{'fraction_non_alphanumeric': 0.03830614227995704, 'fraction_numerical': 0.029969825602209378, 'mean_word_length': 4.734310850439883, 'pattern_counts': {'":': 0, '<': 7, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 9, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Dietary supplementation of finishing pigs with the docosahexaenoic acid-rich microalgae, Aurantiochytrium limacinum: effects on performance, carcass characteristics and tissue fatty acid profile

Objective The aim of this experiment was to evaluate the effect of dietary supplementation with the docosahexaenoic acid (DHA)-rich microalgae, Aurantiochytrium limacinum (AURA) on pig performance, carcass traits, and the fatty acid composition of pork Longissimus lumborum (LL) and backfat. Methods A total of 144 Pig Improvement Company (PIC)×Goland finishing pigs (72 females and 72 castrated males) of mean weight 117.1 (±13.1) kg were blocked by sex and body weight and provided with 0% or 1% AURA in isonutritive and isocaloric diets. A total of 24 pens provided 12 replicates per treatment. Animals were weighed on day 0 and 28 with feed and water intake recorded per pen. After 31 days supplementation (28 days of study and 3 days until the slaughtering date) three animals per pen (n = 72) were slaughtered and the LL and backfat thickness, lean meat content and dressing percentage were recorded for the carcasses. The fatty acid (FA) profile of the LL and backfat was established by direct FA methyl ester synthesis. Results No differences were observed for any performance parameters or carcass traits. Supplementation with AURA resulted in significant changes to the FA profiles of both the LL and backfat with male and female pigs responding differently to supplementation in terms of particular FAs. Overall, pork LL samples had significantly higher eicosapentaenoic acid (p<0.001) and DHA concentrations (p<0.001), and higher omega-3 (n-3) FAs (p<0.001), as well as an increased omega3:omega6 (n-3:n-6) ratio (p = 0.001). For backfat, supplementation resulted in significantly higher amounts of DHA (p<0.001) and n-3 FAs (p<0.001). Conclusion These results indicate that dietary supplementation with 1% AURA over a 31 day period can increase the FA composition of pork LL and backfat, specifically the DHA, with no major impact on growth performance and carcass traits.


INTRODUCTION
Humans and other animals are incapable of synthesising essential fatty acids (FA) endogenously and as such, must obtain them from their diet [1]. The essential FA are split into two categories, omega 6 (n-6) and omega 3 (n-3) FA, for which linoleic acid (LA) and α-linolenic (ALA) are the respective parent compounds [2]. The n-3 FA, in particular eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to have a positive impact on a variety of human health problems including cardiovascular disease and depressive disorders [3].
Oily fish are considered the best dietary source of polyunsaturated FA (PUFA), however the consumption of oily fish is low in western style diets [4]. In addition, the conversion of ALA into EPA and DHA is limited in the human body, and as such the direct consumption of EPA and DHA rich products is considered necessary to meet the recommended daily requirements of each [3]. The enrichment of commonly consumed products with n-3 FA represents a way to increase their consumption without requiring any drastic dietary changes. Plant and marine (algae and fish) oils have been added to animal feed in order to increase the n-3 FA content of animal products. The tissues of monogastric animals, such as pigs, are susceptible to FA alteration through dietary modification [5], making feed supplementation a viable option to increase n-3 FA concentrations in their products. The enrichment of pork with PUFA following dietary supplementation with plant and marine (fish and algae) oils has been successfully demonstrated by a number of authors [6][7][8][9][10][11]. Vossen et al [11], recently showed that supplementation with ALA rich linseed did not increase the concentration of DHA and EPA in pork. As in humans, the conversion of ALA to long chain PUFA is limited in pigs [12]. As such, direct supplementation with DHA and EPA rich sources is required. Despite fish being considered the richest source of EPA and DHA, productivity limitations and the issue of overfishing mean that the n-3 FA requirements of the population cannot be met using fish products alone [13]. As such marine algae represent a rich source of DHA that can be produced at scale in a sustainable manner.
Supplementation with the marine algae Schizochythrum sp. has been shown to increase the levels of EPA and DHA in meat without negatively impacting the productivity of the animals [5,10,14,15]. Little information is available regarding dietary supplementation of pigs with other sources of algae with the potential to be heterotrophically grown, especially in low sodium conditions. Recently, the microalga, Aurantiochytrium limacinum, was fed at low inclusion concentrations, 0.25% and 0.5%, to pigs for the entire grower-finisher phase, 121 days resulting in Longissimus lumborum (LL) and backfat enrichment with DHA [16,17]. However, the commercial application of algae for a prolonged feeding period may not be practical for most farms. Therefore, the aim of this study was to investigate the effect of providing a moderate level of algae supple mentation for the final month prior to slaughter on the potential to enrich DHA in the LL and backfat without influencing the productivity of finishing pigs.

