Milk fatty acid composition, rumen microbial population and animal performance in response to diets rich in linoleic acid supplemented with Piper betle leaves in Saanen goats
R. A. P. Purba A , C. Yuangklang B , S. Paengkoum C D and P. Paengkoum AA School of Animal Technology and Innovation, Institute of Agricultural Technology, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand.
B Department of Agricultural Technology and Environment, Faculty of Sciences and Liberal Arts, Rajamangala University of Technology Isan, Nakhon Ratchasima 30000, Thailand.
C Program in Agriculture, Faculty of Science and Technology, Nakhon Ratchasima Rajabhat University, Nakhon Ratchasima 30000, Thailand.
D Corresponding author. Email: took_sw@yahoo.com
Animal Production Science - https://doi.org/10.1071/AN20182
Submitted: 12 April 2020 Accepted: 6 October 2020 Published online: 11 November 2020
Abstract
Context: Rumen biohydrogenation is an important way to produce conjugated linoleic acid (CLA), especially the rumenic acid isomer. However, CLA is principally synthesised endogenously in lactating mammals by delta 9-desaturase in breast tissue.
Aims: The aim of the study was to evaluate milk fatty acid profile, rumen microbial population and animal performance in response to diets containing sunflower oil either supplemented with or without flavonoids and essential oils from Piper betle L. powder (PP) in dairy goats.
Method: Twelve multiparous Saanen goats (42 ± 1.00 kg; mean ± s.d.) were randomly assigned to two treatment groups in an experiment that lasted for 6 weeks. The two experimental diets formulated as total mixed ration were: control (CTH) diet (containing 0% PP) and DPB diet (CTH diet containing 1.3% PP on a dry-matter basis).
Key results: Inclusion of flavonoids and essential oils from PP in the diet (DPB) did not affect dry-matter intake but resulted in a greater milk yield and altered the composition of milk. Compared with the control diet (CTH), the DPB diet decreased the saturated fatty acid concentration and increased the unsaturated fatty acid concentration in milk. Inclusion of PP decreased the C18:0 production (P < 0.05), resulting in higher C18:1 trans11 and C18:2 cis9 trans11 (P < 0.05) concentrations. Overall, DPB diet increased the total CLA by 1.5-fold, from 1.77 to 2.62 g/100 g fatty acid. The desaturase rate (except desaturase for carbon 18, P < 0.05), and atherogenic and thrombogenic indices were not affected by inclusion of PP in the DPB diet. Moreover, the DPB diet escalated total volatile fatty acid production and altered the volatile fatty acid profile. Compared with goats fed with CTH diet, PP supplementation increased the presence of ruminal Butyrivibrio fibrisolvens by ~5-fold, but the presence of B. proteoclasticus decreased to about 1/11 of the control.
Conclusions: The use of sunflower oil at 17.6 g/kg diet and inclusion of a practical dose of flavonoids and essential oils from Piper betle L. leaves in the diet of dairy goats can be an efficient method to improve milk yield and milk composition, including increasing the CLA concentration of milk.
Implications: These results constitute an alternative strategy to improve milk quality, without negatively affecting animal performance.
Keywords: bacteria, dairy, herbage, lactation, nutritive evaluation.
References
Abbas M, Saeed F, Anjum FM, Afzaal M, Tufail T, Bashir MS, Ishtiaq A, Hussain S, Suleria HAR (2017) Natural polyphenols: an overview. International Journal of Food Properties 20, 1689–1699.| Natural polyphenols: an overview.Crossref | GoogleScholarGoogle Scholar |
AOAC (2005) ‘Official methods of analysis.’ (Eds W Horwitz, GW Latimer) (AOAC International: Gaitherburg, MD, USA)
Berger LM, Blank R, Zorn F, Wein S, Metges CC, Wolffram S (2015) Ruminal degradation of quercetin and its influence on fermentation in ruminants. Journal of Dairy Science 98, 5688–5698.
