].
Corresponding author. E-mail address: [email protected] (J.T. Brenna).
https://doi.org/10.1016/j.plefa.2019.09.002 Received 5 April 2019; Received in revised form 15 July 2019; Accepted 4 September 2019 0952-3278/ © 2019 Elsevier Ltd. All rights reserved.
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Branched chain fatty acids (BCFA) are primarily saturated FA (SFA) with one or more methyl branches [8]. BCFA are major components of vernix caseosa and are constituents of the term newborn gut lumen, being swallowed as vernix particulates of the amniotic fluid [8]. BCFA are bioactive components known to reduce incidence of necrotizing enterocolitis (NEC) in a neonatal rat model [9], have anti-tumor effects on T-cell non-Hodgkin's lymphoma (T-NHL) cell lines [10] and regulate post-embryonic development [11]. BCFA are widely available in the food supply, primarily from dairy and beef, seafood and are also present in human colostrum and breastmilk [8,12–15]. A thorough survey of the fatty acid profile of cow's milk from the US market identified 2% BCFA in milk fat and an estimated daily intake of 500 mg BCFA from dairy and beef [12,13]. The human breastmilk samples collected from regular dairy-consuming and low-dairy-consuming populations showed variability in BCFA content, higher BCFA concentrations found with diary consumption [15]. Recent studies have shown that ruminant foods are rich sources of BCFA, with content and profile of BCFA depending on the activity and composition of rumen microbiota [12,16]. BCFA are a major component of bacterial membranes most notably bacilli [17], as well as Bifidobacterium strains [18]. Many bacterial species have more than 50% BCFA in their membranes, including the potential pro-biotic Sporolactobacillus inulinus that have >90% BCFA [19]. BCFA constitute >90% of the fatty acids (FA) in many species of Bacilli, Lactobacilli and Brevibacterium [17,20]. The Bacillus subtilis synthesize higher proportions of anteiso-15:0 and anteiso-17:0 BCFA due to cold shock response [21]. Yaks of reproductive age and that are lactating can be divided into two types- the full-lactating yak and the half-lactating yak. The yaks in the year of calving that are lactating and suckling a calf are called “fulllactating yaks”. Yaks calved in the previous year that are not pregnant however still suckling their last calf are called Yama or “half-lactating yaks” [22,23]. The yak, even though not pregnant at the onset of winter but still suckling a calf, will continue to lactate through the following warm season and, normally, will be hand milked. The "half-lactating yak" will stop being milked at the end of her second warm season and will then go dry, irrespective of whether the yak is pregnant or not. Half-lactating yak milk yield is about half to two thirds of the yield in the year of calving. The two different lactation periods could have effects on milk composition. It is unknown whether fatty acids, especially BCFA vary in milk between these two lactation periods. Manure is a remote indicator of rumen fermentation and can be compared to milk production from the same animals. There are no studies linking BCFA composition of yak milk and manure from the same animal. Here we tested the hypotheses that concentrations of BCFA in yak milk and manure differ between lactation periods and evaluated gene expression levels of certain genes involved in the biosynthesis and elongation of fatty acids.
Fig. 1. BCFA methyl esters distribution in yak milk and manure (mean ± SD). The iso-13:0, anteiso-13:0 and iso-17:0 are under detection limits in yak milk.
2.2. Fatty acid extraction and analysis Fatty acid methyl esters (FAME) were prepared using a modified one-step digestion and methylation method [24]. FAME mixtures were analyzed quantitatively by Hewlett Packard 5890 series II gas chromatograph-flame ionization detector (GC-FID) using an equal weight mixture for response factor calibration. Detection limits are estimated to be about 0.02% w/w. FAME structural identity was verified by gas chromatography covalent adduct chemical ionization tandem mass spectrometry (GC-CACI-MS/MS) and/or gas chromatography electron ionization tandem mass spectrum (GC-EI-MS/MS) as described earlier [13,25]. The section of a typical chromatogram demonstrating resolution of BCFA from adjacent peaks is presented in Supplementary Materials (Fig. 1). 2.3. RNA isolation and cDNA synthesis Total RNA was isolated from lyophilized yak milk powder samples using the E.Z.N.A. Total RNA kit (Omega Bio–Tek, GA). The quantity and quality of RNA was analyzed by 260/280 nm ratios using a microspectrophotometer (NanoDrop 2000, Thermo Scientific). The total RNA was reverse-transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Life Technologies, NY) according to the manufacturer's instructions. The resulting cDNA was used as template for semi-quantitative real time PCR reactions. 2.4. Semiquantitative RT-PCR We designed gene specific PCR primers using Bos mutus (wild yak) mRNA sequences and the PrimerQuest software from Integrated DNA Technologies (IDT; Coralville, IA). Primer pairs were ordered from IDT. The primer sequences and standardized annealing temperatures of each primer pair are presented in supplementary Table 1 (Table S1). Semiquantitative RT-PCR amplification reactions were run on an Eppendorf gradient thermal cycler (Eppendorf, NY) using EmeraldAmp GT PCR Master Mix (Clontech, CA). PCR products were separated on 2% agarose gel stained with ethidium bromide and bands visualized under UV light. The intensities of the expressed amplicons were quantified densitometrically by ImageJ software (National Institutes of Health, USA). Expression levels of each transcript were normalized to expression values of control Beta-Actin gene.
