Chlorella pyrenoidosa supplementation increased the concentration of unsaturated fatty acids in the rumen fluid of cattle fed a low-quality tropical forage

Diogo Fleury Azevedo Costa Simon Paul Quigley Peter Isherwood Stuart Ross McLennan Xiao Qiao Sun Steven James Gibbs Dennis Paul Poppi About the authors

ABSTRACT

The objective of this study was to evaluate the effects of algae and cottonseed meal (CSM) supplementation on the fatty acid (FA) profile in the rumen fluid (RF) of steers fed a low-quality forage. Five Bos indicus crossbred steers, 187±7.5 kg liveweight (LW; mean±SD), were fed a low crude protein speargrass (Heteropogon contortus) hay as the basal diet alone or supplemented with either Spirulina platensis, Chlorella pyrenoidosa , Dunaliella salina , or CSM in Latin square design. The proportion of individual FA in the RF of steers varied in response to supplement, and these were most likely due to differences in the FA profile in supplements. Steers supplemented with Chlorella pyrenoidosa and CSM had a higher concentration of linoleic acid (C18:2n-6) in RF than unsupplemented steers or steers offered the other supplements, but there was no difference in the concentration in RF in steers supplemented with Chlorella pyrenoidosa and CSM. The concentration of linolenic acid (C18:3n-3) was higher in the RF of unsupplemented steers compared with supplemented steers. Steers receiving Chlorella pyrenoidosa supplementation showed an increase in total unsaturated FA in the RF compared with other supplemented and unsupplemented steers, which if transferred to meat, could have health related benefits to consumers. None of the supplements led to the formation of isomers known to inhibit fat synthesis.

Keywords:
biohydrogenation; conjugated linoleic acid; microalgae; tropical grass

1. Introduction

In recent years, the biotechnology of fuel production has led to an increased use of algae, with a parallel interest in the potential use of de-oiled algae byproducts for animal feeds ( Bryant et al., 2012Bryant, H. L.; Gogichaishvili, I.; Anderson, D.; Richardson, J. W.; Sawyer, J.; Wickersham, T. and Drewery, M. L. 2012. The value of post-extracted algae residue. Algal Research 1:185-193. https://doi.org/10.1016/j.algal.2012.06.001
https://doi.org/10.1016/j.algal.2012.06....
). Algae are mainly targeted as feedstuffs for ruminants because of their fatty acid (FA) content and the health-related benefits to consumers of meat ( Vahmani et al., 2013Vahmani, P.; Fredeen, A. H. and Glover, K. E. 2013. Effect of supplementation with fish oil or microalgae on fatty acid composition of milk from cows managed in confinement or pasture systems. Journal of Dairy Science 96:6660-6670. https://doi.org/10.3168/jds.2013-6914
https://doi.org/10.3168/jds.2013-6914...
) and milk products ( Papadopoulos et al., 2002Papadopoulos, G.; Goulas, C.; Apostolaki, E. and Abril, R. 2002. Effects of dietary supplements of algae, containing polyunsaturated fatty acids, on milk yield and the composition of milk products in dairy ewes. Journal of Dairy Research 69:357-365. https://doi.org/10.1017/S0022029902005599
https://doi.org/10.1017/S002202990200559...
). The protein and fat content vary considerably between algae, and this can have variable effects within the rumen ( Panjaitan et al., 2015Panjaitan, T.; Quigley, S. P.; McLennan, S. R.; Swain, A. J. and Poppi, D. P. 2015. Spirulina ( Spirulina platensis ) algae supplementation increases microbial protein production and feed intake and decreases retention time of digesta in the rumen of cattle. Animal Production Science 55:535-543. https://doi.org/10.1071/AN13146
https://doi.org/10.1071/AN13146...
) and after absorption by the ruminant animal ( Meale et al., 2014Meale, S. J.; Chaves, A. V.; He, M. L. and McAllister, T. A. 2014. Dose-response of supplementing marine algae ( Schizochytrium sp.) on production performance, fatty acid profiles and wool parameters of growing lambs. Journal of Animal Science 92:2202-2213. https://doi.org/10.2527/jas.2013-7024
https://doi.org/10.2527/jas.2013-7024...
; Lamminen et al., 2019Lamminen, M.; Filleau, A. H. B.; Kokkonen, T.; Jaakkola, S. and Vanhatalo, A. 2019. Different microalgae species as a substitutive protein feed for soya bean meal in grass silage based dairy cow diets. Animal Feed Science and Technology 247:112-126. https://doi.org/10.1016/j.anifeedsci.2018.11.005
https://doi.org/10.1016/j.anifeedsci.201...
).

The hypothesis of this study was that a greater concentration of polyunsaturated FA in an algae species would be associated with a greater concentration of total unsaturated FA in the rumen fluid (RF) of ruminants consuming the algae. Therefore, supplementation with algae varying in FA profile, with a high concentration of C18s FA, would result in different concentrations of conjugated linoleic acid isomers, reflecting the FA profile in RF and the extent of biohydrogenation thereof. The objective of this experiment was to evaluate the effects of algae and cottonseed meal (CSM) supplementation on the FA profile in the RF of cattle consuming a low-quality tropical forage.

2. Material and Methods

The experiment was conducted in Gatton, QLD, Australia (27.5551° S, 152.3369° E, and 94 m), and all procedures were performed in accordance with the guidelines of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, reviewed and approved by the institutional Animal Ethics Committee (SAS/236/10/MLA).

2.1. Experimental design, animals and nutritional treatments

The experiment was a 5×5 complete Latin square, incorporating five steers and five nutritional treatments. The experiment consisted of five 21-day experimental periods, with each experimental period consisting of 12 days in outdoor pens and nine days in metabolism crates. Each steer was exposed to a different nutritional treatment within each experimental period.

