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2…Limits of Treatment of Pain in Animals

2…Limits of Treatment of Pain in Animals

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26



Pain Management in Companion Animals



143



It can be difficult to recognize the presence of pain in animals. They cannot

verbalize, and at present there are no objective methods to demonstrate and

quantify the presence of pain. Changes in physiological, metabolic, and neuroendocrine parameters are not just indicative of pain but are also associated with

anxiety and stress. In veterinary medicine, it is preferable to assess behavioral

responses to pain. It requires a good knowledge of the behavioral repertoire of the

species, as well as the individual, in response to pain. In particular, the relief of

abnormal behavior patterns is considered particularly useful and meaningful in this

respect. It can also be useful to estimate the potential level of pain that an animal

may have, following a surgery or a disease, and to apply ‘‘pain scales.’’ These

systems all have limits and it is often necessary to apply various diagnostic

methods in combination, as part of a careful clinical evaluation of the patient.

Analgesic therapy cannot be predetermined, but must be adapted to the type of

pain, the animal species, the individual response to the drug, and to the health

status of the patient. In addition to the classes of analgesics commonly used to

combat acute pain (opioids, NSAIDs, local anesthetics, b2-agonists, and tramadol), we must also consider other molecules (ketamine, anticonvulsants,

antidepressants, GABA-mimetics, etc.), and nonpharmacological approaches (e.g.,

technical and physical rehabilitation, acupuncture, etc.) which can be applied and

combined as part of a multimodal analgesic protocol.

Medication side effects and drug interactions have to be taken into account,

while remembering that the clinical consequences of pain, whether acute or

chronic, are still often more severe than complications due to treatments.



26.3 Medical–Legal Considerations

In recent years, animals have been assigned important emotional, social, and

supportive roles and this has allowed the conception of an animal as ‘‘res’’ (Article

812 of Italian Civil Code) (Passantino 2008) and the recognition, even from a legal

point of view, of its nature as alive, sentient, and able to experience suffering and

pain (EC 2007).

For this reason, many researchers studied animals’ sensitivity and ability to feel

pain (Bianchi et al. 2003; della Rocca and Di Salvo 2008; della Rocca et al. 2009).

Such studies have shown that animals feel pain, as they have all of the anatomic

and functional components necessary for the perception of painful stimuli as both

reflex stimuli and conscious stimuli. It was also shown that fish are sentient and are

capable of feeling pain (Algers et al. 2009). In some fish species, two types of

nociceptors (A-delta and C) were identified to be responsible for the transduction

and conduction of painful stimuli (Braithwaite and Boulcott 2007; Broom 2007;

Hastein 2008; Sneddon 2002; Sneddon et al. 2003). The sensitivity of pet owners

to the suffering of their animals has inspired progress in this area, putting pain

control at the forefront of ‘‘compassionate care’’ (della Rocca and Di Salvo 2008;

Ogilvie 2004). The treatment of pain should be an integral and inalienable part of



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the therapeutic process in the animal patient as it is in the human patient, and must

take into consideration the value and the dignity of the animal.

There is no community or national legislation defining proper conduct of the

veterinarian in the treatment of pain in animals or providing guidelines regarding

the diagnosis or treatment of pain.

At present, the only legal regulations related to pain therapy, concerning narcotic drugs and their storage, are in reference to humans (Anon 1990, 2001, 2006).

Moreover, our country has recently issued a regulation (Anon 2010) relating to

palliative care and pain therapy in the human field, providing an obligation to

report pain level (Article 7 of Law no. 38/2010), the applied analgesic technique,

drugs used, dosages, and the obtained results in the patient’s medical records. This

regulation also considers the simplification of prescribing drugs for pain relief

(Article 10 of Law no. 38/2010).

The same has not been determined for veterinary medicine. These deficiencies

are reflected in clinical practice, where pain management is often superficial or

lacking.

It is, therefore, necessary for the legislation to at least propose a measure aimed

at regulating the use of analgesic drugs in veterinary medicine. It would also be

desirable to formulate guidelines on the minimum requirements in the practice of

pain management, which would aim to standardize medical conduct. These recommendations could be met, modified, or rejected depending on the clinical or

surgical needs of the animal patient, but each case would be supported by an

updated tool, utilizing data from the current literature, and the synthesis of expert

opinion and clinical practice.

In the absence of a legal act, the veterinarian must always make choices

respecting the life and health of the patient, and evaluating the relationship

between costs and benefits, as determined by the code of ethics. It is, therefore,

important to keep the animal’s life free of pain and stress as much as possible. This

objective can be achieved only by the veterinary surgeon who possesses the

appropriate knowledge to prevent and stop suffering.