2. Materials and methods The study was approved by the institutional review board of Qinghai University on animal subjects research.
2.1. Collection of yak milk and manure The milk and manure samples were all collected from the same farm of yak herds located on the plateau altitude of 3200 m in Qilian county of Qinghai Province in China. Fresh milk samples were collected from free range ten full-lactating yaks in October 2015 and eight half-lactating yaks in March 2016. The milk samples were directly milked from the yak and stored in vials covered with ice packs. All the milk samples were lyophilized to yak milk powder and kept in −20 °C for further processing in the laboratory. Manure from the same animals were also collected, labeled and kept in −20 °C until laboratory analysis.
2.5. Statistical analysis Data was analyzed using Microsoft Excel (2010) software. All data in figures are presented as mean ± SD. The difference between full lactation and half lactation was tested using student's t-test. Linear regression model was modelled in Excel and its significance was tested by 17
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Table 1 Fatty acid composition of yak milk from full lactation (n = 10) and half lactation (n = 8) (%, w/w; mean ± SD).
C10:0 C12:0 iso-14:0 C14:0 iso-15:0 Anteiso-15:0 C15:0 iso-16:0 C16:0 C16:1 Anteiso-17:0 C17:0 C17:1 C18:0 C18:1 C18:2 C19:1 C18:3 C20:0 CLA C20:1 C20:2 C22:0 C22:1 C22:4n-6 C24:0 C22:5n-3 Σ BCFA Σ PUFA
Full lactation
Half lactation
p value
1.13 ± 0.28 1.29 ± 0.23 0.32 ± 0.06 7.68 ± 1.02 0.92 ± 0.19 1.09 ± 0.15 1.83 ± 0.18 0.60 ± 0.07 33.26 ± 1.67 2.93 ± 0.50 1.07 ± 0.07 1.44 ± 0.08 0.63 ± 0.05 11.84 ± 2.90 28.70 ± 2.10 1.58 ± 0.25 0.33 ± 0.04 0.89 ± 0.28 0.69 ± 0.13 0.49 ± 0.12 0.46 ± 0.09 0.21 ± 0.04 0.39 ± 0.07 0.10 ± 0.02 0.19 ± 0.04 0.15 ± 0.04 0.29 ± 0.06 4.00 ± 0.46 3.17 ± 0.59
0.95 ± 0.23 1.19 ± 0.15 0.44 ± 0.06 7.09 ± 0.41 0.99 ± 0.08 1.45 ± 0.17 2.35 ± 0.27 1.05 ± 0.16 33.13 ± 3.26 2.54 ± 0.97 1.36 ± 0.13 1.88 ± 0.20 0.90 ± 0.11 11.32 ± 2.70 27.23 ± 2.76 1.93 ± 0.18 0.48 ± 0.05 0.48 ± 0.09 0.76 ± 0.09 0.32 ± 0.10 0.41 ± 0.04 0.24 ± 0.04 0.17 ± 0.06 0.39 ± 0.04 0.94 ± 0.37 0.13 ± 0.04 0.18 ± 0.03 5.29 ± 0.53 3.77 ± 0.39
0.1422 0.2535 0.0003 0.1233 0.3145 0.0001 0.0001 0.0000 0.9108 0.2557 0.0000 0.0000 0.0000 0.6872 0.1892 0.0030 0.0000 0.0006 0.1892 0.0034 0.2037 0.0787 0.0000 0.0000 0.0000 0.3360 0.0001 0.0000 0.0175
ANOVA. Statistical significance was determined by performing student's t-test for gene expression studies. 3. Results 3.1. BCFA in yak milk samples Total BCFA ranged from 3–6% of total FA in yak milk with an overall mean of 4.57 ± 0.82 regardless of lactation periods. BCFA identified in yak milkfat are iso-14:0, iso-15:0, anteiso-15:0, iso-16:0, and anteiso-17:0. The anteiso BCFA constituted about half of the BCFA pool. PUFA are 3.43 ± 0.59 of the total fatty acids. To determine the effect of lactation period on BCFA content, Table 1 summarizes the fatty acid profile of full-lactation and half-lactation yak milk expressed as mean ± SD, respectively. BCFA from half-lactation milk are significantly higher (5.29 ± 0.53) than full-lactation milk (4.00 ± 0.46, p < 0.001). PUFA levels are also significantly higher in half-lactation milk (3.77 ± 0.39) than full-lactation milk (3.17 ± 0.59, p = 0.02). In comparison, conjugated linoleic acid (CLA) is lower in half-lactation milk (0.32 ± 0.10) than full-lactation milk (0.49 ± 0.12, P < 0.0034). Table S2 & S3 present the fatty acid profile of ten yak milk's collected in October, 2015 (full lactation) and eight in March subsequent year (half lactation). Levels of BCFA and PUFA are summarized at the bottom of the two tables.
