Transcript:

[0:03]

Hello, my name is Dr. Cara Cargo Froom, and today I’m going to be discussing capturing equine digestion in an in vitro protocol. So, what is in vitro? I think that’s the first important question we need to ask about today’s talk. In vitro is the simulation of what occurs in a living organism — whether that’s an animal or a microorganism — in a lab setting.

[0:30]

What we’re trying to do is apply physiological properties from an animal in a lab setting, absent of any animal use. A great example of in vitro that a lot of people experience every single day is in vitro fertilization. So in vitro doesn’t just apply to digestion — it can apply to many different areas of animal use or animal studies, because we as humans are animals, too. If we think about reproduction, we can do that naturally, or we can take it and flip it into a lab setting. The same goes for digestion.

[1:08]

What in vitro digestion is trying to do is simulate that in vivo digestion — digestion occurring in a live organism — in a lab setting. Why do we want to study digestibility and fermentability? Compared to other animal species, such as livestock production species like cattle, swine, and poultry, there’s very minimal understanding of the digestibility of even our most common feedstuffs in horses. While some data exists, it’s not nearly as robust as in other species.

[1:52]

Digestibility and fermentability values give us the amount of nutrient that’s actually available to the animal for absorption. Without these values, we only know what we’re offering the animal via the feed composition — for example, the amount of protein, fat, and minerals in an ingredient — but those nutrients are not always available for absorption. These values also tell us how efficiently a feed can be converted into an output, such as milk production, performance, or reproduction. Generally, a more digestible or fermentable feed is higher in quality because it can provide more nutrients, whereas a lower-quality feed requires more to be fed to meet the same nutrient needs.

[3:53]

Digestibility and fermentability are different. Digestibility refers to the breakdown of food by the animal’s own digestive juices and enzymes into smaller subunits for absorption. Fermentability, on the other hand, refers to the breakdown of certain nutrients — such as fiber — by the microbiome in the hindgut. Horses cannot digest fiber themselves, but microbes ferment it, releasing volatile fatty acids and B vitamins that the horse can use. The difference lies in how the nutrients are released: digestibility is via the animal’s enzymes, fermentability is via microbial action.

[5:20]

Before we talk about measuring these, let’s look at their benefits to the horse industry. With these values, nutritionists and horse owners can more accurately meet nutrient requirements. Not all feeds deliver the same nutrients or have the same digestibility and fermentability. By using these coefficients in diet formulation, we can better estimate the nutrients actually available for absorption, which helps avoid overfeeding or underfeeding. This can reduce risks such as obesity or nutrient deficiencies, and it can also be important for managing horses prone to colic, laminitis, or other health concerns.

[7:22]

More precise feeding also reduces the environmental footprint. Overfeeding nutrients results in excess waste — whether fecal or urinary — which adds up across many animals. Better matching of nutrient supply to requirements reduces this waste. Measuring digestibility and fermentability also helps us understand how feed processing and storage affect nutrient content. Processing or long storage can change nutrient quality, and by accounting for this, we can adjust formulations accordingly.

[9:13]

However, there are inherent difficulties when measuring these values in live animals. One big concern is animal welfare, which brings in the three Rs of research: Replacement, Reduction, and Refinement. Replacement asks if we can use another model, such as in vitro, instead of a live animal. Reduction asks if we can use fewer animals overall. Refinement asks if we can improve our process to eliminate invasive or painful procedures, or at least require fewer animals for them.

[10:08]

Animal welfare is a really large concern in our current world — we want animals to be treated in the best way possible. In addition to that, there have been more restrictions placed on animal use in research over the past couple of decades. We need to go through processes to have animal utilization approved by an animal care committee, and they have oversight on what procedures we are and are not allowed to perform on animals. By replacing animals with an in vitro — so an in-lab — system, we remove this animal welfare concern.

[10:44]

There’s also reduced control when we’re using an animal model versus an in-lab model. In the lab, we can closely control all of the parameters of our study — whether that’s the concentration of a solution we’re adding, the specific activity of enzymes, or even how long a food sits in the digestion chamber. This isn’t always controllable in animals: we cannot control gastrointestinal transit time, enzyme activity, hormone signaling and feedback, and other variables. An animal might become sick and need to be removed from the study. Sometimes we can’t get animals to eat, and if they won’t eat, we can’t measure digestion.

[11:44]

It’s also much more challenging to measure digestion in an animal compared to an in-lab setting. For example, there are many approaches to measure digestion in animals. We can use total tract digestion, which measures the difference between what’s eaten and the final fecal output. We can also measure ileal digestibility, which tracks digestion from ingestion to just before food enters the hindgut, sampled at the end of the small intestine. This method is quite invasive, requiring surgical insertion of a cannula — a tube from the small intestine to the outside of the body for sampling — which ties back to our welfare concern. It’s also more complex and costly, so we often revert to total tract digestion, which is less invasive but can be less accurate due to variations in digestion and fermentation.