References

Algers B, Blokhuis HJ, Bøtner A, Broom DM et al (2009) General approach to fish welfare and to

the concept of sentience in fish. EFSA J 954:1–27

Anon (1990). D.P.R. 9 ottobre 1990, n. 309. G.U. 31 ottobre 1990, n. 255—S.O. n. 67

Anon (2001). Legge 8 febbraio 2001, n.12. G.U. 19 febbraio 2001, n 41

Anon (2006). Legge 21 febbraio 2006, n. 49. G.U. 27 febbraio 2006, S.O. n 45

Anon (2010). Legge 15 marzo 2010, n. 38. G.U. 19 marzo 2010, Serie Generale n 65

Bianchi E, Leonardi L, Breghi G, Melanie P (2003). Le scale del dolore come ausilio

nell’interpretazione dello stato algico nel cane. Annali Della Facoltà di Medicina Veterinaria

di, vol LVI. Pisa, pp 267–277

Braithwaite VA, Boulcott P (2007) Pain perception, aversion and fear in fish. Dis Aquat

Organisms 75:131–138



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Broom DM (2007) Cognitive ability and sentience: which aquatic animals should be protected.

Dis Aquat Organisms 75(2):99–108

Della Rocca G, Di Salvo A (2008) Il dolore negli animali: perché è importante trattarlo

Patogenesi e conseguenze cliniche del dolore patologico. Parte 2. Bollettino AIVPA 1:29–35

Della Rocca G, Olivieri E, Di Salvo A, Gogny M (2009) Studio epidemiologico sulla attitudine

dei medici veterinari alla gestione del dolore negli animali da compagnia. Bollettino AIVPA

2:15–21

European Community (2007) Treaty of lisbon amending the treaty on european union and the

treaty establishing the european community, signed at lisbon. Offi J C 306, pp. 1–271

Hastein T (2008) Welfare of fish in aquaculture. Bulletin OIE 2:8–10

Ogilvie GK (2004). Fulfilling the first commandment: Providing analgesia and compassionate

care. In: Proceedings of the 29th World Small Animal Veterinary Congress-WSAVA, October

6–9, Rhodes, pp 30–37

Passantino A (2008) Non-domesticated animals kept for companionship: an overview of the

regulatory requirements in Italy to address animal welfare and human safety concerns. Eur J

Companion Anim Pract 18(2):119–126

Sneddon LU (2002) Anatomical and electrophysiological analysis of the trigeminal nerve in a

teleost fish, Oncorhynchus mykiss. Neurosci Lett 319:167–171

Sneddon LU, Braithwaite VA, Gentle MJ (2003) Do fish have nociceptors? Evidence for the

evolution of a vertebrate sensory system. Proc R Soc London 270:1115–1121



Part IV



Food Inspection



Chapter 27



Increase of TVBN and TMA-N in Skin

and Gills of Sparus aurata During Storage

A. Giuffrida, F. Giarratana, D. Signorino, G. Ziino

and A. Panebianco



Abstract The aim of this work was to assess the increase of total volatile basic

nitrogen (TVBN) and trimethylamine nitrogen (TMA-N) in the skin, gills and

muscle of gilthead seabream (Sparus aurata) during refrigerated storage and to

relate these increases to sensorial scores and spoilage bacteria growth. TVBN and

TMA-N increases in skin and gills were more correlated to the sensorial scores

obtained by the quality index method and to bacterial growth, in comparison to

TVBN and TMA-N of muscle. Since the bacterial load of muscle is very low until

the 168th hour of storage, according to the obtained results, measurement of

TVBN and TMA-N of skin and gills could prove useful for the assessment of the

shelf life of gilthead seabream.



Á



Keywords Gilthead seabream

Total volatile basic nitrogen (TVBN)

Trimethylamine nitrogen (TMA-N) Skin Gills Shelf life



Á



Á



Á



Á



27.1 Introduction

The shelf life of fish is due to several intrinsic and extrinsic factors that can

influence enzyme activity and the behaviour of spoilage bacteria. Some authors

(Gram and Dalgaard 2002) have emphasised the importance of certain bacteria

called specific spoilage organisms (SSO), including Pseudomonas spp., Shewanella putrefaciens and Photobacterium phosphoreum, which are able to exploit

specific metabolites of fish muscle for their growth. Particularly, these bacteria are

able to reduce trimethylamine oxide (TMA-O) into trimethylamine nitrogen

(TMA-N), which causes the specific odour of spoiled fish. The increase of total

A. Giuffrida (&) Á F. Giarratana Á D. Signorino Á G. Ziino Á A. Panebianco

Dipartimento Sanità Pubblica Veterinaria, Università di Messina-Polo

Universitario Annunziata, Messina, Italy

e-mail: agiuffrida@unime.it



C. Boiti et al. (eds.), Trends in Veterinary Sciences,

DOI: 10.1007/978-3-642-36488-4_27, Ó Springer-Verlag Berlin Heidelberg 2013



149



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A. Giuffrida et al.



volatile basic nitrogen (TVBN) during fish storage is related to the activity of SSO

against muscle proteins and for this reason TVBN is considered as a good indicator

of fish spoilage, especially for certain species.