Five Bos indicus crossbred steers, 187±7.5 kg liveweight (LW; mean±SD) were randomly allocated to adjacent individual pens and allocated to one of five nutritional treatments. All steers were offered speargrass ( Heteropogon contortus ) hay ad libitum and were either unsupplemented (control) or supplemented with Spirulina platensis (Spirulina; Phytofoods Australia Pty. Ltd., Labrador, QLD, Australia), Chlorella pyrenoidosa (Chlorella; Phytofoods Australia Pty. Ltd.), Dunaliella salina (Dunaliella; Nutra-Kol, Perth, WA, Australia), or CSM (Riverina Stockfeeds, Murgon, QLD, Australia). Spirulina, Chlorella, and CSM were offered at 0.45 g N/(kg LW.day), whilst Duniella was offered at 4 g DM/(kg LW.day) [equivalent to the amount of Spirulina DM offered/(kg LW.day)]. The amount of Spirulina platensis supplied was similar to that which resulted in the maximum dry matter intake response in Panjaitan et al. (2015)Panjaitan, T.; Quigley, S. P.; McLennan, S. R.; Swain, A. J. and Poppi, D. P. 2015. Spirulina ( Spirulina platensis ) algae supplementation increases microbial protein production and feed intake and decreases retention time of digesta in the rumen of cattle. Animal Production Science 55:535-543. https://doi.org/10.1071/AN13146
https://doi.org/10.1071/AN13146...
. Chlorella pyrenoidosa and CSM were offered at an equivalent amount of N [0.45 g N/(kg LW.day)], whereas Dunaliella salina was offered at an equivalent DM amount [4 g/(kg LW.day)]as that supplied by Spirulina platensis to accommodate its high ash content. Samples of feed offered were collected in duplicate for each run and bulked across the experiment ( Table 1 ).

Table 1
Chemical composition of Heteropogon contortus (speargrass) hay, Spirulina platensis (Spirulina), Chlorella pyrenoidosa (Chlorella), Dunaliella salina (Dunaliella), and cottonseed meal (CSM) 1

2.2. Feeding of hay and supplements

The speargrass hay was chopped to approximately 50 mm in length and offered ad libitum (previous day intake +10% as fed) at 08:00 h each day. Supplement allowances were adjusted based on steer liveweight measured on days 1 and 13 of each experimental run. Supplements were mixed with 300 g molasses and offered daily, prior to hay feeding, at 07:30 h, with the same amount of molasses supplied to control animals. Algae supplements were prepared by mixing the algae and molasses with water to a final concentration of approximately 30% algae (w/w), whereas CSM was offered with the molasses added on top.

2.3. Sample collection and analytical procedures

Samples of hay and supplements were collected daily and bulked within each run. Rumen fluid was collected orally using a stomach tube attached to a hand pump 3 h after feeding on day 21 with unacidified duplicate sub-samples stored at −20 °C until analysis of the FA profile.

Hay and supplement samples were oven-dried to a constant weight at 60 °C and ground through a 1-mm screen (Retsch ZM 200; Haan, Germany). Residual moisture was determined at 105 °C using a drying oven (Industrial 200, Watson Victor Ltd.; Sydney, NSW, Australia) for 24 h and ash determined after incineration at 550 °C for 8 h in a muffle furnace (Modutemp Pty. Ltd.; Perth, WA, Australia). The N content of the hay and supplements was determined using a Nitrogen analyser (Kjeltec, 8400 FOSS; Hillerod, North Zealand, Denmark) based on the Kjeldahl method. The ash-free neutral detergent fibre (aNDFom) and ash-free acid detergent fibre (ADFom) were determined according to Van Soest et al. (1991)Van Soest, P. J.; Robertson, J. B. and Lewis, B. A. 1991. Methods for dietary fiber, neutral detergent fiber, and nonstarch polysaccharides in relation to animal nutrition. Journal of Dairy Science 74:3583-3597. https://doi.org/10.3168/jds.S0022-0302(91)78551-2
https://doi.org/10.3168/jds.S0022-0302(9...
with the use of Gooch crucibles of grade 2 porosity because of the fine particle size of the microalgae. Briefly, for aNDFom analysis, approximately 0.5 g of sample was heated to boiling with 50 mL neutral detergent solution, 0.5 g Na2SO3, and 2 mL of heat stable alpha-amylase (17,400 Liquefon U/mL; FAA, ANKOM Technology; Macedon, USA), and then agitated for 1 h before transferring to a crucible. After draining the solution under vacuum, the samples were washed three times with hot water (the first rinse included a further application of 2 mL alpha-amylase) and twice with acetone. The ADFom was determined essentially the same way, except that the digestion was conducted in 50 mL of acid detergent solution, 2.45 g H2SO4, with no alpha-amylase added to the digestion or washes. Total lipid content was determined according to the method of Hara and Radin (1978)Hara, A. and Radin, N. S. 1978. Lipid extraction of tissues with a low-toxicity solvent. Analytical Biochemistry 90:420-426. https://doi.org/10.1016/0003-2697(78)90046-5
https://doi.org/10.1016/0003-2697(78)900...
. The FA content in hay, supplements, and RF was determined using a method based on Kramer et al. (1997)Kramer, J. K. G.; Fellner, V.; Dugan, M. E. R.; Sauer, F. D.; Mossoba, M. M. and Yurawecz, M. P. 1997. Evaluating acid and base catalysts in the methylation of milk and rumen fatty acids with special emphasis on conjugated dienes and total trans fatty acids. Lipids 32:1219-1228. https://doi.org/10.1007/s11745-997-0156-3
https://doi.org/10.1007/s11745-997-0156-...
using modifications proposed by Sun and Gibbs (2012)Sun, X. Q. and Gibbs, S. J. 2012. Diurnal variation in fatty acid profiles in rumen digesta from dairy cows grazing high-quality pasture. Animal Feed Science and Technology 177:152-160. https://doi.org/10.1016/j.anifeedsci.2012.08.013
https://doi.org/10.1016/j.anifeedsci.201...
, which was described in full by Costa et al. (2019)Costa, D. F. A.; Quigley, S. P.; Isherwood, P.; McLennan, S. R.; Sun, X. Q.; Gibbs, S. J. and Poppi, D. P. 2019. Small differences in biohydrogenation resulted from the similar retention times of fluid in the rumen of cattle grazing wet season C3 and C4 forage species. Animal Feed Science and Technology 253:101-112. https://doi.org/10.1016/j.anifeedsci.2019.05.010
https://doi.org/10.1016/j.anifeedsci.201...
.