Animals, experimental design and diets
The study was carried out at the CERZOO S.r.L. Research Centre (Piacenza, Italy) in compliance with G.L.P guidelines (Directives 2004/9/EC and 2004/10/EC) for the collection, handling and documentation of data. The research protocol and animal care were carried out in accordance with European guidelines on the protection of animals used for scientific purposes (Directive 2010/63/EU). A total of 144 Pig Improvement Company (PIC)×Goland finishing pigs (72 female and 72 castrated males) of mean weight 117.1 (±13.1) kg were recruited for the study. The animals were housed in four growingfinishing rooms, with a total of 24 pens providing 12 replicates (6 female and 6 castrated male) for each of the two treatments. The growing-finishing rooms were equipped with a dynamic ventilation system with heating provided by water aerotherms and positive pressure ventilation achieved by variable speed fans linked to temperature sensors which automatically responded to maintain an appropriate temperature based on the age and temperature of the pigs. Pigs were fed ad libitum via a steel feeder present in each pen and were provided with free access to water. On arrival at the facility, the pigs were allowed to acclimatise for a period of seven days after which they were weighed, sorted by sex and weight and allocated to pens such that each pen contained small medium and heavy, female or castrated male pigs. The pigs were then fed the experimental diets for a period of 31 days (28 days study duration followed by three days until the slaughtering date).

Performance parameters and carcass measurements
Pigs were individually weighed on d 0 and d 28 of the study. Feed and water intake was recorded for each pen from d 0 to d 28. Average daily gain (ADG) and gain:feed (G:F) ratio was calculated per pen. Three pigs per pen were selected based on the mean weight for the treatment group, providing a total of 72 animals for slaughter (18 female and 18 castrated males for each experimental diet). LL and backfat thicknesses were measured, where lean meat content was determined using a Fat-O-Meter (SFK Technology, Herlev, Denmark) optical probe positioned 8 cm to the side of the central line of the carcass between the third and fourth last ribs, in accordance with the Commission Implementing Decision of 24 January 2014 on authorizing methods for grading pig carcasses in Italy (2014/ 38/EU). Carcasses were classified as per the SEUROP carcass classification system (Reg. 2013/1308/EU). LL muscle and backfat Samples were removed from the right side of each animal, within 15 min post-slaughter, between the third and fourth last ribs, placed in vacuum bags and immediately frozen and stored at -18°C until FA analysis.
The FA profiles of carcass samples were determined at the Institute of Food Science and Nutrition (Faculty of Agricultural Sciences, Food and Environment, Catholic University of Sacred Heart, Piacenza, Italy). Samples were thawed, ground and homogenized for subsequent FA profile analysis after direct FA methyl ester synthesis according to the procedure as described by O'Fallon et al [20]. The complete FA profiles included short-chain FAs (C4.0 through C8.0), medium-chain FAs (C10.0 through C15.1), long-chain FAs (C16.0 through C22:6n3), and very long-chain FA (C24:0 and C24:1

Statistical analysis
Data were analysed by the general linear model (GLM) procedure of SAS (2010, release 9.3) using analysis of variance (ANOVA) as the main statistical test. For the performance parameters the pen was the experimental unit with 24 replicates blocked by sex (12 castrated male pens and 12 female pens). For each parameter the GLM was fitted with the following terms: Treatment (Control, AURA); Sex (Castrated male, female); plus the treatment×sex interaction. For the carcass characteristics and the FA profile the pig was the experimental unit with a total of 72 replicates (36 castrated male, 36 female). When a significant treatment×sex interaction was detected, the sexes were analysed separately by GLM to investigate the effect of treatment on either castrated male or female pigs. Students "t" and Tukey tests were used to compare the means of each group. For the ANOVA model, p≤0.05 indicated a significant difference while 0.05<p≤0.10 indicated a trend.