| Ruminal degradation of quercetin and its influence on fermentation in ruminants.Crossref | GoogleScholarGoogle Scholar | 26094220PubMed |
Buccioni A, Pauselli M, Viti C, Minieri S, Pallara G, Roscini V, Rapaccini S, Marinucci MT, Lupi P, Conte G, Mele M (2015) Milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes. Journal of Dairy Science 98, 1145–1156.
| Milk fatty acid composition, rumen microbial population, and animal performances in response to diets rich in linoleic acid supplemented with chestnut or quebracho tannins in dairy ewes.Crossref | GoogleScholarGoogle Scholar | 25434333PubMed |
Calsamiglia S, Busquet M, Cardozo PW, Castillejos L, Ferret A (2007) Invited review: essential oils as modifiers of rumen microbial fermentation. Journal of Dairy Science 90, 2580–2595.
| Invited review: essential oils as modifiers of rumen microbial fermentation.Crossref | GoogleScholarGoogle Scholar | 17517698PubMed |
Castro-Carrera T, Toral PG, Frutos P, McEwan NR, Hervás G, Abecia L, Pinloche E, Girdwood SE, Belenguer A (2014) Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae. Journal of Dairy Science 97, 1661–1669.
| Rumen bacterial community evaluated by 454 pyrosequencing and terminal restriction fragment length polymorphism analyses in dairy sheep fed marine algae.Crossref | GoogleScholarGoogle Scholar | 24440247PubMed |
Chilliard Y, Ferlay A (2004) Dietary lipids and forages interactions on cow and goat milk fatty acid composition and sensory properties. Reproduction, Nutrition, Development 44, 467–492.
| Dietary lipids and forages interactions on cow and goat milk fatty acid composition and sensory properties.Crossref | GoogleScholarGoogle Scholar | 15636165PubMed |
Chilliard Y, Ferlay A, Mansbridge RM, Doreau M (2000) Ruminant milk fat plasticity: nutritional control of saturated, polyunsaturated, trans and conjugated fatty acids. Annales de Zootechnie 49, 181–205.
| Ruminant milk fat plasticity: nutritional control of saturated, polyunsaturated, trans and conjugated fatty acids.Crossref | GoogleScholarGoogle Scholar |
Chilliard Y, Ferlay A, Rouel J, Lamberet G (2003) A review of nutritional and physiological factors affecting goat milk lipid synthesis and lipolysis. Journal of Dairy Science 86, 1751–1770.
| A review of nutritional and physiological factors affecting goat milk lipid synthesis and lipolysis.Crossref | GoogleScholarGoogle Scholar | 12778586PubMed |
Chilliard Y, Glasser F, Ferlay A, Bernard L, Rouel J, Doreau M (2007) Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat. European Journal of Lipid Science and Technology 109, 828–855.
| Diet, rumen biohydrogenation and nutritional quality of cow and goat milk fat.Crossref | GoogleScholarGoogle Scholar |
Clark S, García MBM (2017) A 100-year review: advances in goat milk research. Journal of Dairy Science 100, 10026–10044.
| A 100-year review: advances in goat milk research.Crossref | GoogleScholarGoogle Scholar | 29153153PubMed |
Cobellis G, Yu Z, Forte C, Acuti G, Trabalza-Marinucci M (2016) Dietary supplementation of Rosmarinus officinalis L. leaves in sheep affects the abundance of rumen methanogens and other microbial populations. Journal of Animal Science and Biotechnology 7, 27–34.
| Dietary supplementation of Rosmarinus officinalis L. leaves in sheep affects the abundance of rumen methanogens and other microbial populations.Crossref | GoogleScholarGoogle Scholar | 27123239PubMed |
Correddu F, Lunesu MF, Buffa G, Atzori AS, Nudda A, Battacone G, Pulina G (2020) Can agro-industrial by-products rich in polyphenols be advantageously used in the feeding and nutrition of dairy small ruminants? Animals 10, 131–155
| Can agro-industrial by-products rich in polyphenols be advantageously used in the feeding and nutrition of dairy small ruminants?Crossref | GoogleScholarGoogle Scholar | 32731516PubMed |
Cui K, Guo XD, Tu Y, Zhang NF, Ma T, Diao QY (2015) Effect of dietary supplementation of rutin on lactation performance, ruminal fermentation and metabolism in dairy cows. Journal of Animal Physiology and Animal Nutrition 99, 1065–1073.