2.4. Statistical analysis

The statistical analyses for FA were carried out using the univariate procedure in General Linear Model of SPSS (SPSS for Windows, Version 17.0, SPSS Inc, Chicago, IL, USA), according to the following model:

Y ijk = μ + P i + T j + S k + e ijk ,

in which Yijk is the dependent variable, μ is overall mean, Pi is fixed effect of period (1 to 5), Tj is fixed effect of treatment (1 to 5), Sk is the random effect of steers (1 to 5), and eijk is the residual error. Tukey was used for treatment multiple comparisons. Significance was declared at P<0.05.

3. Results

Palmitic acid (C16:0) was present in the highest proportion FA in the speargrass hay and microalgae supplements but not in CSM, in which C18:2n-6 was the most abundant FA ( Table 2 ). Speargrass and Dunaliella contained the highest proportion of total individual odd- and branched-chain FA, with a low proportion of C15:0anteiso evident in all feeds used. Cottonseed meal and Chlorella had the highest proportion of total unsaturated FA (TUFA) and total C18 (TC18s) FA, with high proportions of linoleic acid (C18:2n-6) in both supplements and a high proportion of alpha-linolenic (C18:3n-3) in Chlorella. The highest proportion of total saturated FA (TSFA) was present in Dunaliella, followed by Spirulina and speargrass hay, which were greater than Chlorella and CSM. The proportion of long-chain FA (LCFA) ( i.e. >C20s) was low for all supplements evaluated in this experiment.

Table 2
Fatty acid profile of Heteropogon contortus (Speargrass) hay, Spirulina platensis (Spirulina), Chlorella pyrenoidosa (Chlorella), Dunaliella salina (Dunaliella), and cottonseed meal (CSM) 1

There was considerable variation in FA profiles in the RF among all treatments ( Table 3 ). Palmitic acid (C16:0) was the most abundant FA in the RF of steers, with the highest proportion in the RF of steers fed Spirulina (P<0.05). Unsupplemented steers had the highest proportion of total odd- and branched-chain FA (TOBCFA) (P<0.05) in the RF and steers supplemented with Chlorella had the lowest proportion, with the microalgae supplemented steers intermediate. A similar pattern was evident for C15:0anteiso, which was present in a higher proportion in RF compared with the feeds offered (P<0.05). The proportions of TUFA and TC18s were highest in the RF of steers supplemented with Chlorella (P<0.05) with no differences detected amongst the other treatments. The proportion of linoleic acid was similar in the RF of steers offered Chlorella and CSM and was higher than in the RF of steers fed the other treatments (P<0.05). Unsupplemented steers had a higher proportion of TSFA in RF than steers supplemented with Chlorella and CSM (P<0.05) but it was not different from that measured in the RF of steers supplemented with Spirulina and Dunaliella. Unsupplemented steers had the highest proportion of alpha-linolenic acid (C18:3n-3) in RF (P<0.05) with no differences in the RF of supplemented animals.

Table 3
Fatty acid profiles in the rumen fluid 1 of steers fed speargrass ( Heteropogon contortus ) hay (control) or supplemented with Spirulina platensis (Spirulina), Chlorella pyrenoidosa (Chlorella), Dunaliella salina (Dunaliella), and cottonseed meal (CSM)

4. Discussion

This experiment compared the effect of supplementation with three species of microalgae and CSM on the profile of FA in the RF of growing steers fed speargrass, a low-quality tropical forage. It was hypothesized that the FA profile in the RF of steers supplemented with microalgae would be different from the profile of steers fed forage alone or supplemented with CSM, and that unsaturated FA intake, through supplementation, could lead to greater concentrations of unsaturated FA in RF. The results indicated that individual FA varied between supplements and that supplementation with Chlorella resulted in a high proportion of unsaturated FA in RF, in particular linoleic acid, which was present in the microalgae used as supplements in this experiment. Interestingly, the high proportion of linoleic acid in CSM did not lead to a higher TUFA in the RF of steers.

The protein in vitro degradation of Chlorella observed in Costa et al. (2016)Costa, D. F. A.; Quigley, S. P.; Isherwood, P.; McLennan, S. R. and Poppi, D. P. 2016. Supplementation of cattle fed tropical grasses with microalgae increases microbial protein production and average daily gain. Journal of Animal Science 94:2047-2058. https://doi.org/10.2527/jas.2016-0292
https://doi.org/10.2527/jas.2016-0292...
was lower than the protein in other supplements. Lamminen et al. (2019)Lamminen, M.; Filleau, A. H. B.; Kokkonen, T.; Jaakkola, S. and Vanhatalo, A. 2019. Different microalgae species as a substitutive protein feed for soya bean meal in grass silage based dairy cow diets. Animal Feed Science and Technology 247:112-126. https://doi.org/10.1016/j.anifeedsci.2018.11.005
https://doi.org/10.1016/j.anifeedsci.201...
observed no differences in apparent total tract digestibility of organic matter and CP when soybean meal was replaced by Spirulina or Chlorella in a diet offered to lactating dairy cows. In their work, it was reported that ruminal microbial N outflow tended (P = 0.053) to be higher on the diet with Spirulina compared with the diet with Chlorella but with no significant differences between dietary treatments ( Lamminen et al., 2019Lamminen, M.; Filleau, A. H. B.; Kokkonen, T.; Jaakkola, S. and Vanhatalo, A. 2019. Different microalgae species as a substitutive protein feed for soya bean meal in grass silage based dairy cow diets. Animal Feed Science and Technology 247:112-126. https://doi.org/10.1016/j.anifeedsci.2018.11.005
https://doi.org/10.1016/j.anifeedsci.201...
). Lower microbial activity could lead to less biohydrogenation in the rumen. Howerver, in corroboration, Costa et al. (2016)Costa, D. F. A.; Quigley, S. P.; Isherwood, P.; McLennan, S. R. and Poppi, D. P. 2016. Supplementation of cattle fed tropical grasses with microalgae increases microbial protein production and average daily gain. Journal of Animal Science 94:2047-2058. https://doi.org/10.2527/jas.2016-0292
https://doi.org/10.2527/jas.2016-0292...
found no differences in microbial protein synthesis among steers supplemented with Spirulina, Chlorella, and CSM. Gulzari et al. (2019)Gulzari, S. O.; Lind, V.; Aasen, I. M. and Steinshamn, H. 2019. Effect of supplementing sheep diets with macroalgae species on in vivo nutrient digestibility, rumen fermentation and blood amino acid profile. Animal 13:2792-2801. https://doi.org/10.1017/s1751731119001502
https://doi.org/10.1017/s175173111900150...
reported lower protein digestibility in sheep supplemented with the macro-algae Saccharina latissima (55%) in comparison with the macro-algae Porphyra spp. (64%), and with soybean meal (66%), but the lower digestibility led to lower production of volatile FA, most likely linked to a lower microbial activity. Gulzari et al. (2019)Gulzari, S. O.; Lind, V.; Aasen, I. M. and Steinshamn, H. 2019. Effect of supplementing sheep diets with macroalgae species on in vivo nutrient digestibility, rumen fermentation and blood amino acid profile. Animal 13:2792-2801. https://doi.org/10.1017/s1751731119001502
https://doi.org/10.1017/s175173111900150...
suggested that phlorotannins present in some algae species may form complexes with protein and fibre, which could not only limit their degradability in the rumen, but also affect the post-ruminal absorption in the intestines. No speculation is made here to indicate that similar mechanisms would be related to the lower in vitro protein degradation of the microalgae Chlorella observed in Costa et al. (2016)Costa, D. F. A.; Quigley, S. P.; Isherwood, P.; McLennan, S. R. and Poppi, D. P. 2016. Supplementation of cattle fed tropical grasses with microalgae increases microbial protein production and average daily gain. Journal of Animal Science 94:2047-2058. https://doi.org/10.2527/jas.2016-0292
https://doi.org/10.2527/jas.2016-0292...
, although their results indicate that something within the composition of Chlorella affects the protein degradation and that could indirectly be affecting the biohydrogenation of FA in the RF of steers fed that algae. Regardless of the latter observation and, most importantly, microbial protein synthesis was not impaired by Chlorella supplementation in their work.