Diet analysis, performance and carcass characteristics
The test article, AURA, used in the study primarily consisted of 70.2 g crude fat/100 g DM biomass composed of a significant level of palmitic acid and DHA, 34.5 g and 18.0 g/100 g DM biomass respectively. Additionally, AURA contained 15.8% crude protein, 3.9% ash, and 2.3% moisture. The EPA accounted for only 0.23 g/100 g DM biomass. The chemical composition, energy value and DHA content of the experimental diets are show in Table 2. The pigs maintained good health throughout the study with no animals being removed or culled for welfare reasons. Treatment with AURA had no effect on ADG, average daily feed intake, average daily water intake, or G:F ratio (Table 3). No differences were observed between the groups in terms of LL thickness, backfat thickness, lean meat content or dressing out (Table 3). Carcass evaluation according to the SEUROP system is provided in Table 4 and indicates the expected difference in lean meat percentage between the sexes.

Fatty acid profile
Supplementation with AURA resulted in significantly higher levels of the following FAs in the pork LL samples: C8:0, C10:0, C14:0, C15:0, C16:0, C18:0, C18:1n9 cis, C18:2n6c, C18:3n3, C20:2, C22:1n9, C24:0, EPA, and DHA (Table 5). Animals supplemented with AURA had higher PUFA, n-3, and n-6 FAs, in addition to an increased n-3:n-6 ratio. The GLM results indicated an interaction between sex and treatment for some FAs and as such the male and female pigs were analysed separately to explore the sex specific effects. The FA profile of the   AURA supplemented female pigs showed increased concentrations of the following FAs: C8:0, C10:0, C12:0, C14:0, C15:0, C16:0, C18:0, C18:1n9 cis, C18:2n6c, C18:3n3, C20:0, C20:1, C20:2, C20:4n6, C22:1n9, EPA, and DHA. In contrast, the FA profile of the AURA supplemented male pigs differed only in terms of a higher EPA and DHA content and a lower C24:0 content in the LL meat. Both male and female pigs had significantly higher n-3 FA levels following AURA supplementation. Female pigs had a greater n-6 content while male pigs did not. As such, the n-3:n-6 ratio for the female pigs did not differ between control and supplemented groups while and increase in the ratio was observed for the male pigs (Table 6).   The FA profile of the backfat indicated significantly higher levels of C22:1n9 and DHA and lower levels of C18:1 trans, and C24:0 in the AURA supplemented animals. Overall a significantly higher n-3 content was found in the backfat in addition to an increased n-3:n-6 ratio. Again, the analysis indicated an interaction between sex and treatment for some FAs and as such each sex was analysed separately. Female pigs had significantly lower C18:1 trans and higher DHA. Male pigs had greater levels of C:10, C15:0, C17:0, C22:1n9, and DHA than the control group. Both male and female pigs had an increased n-3 concentration and a higher n-3:n-6 ratio.   Supplementation with AURA had no effect on the production parameters or on the measured carcass characteristics, which is generally in agreement with other studies that have fed microalgae [5,10,16,17,21], plant oils [22,23] or fish oils [6,24] to manipulate the FA profile of pork products. Incorporation of 1% AURA into the diet of pigs with a mean initial weight of 117.1 (±13.1) kg, for a period of 31 days resulted in significant changes to the FA profile of pork LL and backfat. Pork LL samples from the AURA-fed pigs differed significantly in terms of their EPA and DHA content (p<0.0001), with treated animals having 62% and 247% greater EPA and DHA respectively, than the control group. For the backfat samples, AURA treatment had a significant effect on the level of DHA (with an increase of 218% compared to controls) but did not lead to an increase in EPA concentration. Overall, pork LL and backfat were enriched to a level of 31.2 and 192.4 mg of DHA/100 g respectively. The effect of dietary supplementation of pigs with AURA had previously been investigated by Moran et al [17,18] over longer periods (117 and 121 days), using animals with lower initial weights (approximately 20 to 30 kg) than the current study. In both previous trials, similar increases in LL DHA content (approximately 3X the control) were observed for pigs supplemented with 0.25% AURA. In the current study, a similar level of enrichment was achieved after 31 days of supplementation with 1% AURA. Vossen et al [12], who investigated the effects of dietary supplementation with Schizochytrium (0.3% to 2.2% algae) for a period of 45 days found similar levels of enrichment to the current study, reporting 10 to 20 mg of DHA/100 g of pork longissimus thoracis. Sardi et al [11] also used Schizochytrium to supplement the diet of pigs and demonstrated increases in the DHA content of LL from 100% to 250%. Supplementation with 2.5% algae for a period of 56 or 28 days led to similar levels of LL enrichment (50 mg and 40 mg DHA/100 g LL). A significantly higher LL DHA content was achieved with supplementation at a level of 5% for 28 days (70 mg/100 g LL). These studies indicate that supplementing a higher concentration for a shorter period of time can achieve similar or increased levels of enrichment than longer term supplementation with lower concentrations.

