Pet Food Digestibility Testing: In Vitro vs In Vivo

# Pet Food Digestibility Testing: In Vitro vs In Vivo

When selecting a co-packer, brands often ask: "What is the digestibility of this formula?" The co-packer may give a 90% or 85% number. But how was it measured? In vitro? In vivo? How much do they differ? This guide covers digestibility testing.

Why Digestibility Matters

Digestibility is a core nutritional metric:

• Higher protein digestibility = more absorbed protein = less stool volume;
• Higher metabolizable energy = less food needed per body weight;
• Consumers increasingly value "less stool, better stool."

Per a September 2026 Beijing News report, digestibility has become a core criterion for domestic cat food buyers.

Two Testing Methods

Method 1: In Vitro Simulation

Principle: simulate the GI tract in test tubes —

• Step 1: gastric phase (pH 2, 39°C, pepsin);
• Step 2: intestinal phase (pH 6-7, pancreatin);
• Measure undigested residue weight.

Pros:

• Fast (days);
• Cheap (a few thousand yuan per sample);
• Good for formula screening.

Cons:

• In vitro digestibility is typically **higher than** in vivo (JAST 2026 showed in vitro DM/OM/CP/CF/EE significantly higher than in vivo, p<0.05);
• Cannot reflect individual variation, gut microbiome, feeding behavior.

Method 2: In Vivo Animal Trial

Principle: feed dogs/cats in metabolic cages, collect all feces —

• Feed known intake;
• Collect 5-7 days of feces;
• Measure fecal nutrients, calculate "intake - feces = digestion."

Pros:

• Closest to real conditions;
• AAFCO-recognized animal feeding trial.

Cons:

• Slow (2-4 weeks);
• Expensive (tens of thousands to hundreds of thousands per formula);
• Affected by individual animal variation.

How Much Do In Vitro and In Vivo Differ?

A 2026 JAST study compared two-step in vitro and in vivo digestibility across life stages in mixed-breed dogs:

| Metric | In vitro vs In vivo |

|---|---|

| Dry matter (DM) | In vitro significantly higher |

| Organic matter (OM) | In vitro significantly higher |

| Crude protein (CP) | In vitro significantly higher |

| Ether extract (EE) | In vitro significantly higher |

| Gross energy (GE) | In vitro higher except puppies |

**Key takeaway: in vitro numbers run several percentage points above actual in vivo.** A co-packer's 90% in vitro may be 85%-87% in vivo.

How to Read Co-Packer Data

1. Ask Which Method

• "Is this in vitro or in vivo?"
• If in vitro: "Do you have corresponding in vivo validation?"

2. Look at Conditions

• In vitro: how many steps? Pepsin/pancreatin dose? Temperature? Time?
• In vivo: how many dogs/cats? What breeds? How many days?

3. Look for Third-Party Reports

• Co-packer in-house lab data should be discounted;
• CMA/CNAS-accredited third-party reports are more credible;
• AAFCO animal feeding trial reports are the gold standard.

4. Don't Just Look at One Number

• 90% crude protein digestibility looks good, but if fat digestibility is 80%, something is off;
• Look at the full panel (DM, OM, CP, EE, GE), not just protein.

Practical Recommendations

| Stage | Recommended Test |

|---|---|

| Formula screening | In vitro, quickly filter low-digestibility formulas |

| Prototype validation | In vitro + small in vivo (10-20 animals) |

| Pre-launch finalization | AAFCO animal feeding trial, formal report |

| Per-batch outgoing | Routine nutrition only, no digestibility |

Bottom Line

Digestibility is not one number — it is a testing system. When a co-packer says 90%, ask: in vitro or in vivo? Under what conditions? Third-party or in-house? Use different methods at different stages, and don't let an in vitro number mislead consumers.

For brands needing digestibility validation, **Mixiaoqi pet food OEM/ODM** in Guangzhou supports in vitro simulation and small-batch animal trials from screening to finalization.

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FAQ

**Q: How much do in vitro and in vivo differ?**

A: Typically 3-5 percentage points, with in vitro higher, varying by metric.

**Q: How long does an AAFCO trial take?**

A: Typically 6 months (dogs) or 10 months (cats), covering a life stage.

**Q: Is a co-packer's 90% credible?**

A: Depends on method and source. In vitro 90% ≈ in vivo 85%-87%.