Fig. 2. Manure and milk BCFA correlation reveal a rumen origin of milk BCFA in yak. (a) anteiso-15:0 (r2 = 0.63, p < 0.001); (b) anteiso-17:0 (r2 = 0.63, p < 0.001); (c) total BCFA (r2 = 0.12, p = 0.17).
17:0 are the top two BCFA by weight. A full fatty acid profile of yak manure collected in October, 2015 and in March, 2016 are presented in supplementary Table S4 and Table S5. Fig. 2 shows the correlation between manure and milk BCFA. The two major BCFA, anteiso-15:0 and anteiso-17:0 are explored separately for this purpose. One outlier, animal No.4 in October was excluded because manure sample was high in both anteiso-15:0 and anteiso-17:0, presumably due to post-fermentation or contamination. Both anteiso15:0 and anteiso-17:0 followed a positive linear regression between manure BCFA and milk BCFA (r2 = 0.6, p < 0.001). Total manure and milk BCFA show a weak correlation (Fig. 3c, r2 = 0.1).
3.2. Manure BCFA and its correlation with milk BCFA For each yak milk sample, we also analyzed a sample of the same yak's manure. Fig. 1 shows the BCFA concentrations of yak manure beside yak milk. The total BCFA in yak manure was found to be 14.67 ± 1.21, w/w. BCFA are much higher in manure samples, than the milk samples. BCFA identified in yak manure are iso-13:0, anteiso13:0, iso-14:0, iso-15:0, anteiso-15:0, iso-16:0, iso-17:0 and anteiso-17:0. The iso-13:0, anteiso-13:0 and iso-17:0 present in yak manure are under detection limits in yak milk. In yak manure, anteiso-15:0 and anteiso-
3.3. Expression levels of genes involved in the biosynthesis and elongation of fatty acids Semi-quantitative RT-PCR was performed to test expression levels of certain genes involved in the biosynthesis and elongation of fatty acids (Fig. 3). ELOVL1 enzyme involved in the elongation of saturated C18 to 18
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milk can be accounted for by the grass composition of the pasture, which in turn having influence on the gut microbiota. We found certain differences in BCFA profile in yak manure compared to yak milk. The shorter chain iso-13:0 and anteiso-13:0 and longer chain iso-17:0 are detected only in yak manure. As the fatty acid are metabolized in the rumen before their absorption in ruminants [34], the presence of iso-13:0, anteiso-13:0 and iso-17:0 in yak manure shows that these BCFA are not metabolized in the rumen or they might be of lower gut microbiome origin. The anteiso BCFA are positively correlated with yak milk from the same animal, indicating that these BCFA come from dietary sources. ELOVL1 belongs to a highly conserved microsomal enzyme family that are involved in fatty acid biosynthesis. ELOVL1 elongates straightchain SFA of chain lengths C18:0 to C26:0, with the highest activity toward C20:0 and C22:0 FA [35]. ELOVL1 was found to be ubiquitously expressed in human and mouse tissues [35,36]. ELOVL1 is 7-fold upregulated in full lactation milk compared to half lactation and is well correlated with significant increase in C22:0 levels in full lactation milk samples (Table 1). The protein encoded by MCAT gene is found exclusively in the mitochondrion and the malonyl-CoA-dependent system is capable of synthesizing straight-chain SFA C14-C18 and 20:5n-3 [37,38]. MCAT is 2.8 fold upregulated in full lactation milk compared to half lactation. In the present study we were not able to detect 20:5n3, however, it's elongation product 22:5n-3 is significantly increased in full lactation milk, whereas, straight-chain SFA of chain lengths C14C18 are slightly increased in full lactation milk but are not statistically significant (Table 1). Earlier we have estimated the contribution of BCFA to the nutrition of Americans, using measured and estimated cow's milk intake [13]. Similarly, here we estimate the contribution of BCFA from yak milk to the nutrition of QTP population. One cup (244 g = 8 oz) of yak milk (6% of fat) contains about 14.6 g fat. If we take 4.57% BCFA into the calculations, one cup of yak milk would provide 669 mg BCFA. This number is more than 4 times as cow's whole milk, which yields 158 mg BCFA per cup [13]. There is no data on how much and how frequent QTP habitants consume yak milk. If for example the QTP habitants consume three cups of yak milk, as recommended for dairy consumption in 2015–2020 Dietary Guidelines for Americans, yak milk would contribute 2007 mg of BCFA per capita per day, compared with an average of 500 mg BCFA per capita per day for the Americans. If we add intakes from yak meat and butter, these estimates will rise.
