Effect of dietary krill oil supplementation on horse cell membrane composition

The results of this study show that krill oil supplementation containing 6.6 g eicosapentaenoic acid (EPA) and 4.5 g docosahexaenoic acid (DHA) per appx. 1200 lb significantly and substantially increased the anti-inflammatory fatty acid (FA) composition of the cell membranes while at the same time lowering the inflammatory FA component. Good health starts with healthy cells.
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Effect of dietary krill oil supplementation on horse red blood cell membrane fatty acid composition and blood parameters

J Anim Physiol Anim Nutr (Berl). 2023 May 5. doi: 10.1111/jpn.13828. Online ahead of print.
Authors
Nicole Frost Nyquist 1 , Lena Burri 2 , Rasmus Bovbjerg Jensen 3
Affiliations

1 Department of Paraclinical Sciences, Faculty of Veterinary Sciences, Norwegian University of Life Sciences, Ås, Norway.
2 Aker BioMarine Antarctic AS, Lysaker, Norway.
3 Department of Animal and Aquacultural Sciences, Faculty of Bioscience, Norwegian University of Life Sciences, Ås, Norway.

PMID: 37144326
DOI: 10.1111/jpn.13828

Abstract

Supplementation with marine-derived n-3 long-chain polyunsaturated fatty acids (LC PUFAs), eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) is linked to beneficial health effects in both humans and horses. Krill oil (KO), which is extracted from the Antarctic krill (Euphausia superba), is well documented as a safe and biologically available dietary supplement in humans and several animal species, but there is a lack of documentation regarding its effect as a dietary ingredient for horses. The objective of this study was to test whether KO as a dietary supplement had the ability to raise horse red blood cell (RBC) membrane EPA and DHA, expressed as the n-3 index. Five nonworking Norwegian cold-blooded trotter horse geldings (body weight [BW]: 567 ± 38 kg) were supplemented with KO (10 mL/100 kg BW) for 35 days in a longitudinal study. Blood samples were analysed for RBC membrane fatty acid (FA) profile, haematology and serum biochemistry every 7th day. KO was well accepted by all horses, and no adverse health effects were observed during the 35-day trial period. KO supplementation affected the RBC membrane FA profile by increasing the n-3 index from Day 0 to 35 (Day 0: 0.53% vs. Day 35: 4.05% of total RBC FAs). The observed increase in the sum of EPA and DHA (p < 0.001), total n-3 FAs (p < 0.001) and the reduction of n-6 FAs (p < 0.044) resulted in a lower n-6:n-3 ratio (p < 0.001) by Day 35 of KO supplementation. In conclusion, the RBC n-3 index was increased and the general n-6:n-3 ratio was decreased in horses receiving 35-day dietary KO supplementation.

4 DISCUSSION
In humans (Bilinski et al., 2020), dogs (Burri et al., 2020) and other animals (Burri & Johnsen, 2015), KO has been shown to be a viable option as an n−3 LC PUFA dietary supplement. The primary objective of the current study was to evaluate the effect of a 35-day supplementation of KO on the n−3 index and other RBC membrane FAs in horses. To the knowledge of the authors, this is the first controlled study demonstrating that supplementing horses with a known daily dose of 6.6 g EPA and 4.5 g DHA has the anticipated effects on these blood variables. All horses readily consumed the KO as a top dressing and remained healthy throughout the study. No adverse effects, such as allergic reactions or gastrointestinal issues, were detected by those involved in animal care. These observations are in agreement with studies that involved supplementing horses with other marine-based FA supplements (Pearson et al., 2022). In agreement with earlier studies of KO supplementation in both humans (Bilinski et al., 2020) and dogs (Burri et al., 2020), the daily supplementation with 55 mL KO (supplying 6.6 g EPA and 4.5 g DHA) performed in the current study significantly raised the n−3 index of the horses. Other dietary n−3 PUFA sources, such as fish oils, have also been shown to influence the n−3 index in horses (T. M. Hess et al., 2012; Vineyard et al., 2010).

An increase in the n−3 index was already seen after the first adaptation week. This observation is in agreement with earlier studies on n−3 LC PUFA supplementation in horses. King et al. (2008) measured the greatest plasma concentration change in EPA and DHA by Day 3 of a 28-day supplementation period. The same study observed that a dose-related peak level was reached on Day 7 of n−3 FA supplementation. In the current study, a gradual weekly increase was observed in RBC, EPA and DHA levels from pre-supplementation to Day 35 of supplementation. T. M. Hess et al. (2012) observed similar changes to RBC membrane FA compositions when supplementing with a marine n−3 oil in horses. They reported an increase in EPA from Day 0 to 30 and from Day 30 to 60 but no further increase from Day 60 until Day 90 of supplementation. For DHA, they observed a gradual increase in RBC levels from Day 0 to 90 (T. M. Hess et al., 2012). Other studies have observed plasma DHA reaching a peak on Day 40 of supplementation (Nogradi et al., 2015). Prior to the onset of the current study, the horses were fed a pure forage and grass diet devoid of AA. The observed slight increase in RBS membrane AA level may therefore be seen in relation to the natural level of n−6 AA in KO (Table 2), and similarly, slight increases in AA have been reported for other studies in horses receiving marine oil supplementation (Vineyard et al., 2010).<

