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European Journal of Applied Sciences – Vol. 13, No. 1

Publication Date: February 25, 2025

DOI:10.14738/aivp.131.18377.

Swatland, H. J. (2025). Histochemistry of Anaerobic Glycolysis in Meat Animals. European Journal of Applied Sciences, Vol - 13(1).

465-472.

Services for Science and Education – United Kingdom

Histochemistry of Anaerobic Glycolysis in Meat Animals

H. J. Swatland

Department of Animal Biosciences, University of Guelph

ABSTRACT

This review explains how pH measurements of pork and beef are dominated by the

post mortem metabolism of one histochemical type of myofibre with alkaline

myosin ATPase (fast contraction) and few mitochondria (an anaerobic

specialization). Using glycogenolysis to identify the source of the lactic acid that

acidifies muscles post mortem, these myofibres are involved in a variety of

commercially important phenomena, from PSE (pale, soft, exudative) pork to

electrical stimulation of beef to increase productivity.

Keywords: Anaerobic glycolysis, Meat pH, Meat quality.

INTRODUCTION

When muscle is converted to meat, the sliding thick and thin myofilaments that used to power

muscle contraction become locked together in rigor mortis. Lacking oxygen from the

circulatory system, many myofibres start to obtain energy from their stored glycogen if they

have any, and they accumulate lactate because it cannot be taken to pyruvate to create acetyl

CoA without oxygen. Lactate spreads easily between myofibres. This acidifies the muscle, and

this decline in pH is one of the major factors determining meat quality.

There are two conventional ways to examine the acidification that anaerobic glycolysis creates

in meat. Most commonly, technicians push a glass or solid state pH sensitive electrode into the

meat. There is a lot of history here, from the Danish inventor of the pH scale, Søren Sørensen,

to Arnold Beckman’s electronics that have dominated the world markets for pH meters [1]. But

they all rely on a reference electrode somewhere, and meat does not always have an

appropriate conductivity between the calomel electrode (silver/potassium chloride) and the

reference electrode. Wet meat may give repeatable results, but dry meat may not. An older way

to measure acidification in meat was to emulsify a meat sample in a fluid, taking care to avoid

the problems of dilution and inactivating any further glycolysis [2].

Both methods integrate the activities of countless individual myofibres to produce an average.

But they do not reveal what is happening at the cellular level where different histochemical

types of myofibres may doing different things at different times depending on temperature,

neural activity, levels of stored glycogen and applied treatments such as electrical stimulation

[3].

In this review, the primary objective is to explain what may be happening at the cellular level

with anaerobic glycolysis in meat. There are a few situations where meat may be dominated by

a single histochemical type of myofibre in adult animals, but only after birth or hatching (as in

the case of superficial parts of chicken breast muscle). So, to fully understand anaerobic

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Services for Science and Education – United Kingdom 466

European Journal of Applied Sciences (EJAS) Vol. 13, Issue 1, February-2025

glycolysis in meat we must get down to the cellular level, and this requires histochemistry – a

combination of microscopic histology and biochemistry.

FROM WHOLE MUSCLES TO HISTOCHEMISTRY

Post mortem glycogenolysis in bovine myofibres was detected histologically by Robertson and

Baker in 1933 [4]. Soon after slaughter, Best’s carmine stained the glycogen between

myofibrils, but this was lost in later samples because of post mortem glycogenolysis. The

significance of this discovery was not obvious at the time – Nobel laureates Albert Szent-Györgi

and Hans Krebs were still busy discovering and proving the citric acid cycle, and there were no

pH meters capable of measuring meat pH directly.

Here is an informal introduction to set a background for a story to come. In the late 1800s,

muscle physiologists had tickled the surfaces of frog muscles with stimulatory electrodes and

found that myofibres with a red colour had slower and weaker contractile responses to pulling

up a weight attached to the whole muscle than pale myofibres. This started the whole

nomenclature of red and white myofibres.