| Effect of dietary supplementation of rutin on lactation performance, ruminal fermentation and metabolism in dairy cows.Crossref | GoogleScholarGoogle Scholar | 26053391PubMed |
Das S, Parida R, Sandeep SI, Nayak S, Mohanty S (2016) Biotechnological intervention in betelvine (Piper betle L.): a review on recent advances and future prospects. Asian Pacific Journal of Tropical Medicine 9, 938–946.
| Biotechnological intervention in betelvine (Piper betle L.): a review on recent advances and future prospects.Crossref | GoogleScholarGoogle Scholar | 27794386PubMed |
De Nardi R, Marchesini G, Plaizier JC, Li S, Khafipour E, Ricci R, Andrighetto I, Segato S (2014) Use of dicarboxylic acids and polyphenols to attenuate reticular pH drop and acute phase response in dairy heifers fed a high grain diet. BMC Veterinary Research 10, 277–284.
| Use of dicarboxylic acids and polyphenols to attenuate reticular pH drop and acute phase response in dairy heifers fed a high grain diet.Crossref | GoogleScholarGoogle Scholar | 25425091PubMed |
Dubois V, Breton S, Linder M, Fanni J, Parmentier M (2007) Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. European Journal of Lipid Science and Technology 109, 710–732.
| Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential.Crossref | GoogleScholarGoogle Scholar |
Folch J, Lees M, Stanley SGH (1957) A simple method for the isolation and purification of total lipides from animal tissues. The Journal of Biological Chemistry 226, 497–509.
Frutos P, Hervás G, Giráldez FJ, Mantecón AR (2004) An in vitro study on the ability of polyethylene glycol to inhibit the effect of quebracho tannins and tannic acid on rumen fermentation in sheep, goats, cows, and deer. Australian Journal of Agricultural Research 55, 1125–1132.
| An in vitro study on the ability of polyethylene glycol to inhibit the effect of quebracho tannins and tannic acid on rumen fermentation in sheep, goats, cows, and deer.Crossref | GoogleScholarGoogle Scholar |
Gesteira SM, Oliveira RL, Trajano JDS, Ribeiro CVDM, Costa EIDS, Ribeiro RDX, Pereira ES, Bezerra LR (2019) Fatty acid profile, physicochemical composition and sensorial attributes of salted and sun-dried meat from young Nellore bulls supplemented with condensed tannins. PLoS One 14, e0216047
| Fatty acid profile, physicochemical composition and sensorial attributes of salted and sun-dried meat from young Nellore bulls supplemented with condensed tannins.Crossref | GoogleScholarGoogle Scholar | 31276548PubMed |
Hamzaoui S, Salama AAK, Albanell E, Such X, Caja G (2013) Physiological responses and lactational performances of late-lactation dairy goats under heat stress conditions. Journal of Dairy Science 96, 6355–6365.
| Physiological responses and lactational performances of late-lactation dairy goats under heat stress conditions.Crossref | GoogleScholarGoogle Scholar | 23958010PubMed |
Hosseinzadeh H, Nassiri-Asl M (2014) Review of the protective effects of rutin on the metabolic function as an important dietary flavonoid. Journal of Endocrinological Investigation 37, 783–788.
| Review of the protective effects of rutin on the metabolic function as an important dietary flavonoid.Crossref | GoogleScholarGoogle Scholar | 24879037PubMed |
Jafari S, Meng GY, Rajion MA, Jahromi MF, Ebrahimi M (2016) Manipulation of rumen microbial fermentation by polyphenol rich solvent fractions from papaya leaf to reduce green-house gas methane and biohydrogenation of C18 PUFA. Journal of Agricultural and Food Chemistry 64, 4522–4530.
| Manipulation of rumen microbial fermentation by polyphenol rich solvent fractions from papaya leaf to reduce green-house gas methane and biohydrogenation of C18 PUFA.Crossref | GoogleScholarGoogle Scholar | 27192629PubMed |
Jafari S, Ebrahimi M, Meng GY, Rajion MA, Jahromi MF (2018) Dietary supplementation of papaya (Carica papaya L.) leaf affects abundance of Butyrivibrio fibrisolvens and modulates biohydrogenation of C18 polyunsaturated fatty acids in the rumen of goats. Italian Journal of Animal Science 17, 326–335.