In addition to EPA, DHA and AA, there was a significant increase in myristic (C14:0), palmitelaidic (C16:1n7t), elaidic (C18:1t), arachidic (C20:0) lignoceric (C24:0), nervonic (C24:1n9), docosapentaenoic (C22:5n6) and docosapentaenoic (C22:5n3) FAs. The increase seen for the above-mentioned FAs may only be achievable with a compensatory reduction in other RBC membrane FAs. In the current study, there was a reduction in the levels of palmitic, LA, gamma-linolenic, eicosenoic and eicosadienoic acids from the second to the third weeks of KO supplementation, as well as a reduction in ALA after the first acclimatising week. The overall increase in n−3 PUFA and reduction in n−6 PUFA resulted in an overall reduction in the n−6:n−3 PUFA ratio (Figure 1). This may be of relevance for the health of horses because an increase in n−3 FAs is known to have anti-inflammatory properties, as compared to the more proinflammatory properties of n−6 PUFAs (T. Hess & Ross-Jones, 2014). These results are in accordance with studies conducted with other animals, such as dogs (Dominguez et al., 2021), and confirm studies showing that the increase of n−3 PUFAs and the n−3 index is proportional to the level of n−3 LC PUFAs supplemented into the diet (King et al., 2008; Pearson et al., 2022). The results of the present study show that supplementing horses for 28 days with the given dose of KO and n−3 LC PUFA level may lead to an increased n−3 index, potentially improving inflammatory-related health issues in horses (Nogradi et al., 2015; Woodward et al., 2007).

To the authors knowledge, there are currently no studies that specify the lower level of n−3 LC PUFA supplementation needed to achieve health beneficiary effects in horses. Pearson et al. (2022) reported a dose-dependent rise in horse whole blood n−3 LC PUFA following 6 weeks of dietary supplementing two groups of horses fed 1.8 g EPA and 3.6 g DHA per day, indicating that a lower dose of KO supplementation may have been sufficient to result in measurable changes in plasma n−3 LC PUFA. The daily dose of 6.6 g EPA and 4.5 g DHA fed to the horses in the current study were comparable to the lower dose fed by King et al. (2008) and supplied only a slightly higher level of DHA compared to that indicating beneficial effect for horses with chronic lower airway inflammatory disease (Nogradi et al., 2015). Woodward et al. (2007) found little effect on trot stride length and lameness score in horses treated with 4.6 g EPA + DHA per day, while Manhart et al. (2009) and Ross-Jones et al. (2014) reported that supplementing 34.8 g and 34.2 g EPA + DHA per day respectively, may be advantageous for horses with arthritis and results tended to lower synovial fluid PGE2. Bazzano et al. (2015) reported that a 30-day supplementation of 19.2 g EPA + DHA per day might have beneficial effects on performance when using an erythrocyte osmotic fragility.

In humans, non-dietary factors such as sex and age may influence blood tissue levels of n−3 LC PUFA (de Groot et al., 2019) and sex-related differences in metabolic fate of n−3 LC PUFA in animals supplemented with marine n−3 sources are reported (Ghasemifard et al., 2015). However, earlier studies in horses evaluating the ability of marine n−3 dietary supplements using mares, geldings and/or stallions have not reported considerations regarding potential effects of sex on the metabolism of n−3 LC PUFA (Brinsko et al., 2005; T. M. Hess et al., 2012; Munsterman et al., 2005; Nogradi et al., 2015; O’Connor et al., 2007). Furthermore, S. Elzinga et al. (2014) found no differences in total tract macronutrient digestibility between healthy adult and aged horses, indicating that results of the current study are relevant for horses of all ages and gender.

An analysis of general blood parameters is useful for evaluating general health status and widely used in diagnosis, monitoring response to treatments and evaluating an animal’s metabolic state (Messer, 1995). All the biochemical and haematological parameters measured in the current study were within the reference range specified for horses (NMBU, 2022), confirming that the horses were in good health before and during the KO-supplementation period. The results of the current study are in accordance with both human and animal studies, enabling the conclusion that KO may be considered a safe and well-tolerated n−3 LC PUFA source for horses (Burri et al., 2020; Robertson et al., 2014; Ulven et al., 2011).

One limitation to this study is the lack of a control group. Despite the lack of a control group, the clear increase in the n−3 index from Day 0 to 35 of the trial period may be seen as an effect of the dietary KO supplementation, as prior to Day 0 none of the horses had been fed KO or any other form of n−3 dietary supplement. Another limitation is the length of the study, as the n−3 index may still have increased after 35 days of supplementation. It is not known what level the n−3 index would have reached if the horses were supplemented for a longer time. In a comparable study a plateau was reach within 28 days of supplementation with fish oil, another EPA and DHA rich feedstuff (King et al., 2008). The comparison of KO with fish oil would be of interest in future studies.

5 CONCLUSIONS
The study is the first controlled study demonstrating that supplementing horses with a known daily dose of 6.6 g EPA and 4.5 g DHA from KO alters the RBC membrane FA profile. This was demonstrated by a gradual increase in the n−3 index and decrease in the n−6:n−3 ratio during 35 days of supplementation.

Keywords: DHA; EPA; Euphausia superba; docosahexaenoic acid; eicosapentaenoic acid; equine.