The proportion of TUFA was higher in the RF of steers fed Chlorella, and if transferred to meat, could induce health-related benefits to consumers. However, specifically the proportion of linolenic acid was higher in the RF of control steers, with most of the TUFA in the RF of steers supplemented with Chlorella represented by linoleic acid. Linoleic acid is present in the lipids of the microalgae Spirulina and Chlorella ( Otles and Pire, 2001Otles, S. and Pire, R. 2001. Fatty acid composition of Chlorella and Spirulina microalgae species. Journal of AOAC International 84:1708-1714. ), and C18:2 has been reported to be the main FA in CSM, almost double that of C16:0 ( Mohamed et al., 1988Mohamed, O. E.; Satter, L. D.; Grummer, R. R. and Ehle, F. R. 1988. Influence of dietary cottonseed and soybean on milk production and composition. Journal of Dairy Science 71:2677-2688. https://doi.org/10.3168/jds.S0022-0302(88)79861-6
https://doi.org/10.3168/jds.S0022-0302(8...
). In the current experiment, the concentration of C18:2n-6 was greater for these three supplements, but a significantly greater concentration was only found in the RF of steers fed Chlorella and CSM.

The fresh water microalgae Spirulina and Chlorella and the marine microalgae Dunaliella are the most important commercially produced microalgae grown in open systems ( Radmann et al., 2007Radmann, E. M.; Reinehr, C. O. and Costa, J. A. V. 2007. Optimization of the repeated batch cultivation of microalga Spirulina platensis in open raceway ponds. Aquaculture 265:118-126. https://doi.org/10.1016/j.aquaculture.2007.02.001
https://doi.org/10.1016/j.aquaculture.20...
). These algae species have long-chain unsaturated FA that, when offered to monogastric animals, could alter the FA composition of the meat ( Raes et al., 2004Raes, K.; De Smet, S. and Demeyer, D. 2004. Effect of dietary fatty acids on incorporation of long chain polyunsaturated fatty acids and conjugated linoleic acid in lamb, beef and pork meat: a review. Animal Feed Science and Technology 113:199-221. https://doi.org/10.1016/j.anifeedsci.2003.09.001
https://doi.org/10.1016/j.anifeedsci.200...
), but when offered to cattle, the biohydrogenation process in the rumen could affect the original FA profile ( Costa, 2018Costa, D. F. A. 2018. Cattle responses to small inclusions of lipids in the diet. Applied Food Science Journal 2:21. ). Total unsaturated FA in various lipid sources were only partially protected from biohydrogenation, not directly translating to the final FA profile in the RF ( Costa et al., 2017Costa, D. F. A.; Quigley, S. P.; Isherwood, P.; McLennan, S. R.; Sun, X. Q.; Gibbs, S. J. and Poppi, D. P. 2017. The inclusion of low quantities of lipids in the diet of ruminants fed low quality forages has little effect on rumen function. Animal Feed Science and Technology 234:20-28. https://doi.org/10.1016/j.anifeedsci.2017.09.003
https://doi.org/10.1016/j.anifeedsci.201...
). In the current experiment, Chlorella supplementation was the only treatment that resulted in an increase in TUFA in the RF.