[13:07]

Animal studies are also much longer than in-lab studies. When feeding an animal, we must allow their digestive tract to adapt to the new diet before taking samples, because diet composition influences enzyme secretion and can change the hindgut microbiome. For example, a high-fiber diet requires microbial adjustment to process more fiber; reducing fiber again causes adjustment due to less substrate for microorganisms. We usually allow at least 5–7 days for dietary adaptation before collecting samples, and sampling typically occurs over 2–3 days. This means evaluating one diet in animals can take 1–1.5 weeks.

[14:15]

In the lab, we can do this much more rapidly. Beakers don’t need to adapt to diets — we can add the substrate, digestive solutions, and enzymes immediately, begin digestion, and sample straight away. We don’t need multiple days of sampling to get a representative result. While an in-animal study may take a week or more for one ingredient or diet, an in-lab study can generate the same information in just a couple of days.

[14:57]

Before we talk about how we replicate digestion in the lab, it’s important to understand what digestion in the horse actually is. Every animal digests food into nutrients, but the steps to release or produce those nutrients differ. The first step of digestion actually starts in the mouth. The digestive tract is considered an open system — not technically inside the body — because it’s one continuous passage from start to end.

[15:48]

In the mouth, teeth break food into smaller particles. Smaller particles are more accessible for digestion because there’s greater surface area for digestive juices and enzymes. In addition, saliva is excreted in the mouth. Unlike many species, horses have very little starch digestion in the mouth due to much lower amylase activity than humans or pigs. Instead, saliva acts primarily as a buffer, mixing with food and aiding swallowing.

[16:39]

Once swallowing occurs, food is transported down the esophagus to the stomach via peristalsis — involuntary muscle movements that move food through the gastrointestinal tract. In the stomach, two major secretions occur: hydrochloric acid and the enzyme pepsin. Acid breaks food into smaller particles, while pepsin begins protein digestion. When food mixes with acid, stomach pH drops — generally to between 1 and 2 — which is optimal for pepsin activity.

[17:49]

The time food spends in the stomach can range from 15 minutes to 2 hours, depending on the amount and type of food fed.

[17:59]

Food then enters the small intestine, which is the major site of nutrient digestion and absorption. Remember, digestion is different from fermentation — fermentation occurs in another part of the digestive tract. In the small intestine, most proteins, carbohydrates, and fats are broken down into smaller subunits for absorption. It’s also a major site of mineral and vitamin absorption.

[18:31]

In the intestine, various digestive juices, secretions, and enzymes are released to continue breaking down food. The first secretion as food moves from the stomach into the intestine is bicarbonate. Unlike the stomach, enzymes in the small intestine function better at a more neutral pH. Bicarbonate, a basic solution, is secreted to raise pH from the acidic range to about 6.3–7.6, depending on the intestinal segment. This not only creates the optimal environment for enzymes to function but also protects the gastrointestinal lining from acid damage.

[19:50]

Most enzyme activity occurs in the small intestine. Enzyme groups target different nutrients: amylases break down carbohydrates (specifically starch, not fiber) into glucose, fructose, and galactose for absorption and energy use. A good example of the importance of enzymes is lactose intolerance in humans: lactose, a carbohydrate composed of galactose and glucose, cannot be absorbed intact. People lacking lactase (an enzyme) experience digestive upset because lactose remains unbroken down in the gut.

[21:58]

Other important enzymes include lipases, which break down fats, and proteases, which break down proteins. Through these enzymes’ action, nutrients can be absorbed and used for energy or other bodily functions.

[22:21]

Bile is another important secretion. Produced by the liver, bile is essential for fat digestion and absorption. Horses, unlike humans, dogs, and pigs, do not have a gallbladder, so bile is continuously trickled into the small intestine. This means when feeding a higher-fat diet, horses need a longer adaptation period to handle the increased fat intake.

[23:25]

Finally, hormones play an important role in digestion by acting as feedback and signaling loops. Some hormones trigger enzyme release, while others signal satiety. In in vitro systems, hormones are not included because there is no living organism to require brain signaling or feedback loops.

[24:28]

Once extensively digested in the small intestine, food moves into the hindgut — the cecum and colon. In the cecum, the microbiome (bacteria, yeast, protozoa) ferments fiber that the horse cannot digest itself, producing volatile fatty acids and B vitamins for absorption. The digesta then moves into the colon, where fermentation continues and some mineral and water absorption occurs, though to a lesser extent than in the small intestine.