This did not make much sense when applied to higher vertebrates, most of which have visually

red myofibres with some exceptions like the white myofibres of chicken breast muscle. A

further puzzle was that the powerful pale myofibres in frogs had small plate like neuromuscular

junctions while the slower redder myofibres had expanded grape like junctions.

The next jump forwards in understanding of this subject required the invention of cryostat

methods. Small strips of muscle were frozen in liquid nitrogen, trimmed while frozen, then

sectioned transversely while frozen in a cryostat (a refrigerated microtome). This enabled

some outstanding organic chemists to develop histochemical methods to reveal mitochondria

and fast and slow contracting myofibres based on their myosin ATPase activity. Thus, within

the visually red muscles of higher vertebrates was a mixture of histochemical types of

myofibres, where even the fast contracting fibres were red with some myoglobin. The visually

white muscles like chicken breast muscles had all fast contracting myofibres once they matured

after hatching. But all the myofibres in the major postural muscles of mammals had plate like

neuromuscular junctions. Thus, the major muscles of higher vertebrates like cattle, sheep and

pigs had no really slow myofibres like those in frogs. But there was still a differentiation

between myofibres with plate like neuromuscular junctions– some were specialized for fast,

powerful contractions while others were specialized for slower but more sustainable

contractions. All had some mitochondria and could store glycogen, but only the fast ones had a

high level of enzymes for anaerobic glycolysis, and only the slow ones stored triglyceride

droplets for sustainable aerobic activity.

This was an embarrassing time in biological science communication. Many researchers who

examined cryostat sections of skeletal muscle proposed their own nomenclature that their

competitors would not follow. The problem is still with us today, leaving the rest of us to

attempt a clarification of what histochemical types of myofibres we are writing about as we

attempt to explain the histochemistry of glycogenolysis in meat. Figure 1 shows a simple

nomenclature for histochemical types of bovine myofibres, following the oldest terminology of

red and white myofibres.

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Swatland, H. J. (2025). Histochemistry of Anaerobic Glycolysis in Meat Animals. European Journal of Applied Sciences, Vol - 13(1). 465-472.

URL: http://dx.doi.org/10.14738/aivp.131.18377

Fig. 1: A simple nomenclature for histochemical types of myofibres in beef. White myofibres

(w) are fast contracting with alkaline myofibrillar ATPase and few mitochondria while red

myofibres (r) are slow contracting with acid stable myofibrillar ATPase and many

mitochondria. All myofibres may have myoglobin, but it is strongest in red myofibres. There

are intermediate myofibres (i) with fast contraction and a medium content of mitochondria.

READING TRANSVERSE SECTIONS

In Figure 1 there are intermediate myofibres (i) that are in the process of changing their

histochemistry. Depending upon animal age and weight and the requirements for muscle

contraction, postural muscles get more red myofibers by conversion from white myofibers.

Other muscles may convert red to white myofibers to gain power [5]. In Fig. 1, there is a small

myofibre marked with an asterisk (*). This is a tapered ending of a myofibre embedded in

endomysial collagen. In adjacent serial sections it appeared as a normal diameter myofibre.

Thus, reading transverse sections of muscle to measure glycogen content is difficult. An easy

solution is to ignore intermediate myofibres and tapered endings because, at this plane of

sectioning, their glycogenolysis will not contribute very much to overall lactate production and

muscle pH.

MEASURING GLYCOGENOLYSIS

If identifying histochemical types myofibres is difficult, then measuring their glycogen content

as it disappears to lower muscle pH is a greater challenge. The basic problem is that the

glycogen in a rested animal immediately after slaughter is located in the sarcoplasm between

myofibrils. Measuring glycogen photometrically in myofibres is difficult because of the white

light transmitted through the myofibrils (distributional error in photometry). There are ways

around this problem by measuring at two wavelengths, one at the absorbance maximum of the

Schiff-reagent and one to assess the unabsorbed white light, then comparing the two

mathematically [6].