Other microalgae species are rich in longer chain polyunsaturated FA with 20 and 22 C. Meale et al. (2014)Meale, S. J.; Chaves, A. V.; He, M. L. and McAllister, T. A. 2014. Dose-response of supplementing marine algae ( Schizochytrium sp.) on production performance, fatty acid profiles and wool parameters of growing lambs. Journal of Animal Science 92:2202-2213. https://doi.org/10.2527/jas.2013-7024
https://doi.org/10.2527/jas.2013-7024...
offered Schizochytrium sp, a marine alga rich in docosahexaenoic acid (DHA) (C 22:6n-3), to growing lambs and observed DHA increments on the FA profile of adipose tissue, positively altering carcass characteristics. Pickard et al. (2008)Pickard, R. M.; Beard, A. P.; Seal, C. J. and Edwards, S. A. 2008. Neonatal lamb vigour is improved by feeding docosahexaenoic acid in the form of algal biomass during late gestation. Animal 2:1186-1192. https://doi.org/10.1017/S1751731108001997
https://doi.org/10.1017/S175173110800199...
offered Schizochytrium sp to pregnant ewes in the final weeks of gestation, e.g. 10 to six weeks before birth, and reported that lambs from these ewes stood up faster after birth demonstrating a better vigour compared with lambs born from the ewes of the control treatment. The main FA of Spirulina were the already saturated palmitic acid 16:0 and the polyunsaturated FA (C18:1, C18:2, and C18:3) that could be completely hydrogenated to stearic and monoenoic acids in the rumen ( Polan et al., 1964Polan, C. E.; McNeill, J. J. and Tove, S. B. 1964. Biohydrogenation of unsaturated fatty acids by rumen bacteria. Journal of Bacteriology 88:1056-1064. https://doi.org/10.1128/JB.88.4.1056-1064.1964
https://doi.org/10.1128/JB.88.4.1056-106...
). In the current experiment, the unsaturated FA in Spirulina did not markedly increase the TUFA in the RF and Chlorella was the only treatment that led to increases in unsaturated FA, especially linoleic acid, the main polyunsaturated FA found in RF.

Spirulina and Dunaliella had the greatest concentrations of C16:0 (i.e. 47.6 and 61.2% of total FA, respectively), and this probably resulted in a significantly greater C16:0 proportion in the RF of steers supplemented with Spirulina (i.e. around 37% above the control treatment). Palmitic acid is also the most common saturated FA in plants ( McDonald et al., 2002McDonald, P.; Edwards, R. A.; Greenhalgh, J. F. D. and Morgan, C. A. 2002. Animal nutrition. British Library, Harlow, England. ) and is found in greater concentration in the lipids of protozoa ( Or-Rashid et al., 2007Or-Rashid, M. M.; Odongo, N. E. and McBride, B. W. 2007. Fatty acid composition of ruminal bacteria and protozoa, with emphasis on conjugated linoleic acid, vaccenic acid, and odd-chain and branched-chain fatty acids. Journal of Animal Science 85:1228-1234. https://doi.org/10.2527/jas.2006-385
https://doi.org/10.2527/jas.2006-385...
). However, there was no difference in the proportion of palmitic acid in the RF of steers supplemented with Chlorella, Dunaliella, CSM, or unsupplemented steers.

Meat from ruminants have a higher conjugated linoleic acid (CLA) content than meat from monogastric animals ( Schimid et al., 2006Schmid, A.; Collomb, M.; Sieber, R. and Bee, G. 2006. Conjugated linoleic acid in meat and meat products: A review. Meat Science 73:29-41. https://doi.org/10.1016/j.meatsci.2005.10.010
https://doi.org/10.1016/j.meatsci.2005.1...
). One of the main isomers of linoleic acid, CLA c 9, t 11, rumenic acid, was significantly lower in the RF of steers supplemented with Spirulina or Chlorella. Rumenic acid has been reported to have no effect on milk fat content or yield ( Bauman et al., 2008Bauman, D. E.; Perfield, J. W.; Harvatine, K. J. and Baumgard, L. H. 2008. Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model. The Journal of Nutrition 138:403-409. https://doi.org/10.1093/jn/138.2.403
https://doi.org/10.1093/jn/138.2.403...
) and is typically the dominant CLA in forage-based ruminant products ( Kay et al., 2004Kay, J. K.; Mackle, T. R.; Auldist, M. J.; Thomson, N. A. and Bauman, D. E. 2004. Endogenous synthesis of cis- 9, trans- 11 conjugated linoleic acid in dairy cows fed fresh pasture. Journal of Dairy Science 87:369-378. https://doi.org/10.3168/jds.S0022-0302(04)73176-8
https://doi.org/10.3168/jds.S0022-0302(0...
), including the adipose tissue of ruminants, where it accounts for 75-90% of the CLA isomers ( Bauman et al., 2008Bauman, D. E.; Perfield, J. W.; Harvatine, K. J. and Baumgard, L. H. 2008. Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model. The Journal of Nutrition 138:403-409. https://doi.org/10.1093/jn/138.2.403
https://doi.org/10.1093/jn/138.2.403...
).

It appears that none of the supplements in the current experiment would produce a FA profile that could lead to high concentrations of isomers known to inhibit fat synthesis, since rumenic acid only decreased with inclusion of all supplements in the diet. Rumenic acid is an intermediate of C18:2n-6 biohydrogenation ( Kay et al., 2004Kay, J. K.; Mackle, T. R.; Auldist, M. J.; Thomson, N. A. and Bauman, D. E. 2004. Endogenous synthesis of cis- 9, trans- 11 conjugated linoleic acid in dairy cows fed fresh pasture. Journal of Dairy Science 87:369-378. https://doi.org/10.3168/jds.S0022-0302(04)73176-8
https://doi.org/10.3168/jds.S0022-0302(0...
), and the relative differences viewed against the content in feeds were expected to be higher. The hydrogenation of linoleic acid is often incomplete, ranging from 70 up to 95% ( Doreau and Ferlay, 1994Doreau, M. and Ferlay, A. 1994. Digestion and utilisation of fatty acids by ruminants. Animal Feed Science and Technology 45:379-396. https://doi.org/10.1016/0377-8401(94)90039-6
https://doi.org/10.1016/0377-8401(94)900...
). The high linoleic acid proportion observed in the RF of steers supplemented with Chlorella and CSM could be due to an escape from ruminal biohydrogenation, or due to a slow rate of this process. In vitro rates of disappearance have been reported ranging from 23.6 up to 44.6%/h ( Jouany et al., 2007Jouany, J. P.; Lassalas, B.; Doreau, M. and Glasser, F. 2007. Dynamic features of the rumen metabolism of linoleic acid, linolenic acid and linseed oil measured in vitro. Lipids 42:351-360. https://doi.org/10.1007/s11745-007-3032-x
https://doi.org/10.1007/s11745-007-3032-...
), and the RF from steers was collected approximately 3 h after feeding.