[25:48]

Any remaining undigested material, along with waste products, moves into the rectum, where it collects until excreted as feces. Overfeeding nutrients can lead to excess nutrient waste in feces, contributing to environmental impacts.

[26:19]

Now that we’ve reviewed how digestion occurs in the horse, we can look at how to replicate it in an in vitro (in-lab) system. There are many approaches: static digestion methods (such as pH drop, pH stat, two-step digestion using pepsin and pancreatin, and two-step digestion with gut fermentation) and dynamic methods.

[27:12]

Dynamic methods more closely simulate the digestive tract than static methods, as they attempt to replicate processes like peristalsis, digesta mixing, pH control, and enzyme secretion. They use computer models to determine when to add enzymes, when to move food from one compartment to another, and when to adjust acid-base balance. This offers more control than static methods but still avoids live animal use.

[28:06]

However, with our dynamic systems comes more complexity as well as additional cost. In certain studies — especially human in vitro studies compared to in vivo studies done in rats — there’s a relatively high correlation between these two values for static methods. So, while dynamic methods try to best simulate in vivo digestion, static methods still have their place, are simpler to implement, and are often more cost-effective.

[28:38]

Because there are so many different methodologies for in vitro digestion, we’re going to focus on static methods and tie in those steps to their in vivo counterparts in horse digestion. The first method we’ll go over is pH drop digestion. This is one of the simpler in vitro methods to implement, because it doesn’t try to replicate different digestive compartments such as the stomach, small intestine, or hindgut. Instead, we measure the change in pH of a solution over time — starting from a basic pH and dropping to a lower pH.

[29:22]

We start with a food substrate in a solution at pH 8. We then add a specific enzyme mix to start digestion. After letting the mixture sit for about 10 minutes, we measure the drop in pH as nutrients are released during digestion. By plotting this change over time, we can use regression equations to calculate digestibility. In this case, we aren’t directly measuring nutrient intake versus nutrient output — we’re using the graph and its regression line to determine digestibility.

[30:25]

pH stat digestion is similar to pH drop in that it doesn’t replicate the animal’s digestive compartments. However, instead of monitoring pH decrease, we maintain pH at a set level — typically pH 8 — throughout digestion. This is done by titrating in a solution to keep pH constant while an enzyme solution digests the substrate. We then plot the amount of solution consumed to maintain that pH and use regression equations to determine digestibility.

[31:40]

Some in vitro systems aim to mimic different digestive compartments. One such method is the pepsin–pancreatin method, which simulates the stomach and small intestine. The gastric phase involves mixing hydrochloric acid and pepsin with the substrate to begin protein digestion. The intestinal phase begins by neutralizing the acidic solution — similar to bicarbonate action in the small intestine — then adding pancreatin (a cocktail of amylase, lipase, and protease) to continue enzymatic digestion. Digestibility is then determined by analyzing the digested substrate and comparing it to the original nutrient concentration.

[33:54]

The two-step digestion method also simulates gastric and small intestinal phases, but may differ from the pepsin–pancreatin method in the solutions used, enzyme cocktails, or digestion times.

[34:31]

We can also represent hindgut fermentation — the fermentation in the cecum and colon — in the lab. Unlike the previous methods, these do not use enzymes. Instead, samples from the cecum, feces, or both are taken to isolate specific bacteria and microorganisms into what’s called an inoculum. This inoculum is added to the substrate, mixed, and allowed to ferment for about 48 hours in a closed system. Gas production during fermentation is measured, since gas is a byproduct of microbial fermentation alongside volatile fatty acids and vitamins. This gas production data is then used in regression equations to calculate fermentability.

[36:04]

So we talked about regression equations for pH drop and pH stat, but we can also apply regression equations to determine fermentability. Now I’d like to address some of the advantages and limitations that come along with static methods. While there are benefits to using these methods, there’s only so much of digestion we can capture in them.

[36:22]

One of the first advantages of in vitro digestion is that we have no animal welfare concerns — no ethical concerns about using this approach. This means we don’t need animal use approval, and studies can begin as soon as you have the correct equipment, solutions, and feed ingredients. It’s also extremely reproducible — both within the same lab and across different labs — which is essential for reliability. We also have a high level of control, allowing us to monitor every parameter, pinpoint errors, and adjust procedures to understand how each factor influences the outcome.

[37:59]

Another advantage is that in vitro methods are rapid and relatively simple. With the proper equipment and chemicals, they’re easy to teach and much faster than in vivo trials, which require dietary adaptation, extended sampling, and longer analysis times. Most in vitro digestions take hours to a few days, rather than the weeks sometimes needed for animal studies. They’re also cost-effective since there are no expenses for feeding, housing, or caring for animals, which can be significant in large-scale research.

