Combining in vivo and in vitro approaches to investigate the effect of sainfoin on strongyle infection, immunity and large intestine ecosystem of horses.

In horses fed a high-starch diet for 84 days, adding sainfoin pellets did not reduce strongyle egg shedding or broadly improve gut health markers, although it modestly increased plasma acetate and shifted some gut bacteria. In vitro, aqueous extracts of sainfoin pellets inhibited parasite eggs and larvae, with potency varying between products, suggesting effects depend on polyphenol content and warrant prior screening.

What the study set out to test

• Whether long-term inclusion of dehydrated sainfoin (Onobrychis viciifolia) pellets in a high-starch diet reduces strongyle infection and affects intestinal health in horses.
• Whether different commercial sainfoin pellets differ in their direct anti-parasitic potency in vitro, and if such differences could explain inconsistent in vivo results.

Why this matters

• Sainfoin is rich in polyphenols (notably condensed tannins) that show anti-parasitic activity in ruminants, but evidence in horses is mixed.
• Diet composition can modify sainfoin’s effects; prior short-term work suggested sainfoin slowed the rise of egg shedding under high-starch feeding over 21 days.
• Identifying when and which sainfoin products work could offer a nutrition-based adjunct to parasite control and help slow resistance to anthelmintic drugs.

Study design and methods

• Population: 16 horses with a history of strongyle egg excretion.
• Allocation and diets: Horses were assigned to two groups for 84 days on high-starch rations containing either:
  • Sainfoin pellets (SF), or
  • Control pellets (CONT; sunflower and hay based).
• In vivo outcomes:
  • Parasitology: Fecal strongyle egg counts (egg shedding dynamics).
  • Gut microbiome: Relative abundance of bacterial taxa; summary measures of diversity, predicted function, and activity.
  • Host physiology: Plasma acetate (a short-chain fatty acid), plasma lipopolysaccharides (LPS; gut barrier marker), and complete blood count (CBC; general health).
• In vitro assays on two sainfoin pellet products:
  • Egg Hatch Test (EHT): Inhibition of egg hatching across extract concentrations.
  • Larval Migration Inhibition Test (LMIT): Impairment of third-stage larval movement through a mesh after exposure to extracts.
  • Extracts: Aqueous preparations tested at multiple concentrations to reflect water-soluble bioactives relevant to the hindgut environment.
• Statistics: Group comparisons with reported P values; significance generally set at P < 0.05.

Main results

• Fecal egg counts: No reduction with sainfoin over 84 days (P = 0.671).
• Gut microbiota:
  • Taxa-level shifts: Sainfoin induced modest, taxa-specific changes (e.g., Oscillibacter showed differences at some time points).
  • Global metrics: No significant changes in overall diversity, predicted function, or activity (all P > 0.05).
• Host markers:
  • Plasma acetate increased with sainfoin (P = 0.032).
  • No significant differences in plasma LPS or CBC indices (all P > 0.05).
• In vitro anti-parasitic activity:
  • Both sainfoin products inhibited egg hatching and larval migration (all P < 0.05).
  • One product was consistently more potent than the other (EHT: all P < 0.05; LMIT: P = 0.008).

How to interpret these findings

• Lack of in vivo FEC reduction:
  • Sainfoin, as used here within a high-starch ration, did not translate its in vitro activity into fewer eggs shed over three months.
  • Plausible reasons include insufficient dose or bioavailability of active polyphenols, degradation or complexation in the hindgut, product-to-product variability, and the modulating effect of a high-starch diet on microbial metabolism and tannin chemistry.
• Microbiome and acetate:
  • Taxon-specific shifts without global functional change suggest subtle community rebalancing rather than wholesale remodeling.
  • Higher plasma acetate implies altered fermentation end-products, but clinical significance is uncertain given no accompanying improvements in LPS or CBC.
• In vitro potency differences:
  • Direct anti-parasitic effects varied between sainfoin products, consistent with differences in polyphenolic concentration and composition (e.g., condensed tannin content and structure).
  • This variability likely contributes to mixed in vivo results across studies and underscores the value of batch/product screening.
• Overall implication:
  • Sainfoin’s anti-parasitic potential in horses appears conditional—dependent on the product’s polyphenol profile, dose, and dietary context.

Strengths and limitations

• Strengths:
  • Combined in vivo and in vitro approaches enable mechanistic interpretation.
  • Moderate intervention duration (84 days) under a defined high-starch diet.
  • Multiple outcome domains: parasitology, microbiota, and host health markers.
• Limitations:
  • Small sample size (n = 16) limits power to detect modest FEC changes.
  • No reported quantification of pellet polyphenol/tannin profiles to link chemistry with efficacy.
  • High-starch background may mask or counteract sainfoin effects seen under forage-based diets.
  • FECs are variable and may not reflect worm burden or species composition; larval culture/speciation not described.
  • Dose-response and pelleting/processing effects were not tested in vivo.

Practical takeaways for horse owners and veterinarians

• Do not expect sainfoin pellets to reliably lower strongyle egg shedding when simply added to a high-starch ration over several months.
• Continue targeted selective deworming based on fecal egg counts and evidence-based anthelmintics.
• If considering sainfoin as a supportive strategy:
  • Prefer products with documented higher in vitro activity or verified higher polyphenol/condensed tannin content.
  • Monitor FECs regularly to assess any individual response.
  • Consider overall diet composition, as effects may be context-dependent.

How the in vitro tests relate to real-world effects

• Egg Hatch Test (EHT): Measures the ability of extracts to prevent eggs from hatching into L1 larvae; lower hatch rates indicate direct ovicidal activity.
• Larval Migration Inhibition Test (LMIT): Assesses whether L3 larvae lose motility or structural integrity, reducing their ability to migrate through a sieve; reduced migration implies larvicidal or paralytic effects.
• Translation caveat: Potency in aqueous assays confirms bioactivity but does not guarantee in vivo efficacy, which depends on dose delivered to the hindgut, compound stability, and host–microbe interactions.

Future research directions

• Quantify and standardize sainfoin pellet chemistry (e.g., condensed tannin content, mean degree of polymerization, prodelphinidin/procyanidin ratio) and relate it to in vitro and in vivo outcomes.
• Test different doses and formulations, and compare high-starch versus forage-based diets.
• Use larger cohorts, longer follow-up, and include larval cultures/speciation to detect species-specific effects.
• Assess additional gut health markers (e.g., fecal calprotectin, permeability assays) and multi-omics readouts of microbiome function.
• Investigate processing effects (pelleting, dehydration) on polyphenol bioavailability, and perform batch-to-batch in vitro screening to guide product selection.

Bottom line

• Under a high-starch diet, sainfoin pellets did not reduce strongyle egg shedding over 84 days, despite clear in vitro anthelmintic activity that varied by product.
• Efficacy likely hinges on the specific polyphenolic profile and dietary context; preliminary in vitro testing and product standardization may be key to achieving in vivo benefits.