Various isomers result from ruminal biohydrogenation, and a portion of these CLA escape from the rumen and affect lipid metabolism in the mammary gland and subcutaneous and intramuscular fat ( Bauman et al., 2011Bauman, D. E.; Harvatine, K. J. and Lock, A. L. 2011. Nutrigenomics, rumen-derived bioactive fatty acids, and the regulation of milk fat synthesis. Annual Review of Nutrition 31:299-319. https://doi.org/10.1146/annurev.nutr.012809.104648
https://doi.org/10.1146/annurev.nutr.012...
). More importantly, the t 10 c 12 isomer of CLA can markedly inhibit FA synthesis in all three tissue types, although a much greater dose is required to inhibit body fat accretion ( Smith et al., 2008Smith, S. B.; Kawachi, H.; Choi, C. B.; Choi, C. W.; Wu, G. and Sawyer, J. E. 2008. Cellular regulation of bovine intramuscular adipose tissue development and composition. Journal of Animal Science 87:E72-E82. https://doi.org/10.2527/jas.2008-1340
https://doi.org/10.2527/jas.2008-1340...
) than a reduction in milk fat ( Bauman et al., 2008Bauman, D. E.; Perfield, J. W.; Harvatine, K. J. and Baumgard, L. H. 2008. Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model. The Journal of Nutrition 138:403-409. https://doi.org/10.1093/jn/138.2.403
https://doi.org/10.1093/jn/138.2.403...
). Bauman et al. (2008)Bauman, D. E.; Perfield, J. W.; Harvatine, K. J. and Baumgard, L. H. 2008. Regulation of fat synthesis by conjugated linoleic acid: Lactation and the ruminant model. The Journal of Nutrition 138:403-409. https://doi.org/10.1093/jn/138.2.403
https://doi.org/10.1093/jn/138.2.403...
reported that a dose of 2.5 g/day of t 10 c 12 CLA was required to achieve a 25% reduction in milk fat, and the maximum inhibition was 50%, and Smith et al. (2008)Smith, S. B.; Kawachi, H.; Choi, C. B.; Choi, C. W.; Wu, G. and Sawyer, J. E. 2008. Cellular regulation of bovine intramuscular adipose tissue development and composition. Journal of Animal Science 87:E72-E82. https://doi.org/10.2527/jas.2008-1340
https://doi.org/10.2527/jas.2008-1340...
indicated that doses 20 times higher would be required to inhibit fat synthesis in tissues such as subcutaneous and intramuscular fat. The extent of formation of this specific isomer within the rumen of steers in the current experiment could not be accurately measured with the method utilised. In spite of that, the proportion of rumenic acid decreased in the RF of steers supplemented with Spirulina, Chlorella, and CSM when compared with the control treatment. If these treatments reduced the production of the main CLA isomer in the RF, possibly the other isomers would also decrease, and no effects in adipose fat synthesis could be expected in growing cattle fed these supplements.

The same pattern of CLA decrease was described by Noci et al. (2005)Noci, F.; Monahan, F. J.; French, P. and. Moloney, A. P. 2005. The fatty acid composition of muscle fat and subcutaneous adipose tissue of pasture-fed beef heifers: Influence of the duration of grazing. Journal of Animal Science 83:1167-1178. https://doi.org/10.2527/2005.8351167x
https://doi.org/10.2527/2005.8351167x...
, who reported that with the substitution of a pasture-based diet rich in linolenic (with an increased ruminal production of C18:1 t 11) for a silage/concentrate-based diet rich in linoleic acid, there was a decrease in total CLA. The proportion of linoleic acid was significantly higher in the RF of steers supplemented with Chlorella and CSM. Lamminen et al. (2019)Lamminen, M.; Filleau, A. H. B.; Kokkonen, T.; Jaakkola, S. and Vanhatalo, A. 2019. Different microalgae species as a substitutive protein feed for soya bean meal in grass silage based dairy cow diets. Animal Feed Science and Technology 247:112-126. https://doi.org/10.1016/j.anifeedsci.2018.11.005
https://doi.org/10.1016/j.anifeedsci.201...
also reported a higher proportion of linoleic acid in RF of lactating dairy cows fed Chlorella in comparison with Spirulina diet. The proportion of linolenic was almost five times higher in the RF of unsupplemented steers compared with those supplemented with Chlorella. The FA profile of the tropical forage utilised in this experiment differs greatly from results seen for temperate forages. Chilliard et al. (2001)Chilliard, Y.; Ferlay, A. and Doreau, M. 2001. Effect of different types of forages, animal fat or marine oils in cow's diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids. Livestock Production Science 70:31-48. https://doi.org/10.1016/S0301-6226(01)00196-8
https://doi.org/10.1016/S0301-6226(01)00...
reported that temperate forages have 55-65% of their FA composed of linolenic acid, which was also observed in the work of Costa et al. (2019)Costa, D. F. A.; Quigley, S. P.; Isherwood, P.; McLennan, S. R.; Sun, X. Q.; Gibbs, S. J. and Poppi, D. P. 2019. Small differences in biohydrogenation resulted from the similar retention times of fluid in the rumen of cattle grazing wet season C3 and C4 forage species. Animal Feed Science and Technology 253:101-112. https://doi.org/10.1016/j.anifeedsci.2019.05.010
https://doi.org/10.1016/j.anifeedsci.201...
using ryegrass ( Lolium perenne ). The speargrass hay in the current experiment had a much lower (4% of total FA) proportion of linolenic acid than reported in temperate pastures. O'Kelly and Reich (1976)O'Kelly, J. C. and Reich, H. P. 1976. The fatty-acid composition of tropical pastures. Journal of Agricultural Science 86:427-429. https://doi.org/10.1017/S0021859600054915
https://doi.org/10.1017/S002185960005491...
analysed a variety of tropical grasses, and linolenic acid ranged from approximately 13% for speargrass up to 36% of total FA for Dichanthium sericeum . The lowest value observed for speargrass in their study was three times higher than the one observed in the current experiment; however, they analysed a fresh grass sample, whereas ours was a late-vegetative stage plant made into hay. Doreau and Poncet (2000)Doreau, M. and Poncet, C. 2000. Ruminal biohydrogenation of fatty acids originating from fresh or preserved grass. Reproduction Nutrition Development 40:201. emphasised that hay processing tends to decrease the FA concentration, in particular the proportion of linolenic acid. Speargrass hay had 15% of FA comprised of linoleic acid. The ratio between linoleic and linolenic between tropical and temperate grasses is also different with temperate forages having a greater proportion of linolenic acid ( Chilliard et al., 2001Chilliard, Y.; Ferlay, A. and Doreau, M. 2001. Effect of different types of forages, animal fat or marine oils in cow's diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids. Livestock Production Science 70:31-48. https://doi.org/10.1016/S0301-6226(01)00196-8
https://doi.org/10.1016/S0301-6226(01)00...
). The concentration of linoleic acid in speargrass hay utilised in the current experiment was almost four times greater than linolenic acid, and, interestingly, an inversion was observed in the RF of these steers, in which the concentration of linoleic acid was 2.5 times greater, indicating extensive ruminal biohydrogenation in this experiment.