| Dietary supplementation of papaya (Carica papaya L.) leaf affects abundance of Butyrivibrio fibrisolvens and modulates biohydrogenation of C18 polyunsaturated fatty acids in the rumen of goats.Crossref | GoogleScholarGoogle Scholar |
Joch M, Kudrna V, Hakl J, Božik M, Homolka P, Illek J, Tyrolová Y, Výborná A (2019) In vitro and in vivo potential of a blend of essential oil compounds to improve rumen fermentation and performance of dairy cows. Animal Feed Science and Technology 251, 176–186.
| In vitro and in vivo potential of a blend of essential oil compounds to improve rumen fermentation and performance of dairy cows.Crossref | GoogleScholarGoogle Scholar |
Kaps M, Lamberson WR (2004) ‘Biostatistics for animal science.’ (CABI: Wallingford, UK)
Khiaosa-ard R, Kreuzer M, Leiber F (2015) Apparent recovery of C18 polyunsaturated fatty acids from feed in cow milk: a meta-analysis of the importance of dietary fatty acids and feeding regimens in diets without fat supplementation. Journal of Dairy Science 98, 6399–6414.
| Apparent recovery of C18 polyunsaturated fatty acids from feed in cow milk: a meta-analysis of the importance of dietary fatty acids and feeding regimens in diets without fat supplementation.Crossref | GoogleScholarGoogle Scholar | 26142845PubMed |
Kim ET, Guan LL, Lee SJ, Lee SM, Lee SS, Lee ID, Lee SK, Lee SS (2015) Effects of flavonoid-rich plant extracts on in vitro ruminal methanogenesis, microbial populations and fermentation characteristics. Asian-Australasian Journal of Animal Sciences 28, 530–537.
| Effects of flavonoid-rich plant extracts on in vitro ruminal methanogenesis, microbial populations and fermentation characteristics.Crossref | GoogleScholarGoogle Scholar | 25656200PubMed |
Lourenço M, Cardozo PW, Calsamiglia S, Fievez V (2008) Effects of saponins, quercetin, eugenol, and cinnamaldehyde on fatty acid biohydrogenation of forage polyunsaturated fatty acids in dual-flow continuous culture fermenters. Journal of Animal Science 86, 3045–3053.
| Effects of saponins, quercetin, eugenol, and cinnamaldehyde on fatty acid biohydrogenation of forage polyunsaturated fatty acids in dual-flow continuous culture fermenters.Crossref | GoogleScholarGoogle Scholar | 18567728PubMed |
Lourenço M, Ramos-Morales E, Wallace RJ (2010) The role of microbes in rumen lipolysis and biohydrogenation and their manipulation. Animal 4, 1008–1023.
| The role of microbes in rumen lipolysis and biohydrogenation and their manipulation.Crossref | GoogleScholarGoogle Scholar | 22444606PubMed |
Ma T, Chen DD, Tu Y, Zhang NF, Si BW, Diao QY (2017) Dietary supplementation with mulberry leaf flavonoids inhibits methanogenesis in sheep. Animal Science Journal 88, 72–78.
| Dietary supplementation with mulberry leaf flavonoids inhibits methanogenesis in sheep.Crossref | GoogleScholarGoogle Scholar | 27112278PubMed |
Maeda H, Fujimoto C, Haruki Y, Maeda T, Kokeguchi S, Petelin M, Arai H, Tanimoto I, Nishimura F, Takashiba S (2003) Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria. FEMS Immunology and Medical Microbiology 39, 81–86.
| Quantitative real-time PCR using TaqMan and SYBR Green for Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, tetQ gene and total bacteria.Crossref | GoogleScholarGoogle Scholar | 14557000PubMed |
Makkar HPS, McSweeney CS (2005) ‘Methods in gut microbial ecology for ruminants.’ (Springer: Dordrecht, The Netherlands)
Mamuad LL, Lee SS, Lee SS (2019) Recent insight and future techniques to enhance rumen fermentation in dairy goats. Asian–Australasian Journal of Animal Sciences 32, 1321–1330.