Fig. 3. Expression levels of genes involved in the biosynthesis and elongation of fatty acids from full lactation and half lactation yak milk samples. Gene expression levels are measured by semi-quantitative real-time PCR and normalized to the expression levels of house keeping β-actin. Values represent the means ± SD. *P < 0.05.
C26 acyl-CoA substrates and MCAT enzyme involved in the transfer of a malonyl group to the mitochondrial acyl carrier protein are significantly upregulated in full-lactation milk (Fig. 3). No significant changes in the expression levels of FASN, which catalyzes the conversion of Acetyl-CoA to palmitate, FADS2 and FADS1, fatty acid desaturases that introduce double bonds into fatty acyl chains and ELOVL6, enzyme involved in the elongation of saturated C12 to C16 acyl-CoA substrates (Fig. 3). 4. Discussion The BCFA content in foods is low-1–3% of total FA, compared to other saturates, and it is not routinely reported in literature [26]. One more obstacle is their quantification complicated by predominant straight-chain SFA and monounsaturated FA (MUFA) peaks in gas chromatography analyses [27]. In this study total BCFA ranged from 3 to 6% in yak milk and over 14% in yak manure. Our data show yak milk has higher 3 to 6% BCFA content when compared to 1.6 to 3.1% in cow, moose, goat and camel milk's [28,29]. Earlier reports of BCFA in dairy products contained both iso- and anteiso-type with chain length from 14 to 17 carbons, with very little iso-18:0 [12,26]. The odd chain anteiso15:0 and anteiso-17:0 comprised half of the total BCFA concentrations in cow milk–based dairy products [12,26]. BCFA in yak milk consisted of both iso- and anteiso-type with chain length from 14 to 17 carbons, iso-18:0 was under detection limits. In addition, the odd chain anteiso15:0 and anteiso-17:0 comprised more than half of the total BCFA in yak milk, similar in lines with human, goat and cow milk [13,30]. In goat milk, BCFA content was shown to depend on the forage concentration ratio and the forage level [29]. In QTP region the grass growing season is very short, lasting from May to October [31]. In QTP, the month of March is considered the beginning of the spring season with the pasture consisting of Achnatherum splendens and Carex qinghaiensis species. The Achnatherum splendens grow only during the months of March to May, there is no A. splendens pasture in October. The C. qinghaiensis grows in all seasons, however, its herbage height is 12 cm in March and April, significantly higher than in other months. In A. splendens pasture the dry matter, organic matter and acid-detergent fiber content was found to be higher than all other pastures [31]. It has also been shown that changes in diet composition induce changes in rumen bacterial populations [29,32]. In ruminants, the gut microbiota was shown to be influenced by several host and environmental factors, such as diet, age, the general host health, geographical location, season, and feeding regimen [33]. The higher BCFA content in half-lactation
5. Conclusions We report for the first time the profile and amount of BCFA in the yak milk and manure. Milk BCFA had chain lengths of 14–17 carbons, whereas, manure BCFA had chain lengths of 13–17 carbons which includes both iso- and anteiso-BCFA. The anteiso-BCFA are found to be more than half of the total BCFA in yak milk. The half-lactation yak milk contained higher levels of BCFA than the full-lactation milk. ELOVL1 and MCAT are significantly upregulated in full-lactation milk. BCFA in yak manure positively correlated with yak milk from the same animal, indicating that these BCFA come from dietary sources. CRediT authorship contribution statement Wancheng Sun: Conceptualization, Data curation, Formal analysis, Resources, Validation, Writing - original draft, Writing - review & editing. Yihao Luo: Data curation, Formal analysis, Validation, Writing - review & editing. Dong Hao Wang: Validation, Writing - original draft, Writing - review & editing. Kumar S.D. Kothapalli: Conceptualization, Resources, Validation, Writing - original draft, Writing - review & editing. J. Thomas Brenna: Conceptualization, Resources, Validation, Writing - original draft, Writing - review & editing. 19
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Declaration of Competing Interest
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