Odd- and branched-chain fatty acids in the rumen are almost completely derived from bacterial synthesis ( O'Kelly and Spiers, 1991O'Kelly, J. C. and Spiers, W. G. 1991. Influence of host diet on the concentrations of fatty acids in rumen bacteria from cattle. Australian Journal of Agricultural Research 42:243-252. https://doi.org/10.1071/AR9910243
https://doi.org/10.1071/AR9910243...
; Kim et al., 2005Kim, E. J.; Sanderson, R.; Dhanoa, M. S. and Dewhurst, R. J. 2005. Fatty acid profiles associated with microbial colonization of freshly ingested grass and rumen biohydrogenation. Journal of Dairy Science 88:3220-3230. https://doi.org/10.3168/jds.S0022-0302(05)73005-8
https://doi.org/10.3168/jds.S0022-0302(0...
). The proportion of TOBCFA in the RF of steers supplemented with CSM, Spirulina, and Chlorella was lower than unsupplemented and Dunaliella-supplemented steers. A higher proportion of the odd- and branched-chain FA C15:0anteiso was reported for acetate-producing bacteria ( Vlaeminck et al., 2004Vlaeminck, B.; Fievez, V.; Van Laar, H. and Demeyer, D. 2004. Rumen odd and branched chain fatty acids in relation to in vitro rumen volatile fatty acid productions and dietary characteristics of incubated substrates. Journal of Animal Physiology and Animal Nutrition 88:401-411. https://doi.org/10.1111/j.1439-0396.2004.00497.x
https://doi.org/10.1111/j.1439-0396.2004...
). In this study, as the results observed for TOBCFA, steers supplemented with CSM, Spirulina, or Chlorella had a lower proportion of C15:0anteiso in the RF than unsupplemented steers. All microalgae had extremely low fibre content, and CSM also has a low ADFom and aNDFom contents relative to speargrass ( Table 1 ).

The RF of steers supplemented with CSM and Chlorella had reduced proportion of TSFA in the RF, and Chlorella led to a corresponding increase in TUFA concentration, compared with the control. This is most likely a direct result from FA of dietary origin, since it appears that microbes are not capable of synthesizing polyunsaturated FA ( O'Kelly and Spiers, 1991O'Kelly, J. C. and Spiers, W. G. 1991. Influence of host diet on the concentrations of fatty acids in rumen bacteria from cattle. Australian Journal of Agricultural Research 42:243-252. https://doi.org/10.1071/AR9910243
https://doi.org/10.1071/AR9910243...
). Morais and Costa (2008)Morais, M. G. and Costa, J. A. V. 2008. Perfil de ácidos graxos de microalgas cultivadas com dióxido de carbono. Ciências e Agrotecnologia 32:1245-1251. https://doi.org/10.1590/S1413-70542008000400032
https://doi.org/10.1590/S1413-7054200800...
reported that Chlorella sp. had the greatest concentration of TUFA (72% of total lipids), whilst Spirulina had the greatest TSFA, i.e. 81.6% of total lipids, which agreed with the present results. The greater concentration of TUFA in the diet would increase the concentration of TUFA in milk or meat if they bypassed the rumen either unaffected by the rumen microbes or by incorporation into microbial FA with no structural changes, i.e. chain elongation or hydrogenation. Safflower oil increased the concentrations of TUFA in rumen bacteria without altering their concentration of TSFA ( O'Kelly and Spiers, 1991O'Kelly, J. C. and Spiers, W. G. 1991. Influence of host diet on the concentrations of fatty acids in rumen bacteria from cattle. Australian Journal of Agricultural Research 42:243-252. https://doi.org/10.1071/AR9910243
https://doi.org/10.1071/AR9910243...
). It has been suggested that polyunsaturated FA inhibit the reduction of C18:1 to 18:0, giving rise to a high C18:1 t content ( Bessa et al., 2007Bessa, R. J. B.; Alves, S. P.; Jerónimo, E.; Alfaia, C. M.; Prates, J. A. M. and Silva, J. S. 2007. Effect of lipid supplements on ruminal biohydrogenation intermediates and muscle fatty acids in lambs. European Journal of Lipid Science and Technology 109:868-878. https://doi.org/10.1002/ejlt.200600311
https://doi.org/10.1002/ejlt.200600311...
; Or-Rashid et al., 2008Or-Rashid, M. M.; Kramer, J. K. G.; Wood, M. A. and McBride, B. W. 2008. Supplemental algal meal alters the ruminal trans- 18:1 fatty acid and conjugated linoleic acid composition in cattle. Journal of Animal Science 86:187-196. https://doi.org/10.2527/jas.2007-0085
https://doi.org/10.2527/jas.2007-0085...
). A significant reduction of C18:0 was observed for CSM and Spirulina, but there were no significant increases in any of the C18:1 isomers. When supplements rich in polyunsaturated FA, such as Chlorella and CSM (i.e. 55.6 and 72.6 % of total FA, respectively), are utilised, there is a reduced yield of C18:0 ( Bessa et al., 2007Bessa, R. J. B.; Alves, S. P.; Jerónimo, E.; Alfaia, C. M.; Prates, J. A. M. and Silva, J. S. 2007. Effect of lipid supplements on ruminal biohydrogenation intermediates and muscle fatty acids in lambs. European Journal of Lipid Science and Technology 109:868-878. https://doi.org/10.1002/ejlt.200600311
https://doi.org/10.1002/ejlt.200600311...
) and an accumulation of the intermediate C18:1 t 11 ( Chilliard et al., 2001Chilliard, Y.; Ferlay, A. and Doreau, M. 2001. Effect of different types of forages, animal fat or marine oils in cow's diet on milk fat secretion and composition, especially conjugated linoleic acid (CLA) and polyunsaturated fatty acids. Livestock Production Science 70:31-48. https://doi.org/10.1016/S0301-6226(01)00196-8
https://doi.org/10.1016/S0301-6226(01)00...
). This same pattern, not observed here, has been observed for increasing levels of other algae used as supplement ( Boeckaert et al., 2008Boeckaert, C.; Vlaeminck, B.; Fievez, V.; Maignien, L.; Dijkstra, J. and Boon, N. 2008. Accumulation of trans C18:1 fatty acids in the rumen after dietary algal supplementation is associated with changes in the Butyrivibrio community. Applied and Environmental Microbiology 74:6923-6930. https://doi.org/10.1128/aem.01473-08
https://doi.org/10.1128/aem.01473-08...
; Or-Rashid et al., 2008Or-Rashid, M. M.; Kramer, J. K. G.; Wood, M. A. and McBride, B. W. 2008. Supplemental algal meal alters the ruminal trans- 18:1 fatty acid and conjugated linoleic acid composition in cattle. Journal of Animal Science 86:187-196. https://doi.org/10.2527/jas.2007-0085
https://doi.org/10.2527/jas.2007-0085...
), which can be associated with changes in the microbial community ( Boeckaert et al., 2008Boeckaert, C.; Vlaeminck, B.; Fievez, V.; Maignien, L.; Dijkstra, J. and Boon, N. 2008. Accumulation of trans C18:1 fatty acids in the rumen after dietary algal supplementation is associated with changes in the Butyrivibrio community. Applied and Environmental Microbiology 74:6923-6930. https://doi.org/10.1128/aem.01473-08
https://doi.org/10.1128/aem.01473-08...
). In the current experiment, significant reductions in C18:0 proportion were observed in RF of steers supplemented with CSM and Spirulina. However, no significant differences in accumulation of C18:1 t 10 and C18:1 t 11 were observed for any of the treatments, except for the Dunaliella supplement. The latter supplement, interestingly, caused a reduction of this intermediate in the RF, which was most likely related to the basal forage, considering that the intake of Dunaliella, high in Na, was very low ( Costa et al., 2016Costa, D. F. A.; Quigley, S. P.; Isherwood, P.; McLennan, S. R. and Poppi, D. P. 2016. Supplementation of cattle fed tropical grasses with microalgae increases microbial protein production and average daily gain. Journal of Animal Science 94:2047-2058. https://doi.org/10.2527/jas.2016-0292
https://doi.org/10.2527/jas.2016-0292...
).