Mandalari G, Bennett RN, Bisignano G, Trombetta D, Saija A, Faulds CB, Gasson MJ, Narbad A (2007) Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry. Journal of Applied Microbiology 103, 2056–2064.
| Antimicrobial activity of flavonoids extracted from bergamot (Citrus bergamia Risso) peel, a byproduct of the essential oil industry.Crossref | GoogleScholarGoogle Scholar | 18045389PubMed |
McSweeney CS, Palmer B, McNeill DM, Krause DO (2001) Microbial interactions with tannins: nutritional consequences for ruminants. Animal Feed Science and Technology 91, 83–93.
| Microbial interactions with tannins: nutritional consequences for ruminants.Crossref | GoogleScholarGoogle Scholar |
NRC (2007) ‘Nutrition requirements of small ruminants: sheep, goats, cervids, and New World camelids.’ (The National Academies Press: Washington, DC, USA)
Olagaray KE, Bradford BJ (2019) Plant flavonoids to improve productivity of ruminants: a review. Animal Feed Science and Technology 251, 21–36.
| Plant flavonoids to improve productivity of ruminants: a review.Crossref | GoogleScholarGoogle Scholar |
Oskoueian E, Abdullah N, Oskoueian A (2013) Effects of flavonoids on rumen fermentation activity, methane production, and microbial population. BioMed Research International 2013, 8–16.
| Effects of flavonoids on rumen fermentation activity, methane production, and microbial population.Crossref | GoogleScholarGoogle Scholar |
Paengkoum P (2019) ‘Applied goat nutrition.’ (Korat Marketing and Production: Nakhon Ratchasima, Thailand)
Pariza MW (2004) Perspective on the safety and effectiveness of conjugated linoleic acid. The American Journal of Clinical Nutrition 79, 1132S–1136S.
| Perspective on the safety and effectiveness of conjugated linoleic acid.Crossref | GoogleScholarGoogle Scholar | 15159246PubMed |
Patra AK, Geiger S, Braun HS, Aschenbach JR (2019) Dietary supplementation of menthol-rich bioactive lipid compounds alters circadian eating behaviour of sheep. BMC Veterinary Research 15, 352–361.
| Dietary supplementation of menthol-rich bioactive lipid compounds alters circadian eating behaviour of sheep.Crossref | GoogleScholarGoogle Scholar | 31638982PubMed |
Potu RB, AbuGhazaleh AA, Hastings D, Jones K, Ibrahim SA (2011) The effect of lipid supplements on ruminal bacteria in continuous culture fermenters varies with the fatty acid composition. Journal of Microbiology 49, 216–223.
| The effect of lipid supplements on ruminal bacteria in continuous culture fermenters varies with the fatty acid composition.Crossref | GoogleScholarGoogle Scholar |
Poudel P, Froehlich K, Casper DP, St-Pierre B (2019) Feeding essential oils to Neonatal holstein dairy calves results in increased ruminal prevotellaceae abundance and propionate concentrations. Microorganisms 7, 120–134.
| Feeding essential oils to Neonatal holstein dairy calves results in increased ruminal prevotellaceae abundance and propionate concentrations.Crossref | GoogleScholarGoogle Scholar |
Purba RAP, Paengkoum P (2019) Bioanalytical HPLC method of Piper betle L. for quantifying phenolic compound, water-soluble vitamin, and essential oil in five different solvent extracts. Journal of Applied Pharmaceutical Science 9, 033–039.