DISCUSSION
In contrast to the current study, Sardi et al [11] found no increase in the EPA content of LL or backfat. In a seperate study, in which pigs were supplemented with a large dose of 9,400 mg DHA/d, the authors showed a significant increase in both EPA and DHA content of both the LL and backfat [21]. The differences in the EPA content of LL and backfat observed in the current study could be influenced by a number of factors. Firstly, the concentration of ALA, the parent compound of EPA and DHA, differed significantly between the control and treatment LL samples but did not differ in the backfat samples. Smink et al [13] demonstrated that higher concentrations of ALA significantly increased EPA levels in intramuscular fat, but had no effect in the backfat, which may explain the in-creased concentration of EPA in pork LL, but its absence from the backfat in the current study. In addition, following dietary DHA supplementation of pigs, the expression of FA synthesis genes has been shown to increase significantly, in the muscle and liver tissue but not in fat [21]. Secondly, the supplemental levels of ALA and EPA detected in AURA were relatively low (0.02 and 0.23 g/100 g DM, respectively). As n-3 FAs are preferentially incorporated into phospholipids over triacylglycerol, the low level of supplementation with ALA and EPA might help explain their relative increases in the phospholipid rich muscle as opposed to the triacylglycerol rich backfat [25]. However, it is unlikely that these are the only causes for the increased concentration of EPA in the LL. Vossen et al [12], also observed an increase in the concentration of EPA that could not have come directly from the diet. The authors suggested that higher EPA concentrations in algae supplemented pigs could be attributed to DHA retro-conversion, a minor metabolic pathway which involves one cycle of β-oxidation [26].
Both previous studies by Moran and colleagues [17,18], indicated that higher levels of AURA supplementation (0.5%) may affect the sexes differently. While the LL DHA content of both males and females significantly increased relative to the controls, the LL of the males was enriched to a higher degree (5× vs 4.5× and 4× vs 2.9×; [17], [18], respectively). In contrast, no treatment×sex interaction was detected for the pork LL or backfat DHA content in the current study, indicating that the sexes responded similarly under these conditions. However, treatment×sex interactions were observed for a number of other FAs. In the LL samples of female pigs, the FA composition was significantly different from the control for 17 FAs, while only three significant differences in the male FA profile were observed. In the backfat of female pigs, the only changes detected involved c18:1 trans and DHA, while in male pigs the level of five different FAs differed from the control animals. The FA composition of pigs is mainly influenced by nutrition, however, other factors such as genotype, sex, age and the total amount of carcass fat also impact the individual FA composition [25]. In humans and mice, sex hormones are thought to play a role in the metabolism of n-3 PUFAs [27]. Higher DHA concentrations in the tissues and plasma of women have been attributed to greater levels of DHA synthesis [28]. These studies indicate that the sex hormones may influence FA metabolism and could explain the difference in response to supplementation observed in this study. Overall, EPA and DHA both increased to similar degrees in male and female pigs. In pork LL, an increase in the n-3:n-6 ratio was not observed for the female pigs, likely due to the increase in the level of n-6 FA observed in this group. For the backfat however, no significant increase in n-6 FAs led to similar n-3:n-6 ratios for the both males and females.

CONCLUSION
These results indicate that dietary supplementation with 1% AURA over a 31-day period can increase the FA composition of pork LL and backfat, specifically the DHA, with no major impact on growth performance and carcass traits. Both female and castrated male pigs responded similarly to AURA supplementation in terms of the DHA content of the LL and backfat but differed in terms of a number of other FAs. These results support the use of AURA as an effective, sustainable method by which to increase the n-3 content of pork.

CONFLICT OF INTEREST
Funding for this work was provided by Alltech SARL (France). The author Moran CA and Keegan JD are employees of Alltech which produces and markets ALL-G-RICH, the commercial product used in this study.