Alpha-linolenic acid (C18:3n-3) was present in high concentrations in the Chlorella supplement and was significantly greater in the RF of steers supplemented with this source. Free FA, such as C18:3n-3 and C18:2n-6, are often incompletely biohydrogenated, converting into C18:0 and monounsaturated isomers ( Scollan et al., 2001Scollan, N. D.; Dhanoa, M. S.; Choi, N. J.; Maeng, W. J.; Enser, M. and Wood, J. D. 2001. Biohydrogenation and digestion of long chain fatty acids in steers fed on different sources of lipid. Journal of Agricultural Science 136:345-355. https://doi.org/10.1017/S0021859601008796
https://doi.org/10.1017/S002185960100879...
).

The concentration of LCFA was usually below 1% of total FA in the supplements and the RF of supplemented animals. Speargrass hay had a greater concentration of LCFA than the microalgae and CSM supplements used in the present study, and this was reflected in greater concentrations in the RF of unsupplemented steers, but they were still very low values. Some marine algae are rich sources of specific long chain FA, such as DHA C22:6n-3 in Schizochytrium sp. (as much as 39% of total FA; Jiang et al., 2004Jiang, Y.; Fan, K. W.; Wong, R. T. and Chen, F. 2004. Fatty acid composition and squalene content of the marine microalga Schizochytrium mangrovei . Journal of Agricultural and Food Chemistry 52:1196-2000. https://doi.org/10.1021/jf035004c
https://doi.org/10.1021/jf035004c...
; Hauvermale et al., 2006Hauvermale, A.; Kunera, J.; Rosenzweiga, B.; Guerra, D.; Diltz, S. and Metza, J. G. 2006. Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase. Lipids 41:739-747. https://doi.org/10.1007/s11745-006-5025-6
https://doi.org/10.1007/s11745-006-5025-...
) or in Crypthecodinium cohnii (68.9% of total FA; Pickard et al., 2008Pickard, R. M.; Beard, A. P.; Seal, C. J. and Edwards, S. A. 2008. Neonatal lamb vigour is improved by feeding docosahexaenoic acid in the form of algal biomass during late gestation. Animal 2:1186-1192. https://doi.org/10.1017/S1751731108001997
https://doi.org/10.1017/S175173110800199...
). However, the fresh algae Chlorella and Spirulina have much lower concentrations of those LCFA. In particular, C22:6 DHA was present in very low concentration in speargrass hay sample, i.e. 0.53% of total FA and close to zero for all supplements utilized in this study. Therefore, the results of the present experiment are more likely to reflect the general FA profile of the feeds than any ruminal function characteristic.

5. Conclusions

Chlorella pyrenoidosa supplementation results in a higher proportion of unsaturated fatty acids in rumen fluid of steers. Supplementation with microalgae sources and CSM decrease rumenic acid (C18:2 c 9, t 11), the main conjugated linoleic acid isomer, indicating that it is unlikely that any of the supplements tested would have differential effects on other isomers directly related to fat synthesis.

Acknowledgments

The authors acknowledge Meat and Livestock Australia for funding this research. The first author acknowledges financial support from the International Postgraduate Research Scholarship (IPRS) and living allowance provided by UQ Research Scholarship (UQRS).

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Publication Dates

  • Publication in this collection
    25 Sept 2020
  • Date of issue
    2020

History

  • Received
    29 Mar 2020
  • Accepted
    14 July 2020
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