| Bioanalytical HPLC method of Piper betle L. for quantifying phenolic compound, water-soluble vitamin, and essential oil in five different solvent extracts.Crossref | GoogleScholarGoogle Scholar |
Purba RAP, Paengkoum P, Paengkoum S (2020a) The links between supplementary tannin levels and conjugated linoleic acid (CLA) formation in ruminants: a systematic review and meta-analysis. PLoS One 15, e0216187
| The links between supplementary tannin levels and conjugated linoleic acid (CLA) formation in ruminants: a systematic review and meta-analysis.Crossref | GoogleScholarGoogle Scholar | 32168348PubMed |
Purba RAP, Paengkoum S, Yuangklang C, Paengkoum P (2020b) Flavonoids and their aromatic derivatives in Piper betle powder promote in vitro methane mitigation in a variety of diets. Ciência e Agrotecnologia 44, e012420
| Flavonoids and their aromatic derivatives in Piper betle powder promote in vitro methane mitigation in a variety of diets.Crossref | GoogleScholarGoogle Scholar |
Purba RAP, Yuangklang C, Paengkoum P (2020c) Enhanced conjugated linoleic acid and biogas production after ruminal fermentation with Piper betle L. supplementation. Ciência Rural 50, e20191001
| Enhanced conjugated linoleic acid and biogas production after ruminal fermentation with Piper betle L. supplementation.Crossref | GoogleScholarGoogle Scholar |
Schogor ALB, Palin M, dos Santos GT, Benchaar C, Lacasse P, Petit HV (2013) Mammary gene expression and activity of antioxidant enzymes and oxidative indicators in the blood, milk, mammary tissue and ruminal fluid of dairy cows fed flax meal. British Journal of Nutrition 110, 1743–1750.
| Mammary gene expression and activity of antioxidant enzymes and oxidative indicators in the blood, milk, mammary tissue and ruminal fluid of dairy cows fed flax meal.Crossref | GoogleScholarGoogle Scholar |
Shingfield KJ, Bonnet M, Scollan ND (2013) Recent developments in altering the fatty acid composition of ruminant-derived foods. Animal 7, 132–162.
| Recent developments in altering the fatty acid composition of ruminant-derived foods.Crossref | GoogleScholarGoogle Scholar | 23031638PubMed |
Spek JW, Dijkstra J, Duinkerken GV, Bannink A (2013) A review of factors influencing milk urea concentration and its relationship with urinary urea excretion in lactating dairy cattle. The Journal of Agricultural Science 151, 407–423.
| A review of factors influencing milk urea concentration and its relationship with urinary urea excretion in lactating dairy cattle.Crossref | GoogleScholarGoogle Scholar |
Tian XZ, Xin H, Paengkoum P, Paengkoum S, Ban C, Sorasak T (2019) Effects of anthocyanin-rich purple corn (Zea mays L.) stover silage on nutrient utilization, rumen fermentation, plasma antioxidant capacity, and mammary gland gene expression in dairy goats. Journal of Animal Science 97, 1384–1397.
| Effects of anthocyanin-rich purple corn (Zea mays L.) stover silage on nutrient utilization, rumen fermentation, plasma antioxidant capacity, and mammary gland gene expression in dairy goats.Crossref | GoogleScholarGoogle Scholar |
Van Soest PJ, Robertson JB, Lewis BA (1991) Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74, 3583–3597.
| Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition.Crossref | GoogleScholarGoogle Scholar | 1660498PubMed |
Wallace Initial (2004) Antimicrobial properties of plant secondary metabolites. Proc Nutr Soc 63, 621–629.
| Antimicrobial properties of plant secondary metabolites.Crossref | GoogleScholarGoogle Scholar |
Winkler A, Gessner D, Koch C, Romberg FJ, Dusel G, Herzog E, Most E, Eder K (2015) Effects of a plant product consisting of green tea and curcuma extract on milk production and the expression of hepatic genes involved in endoplasmic stress response and inflammation in dairy cows. Archives of Animal Nutrition 69, 425–441.
| Effects of a plant product consisting of green tea and curcuma extract on milk production and the expression of hepatic genes involved in endoplasmic stress response and inflammation in dairy cows.Crossref | GoogleScholarGoogle Scholar | 26490199PubMed |
Yu Z, Morrison M (2004) Improved extraction of PCR-quality community DNA from digesta and fecal samples. BioTechniques 36, 808–812.
| Improved extraction of PCR-quality community DNA from digesta and fecal samples.Crossref | GoogleScholarGoogle Scholar | 15152600PubMed |