[39:22]

However, there are inherent limitations. One of the biggest is that we can’t capture the dynamic, ever-changing nature of digestion. Factors like gut transit time, enzyme activity, and nutrient secretion vary with diet, meal size, and other conditions — but in a static system, enzyme activities and incubation times are standardized. Static systems also don’t capture nutrient absorption; there’s no removal of nutrients from the system into circulation as happens in a living animal. Physiological feedback loops, such as hormonal regulation of digestion and satiety, are also absent.

[42:11]

Another limitation is that while we can use inocula to capture fermentation, we’re missing most of the gut microbiome — especially microorganisms from the small intestine, stomach, and mouth. The hindgut microbiota in in vitro models is also less diverse than in a live animal.

[42:55]

To ensure in vivo can be reliably replicated in vitro, we try to follow five steps: (1) use digestive enzymes in the correct sequence that occurs in the animal (e.g., pepsin in the stomach, not amylase or lipase); (2) match physiological conditions like temperature and pH to those of the animal; (3) replicate mixing to mimic peristalsis; (4) match gut transit times for each compartment; and (5) consider partial removal of digesta between compartments to somewhat mimic nutrient movement and absorption, even though we can’t fully replicate absorption in the lab.

[45:45]

So, I’ve thrown a lot of science at you now, but let’s talk about how this applies to the equine industry. How can we fit in vitro characterization, digestion, and fermentability of feedstuffs into the equine sector? I think the first thing to address is digestion, fermentation, and overall food degradation.

[46:14]

So digestion and fermentation fall under food degradability — how a food is broken down. One reason degradability is of interest is because it can help us better formulate diets, and we can also use this information as inputs for equine models. Dr. Emily Lehman talked about developing an equine model for digestion, and this is where my project fits into the bigger picture.

[46:48]

Feed degradability is important because many factors can influence it. Feed degradability can be affected by the feed characteristics and composition of the ingredient you offer a horse. Going further back, feed composition and characteristics themselves can be influenced by crop management, geographic origin (e.g., Canada, Europe), harvest and storage conditions, soil and nutrient composition, and plant variety. Even within the same plant species, varieties can differ greatly in nutrient composition — for example, kabuli vs. desi chickpeas vary in protein, minerals, and soluble fiber content.

[48:32]

As we discussed earlier, digestion and fermentation coefficients (feed degradability values) can be used directly in feed formulation or as inputs for computer models to predict digestion without invasive animal use. The overarching goal of this study is to support the development of a mechanistic model for the equine sector to advance equine nutrition. My project focuses specifically on in vitro evaluation of feedstuffs to determine digestibility, fermentability, and overall feed degradation.

[49:22]

The specific goals are to assess existing in vitro protocols for both equines and other species, select the most appropriate approach from the literature, and then apply that method to measure digestibility, fermentability, and degradation in both the foregut (stomach and small intestine) and hindgut. These results will serve as inputs for the equine digestion model.

[50:21]

For my study, I must consider: (1) the digestive physiology of the horse (e.g., reduced amylase activity, constant bile trickle, gut transit time); (2) acid–base balance (ensuring correct pH for each compartment); (3) enzyme type, activity, and timing of addition; and (4) how to represent the gut microbiome in a lab setting.

[51:44]

Sub-objectives include: first, reviewing the literature on in vitro digestion in equines and other species, and selecting the best approach to simulate gastric and small intestinal digestion — currently leaning toward a two-step or pepsin–pancreatin method. Next, we will apply that method to common North American horse feed ingredients to determine digestibility and degradation rates. Then, we will assess hindgut fermentation methods, choose or adapt one, and apply it to the same ingredients to measure fermentation and nutrient degradation.

[53:33]

Revisiting why this is important: understanding how digestible and fermentable an ingredient is can improve ration balancing, reduce environmental impact, deliver nutrients more effectively, account for disease management, and potentially reduce disease risk (e.g., laminitis). Using these values in digestion models can also help optimize performance — Dr. Emily Lehman’s work explains how modeling integrates these inputs into the bigger picture.

[54:41]

Achieving this project requires collaboration among experts from different backgrounds: nutrition and in vitro digestion (my role), animal modeling, computer science, and engineering — as well as industry partners who ensure the work meets stakeholder needs. By working with industry, we can make sure the outcomes are useful not just academically, but also for horse owners, trainers, and feeders.

[55:45]

I appreciate the time you’ve spent listening to me, and I hope you’ve learned something interesting — whether about digestion, applying it in a lab, or its relevance to the equine industry. If you have questions, please reach out. We’re always happy to discuss our research and future directions, and I look forward to updating you on the next steps and potential outcomes. Thank you.