Several studies have been carried out to assess the relationships between SSO

behaviour, sensorial parameters and chemical indicators such as TMA-N and

TVBN, especially for species widely exploited in aquaculture as Sparus aurata

(Giuffrida et al. 2005; Huidobro et al. 2000; Koutsoumanis and Nychas 2000;

Lougovois et al. 2003; Marrone et al. 2007). In this species, however, the increase

of TVBN and TMA-N during refrigerated storage did not always correlate to

sensorial decay (quality index method, QIM and score) or to the growth of SSO. It

is well-known that the bacterial load of fish muscle can be very low for several

days of refrigerated storage, especially in fish with thick skin like gilthead seabream. Conversely, skin and gills normally have a high bacterial load that affects

sensorial parameters early. These aspects could explain the low correlation

between muscle chemical indicators and sensorial or microbiological parameters.

Thus, the aim of this work was to study the increase of TVBN and TMA-N in the

skin and gills of specimens of Sparus aurata during refrigerated storage, and at the

same time to evaluate the relationship between these chemical indicators, spoilage

flora and the QIM scores.



27.2 Materials and Methods

This study was carried out using 21 specimens of Sparus aurata obtained from a

Sicilian aquaculture plant. Fish, after harvest, were freighted to the laboratory

within 2 h and submitted to analytical and sensorial evaluation. The former was

carried out after 0, 120, 168, 216 and 288 h of storage at 6 °C and included the

sterile sampling of gills, skin and muscle from three specimens. These samples

were cultured onto Levine Iron Agar at 25 °C for 72 h, after 10-fold dilutions in

peptone water. An aliquot of each subsample was used for the determination of

TVBN and TMA-N by the microdiffusion method in Conway cells, according to

Mahmud et al. (2007). The sensorial evaluation was carried out at the same time

intervals using the quality index method (Huidobro et al. 2000). Microbial,

chemical and sensorial data were statistically evaluated by linear regression tests.



27.3 Results

Figure 27.1a shows the spoilage bacteria trends on skin, gills and muscle during

storage. Of note is the quick growth on skin and gills and the slight growth in

muscle until the end of storage, when the fish were considered completely spoiled.

The partial (QIMskin-eye, QIMgills and QIMmuscle) and total scores of QIM

(Fig. 27.1b) appear in agreement to the bacterial trends, showing a rejection

sensorial point when the concentration of the spoilage flora reaches values [ Log



27



Increase of TVBN and TMA-N in Skin and Gills of Sparus aurata During Storage



151



Fig. 27.1 Trends of SSO (a), QIM (b), TVBN (c) and TMA-N (d) in skin (filled circle), gills

(filled squre) and muscle (filled triangle) during storage. The QIM total scores (b) are indicated

by the star symbol



7 cfu/g. Figure 27.1c and d shows TVBN and TMA-N trends for skin, gills and

muscle during storage. The concentration of muscle TVBN almost remained

constant until the 166th hour, while the values for skin and gills progressively

increased along the entire storage period. Concerning TMA-N values (Fig. 27.1d),

only in the gills did the increase appear to correlate with prolonged storage period;

on the contrary, the muscle values had a fluctuating trend, and the skin values

showed a moderate increase. All the above-mentioned aspects are confirmed by

regression tests which are summarised in Table 27.1.



Table 27.1 Coefficient of determination R2 for TVBN and TMA-N versus QIM scores and SSO

counts

TVBN

TVBN

TVBN

TMA-N

TMA-N

TMA-N

gills

skin

muscle

gills

skin

muscle

QIM

SSO gills

SSO skin

SSO

muscle



0.8404

0.6564







0.8797



0.6462





0.6985





0.4686



0.9479

0.8452







0.8714



0.9297





0.6439





0.5221



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A. Giuffrida et al.



27.4 Discussion

These results confirm the low significance of muscle TVBN and TMA-N values to

assess the shelf life of gilthead seabream, according to Koutsoumanis and Nychas

(2000) and Marrone et al. (2007). Conversely, these chemical indicators on gills

and skin could be useful as their increase appeared correlated to the storage

duration. These aspects could be due to the high level of bacterial contamination in

these sites, and at the same time to the accumulation on skin and gills of several

mucoproteins and excretion compounds. It is known that, for some species, the

potential accumulation of TMA-O on skin and gills is to regulate hydro-osmotic

homeostasis (Wood 1993). Therefore, our results appear worthy of further investigations, especially in order to utilise a more correct application of Regulations

853/2003 and 2074/2005 EC concerning the use of these chemical indicators.



References

Giuffrida A, Ziino G, Pennisi L, Panebianco A, Donato G (2005) Valutazioni comparative sulla

conservabilità di Sparidi allevati. Ind Alim 44:381–386

Gram L, Dalgaard P (2002) Fish spoilage bacteria-problems and solution. Current Op in Biotec

13:262–266

Huidobro A, Pastor A, Tejada M (2000) Quality index method developed for Raw Gilthead

Seabream (Sparus aurata). J Food Sci 65:1202–1205

Koutsoumanis K, Nychas GJE (2000) Application of a systematic experimental procedure to

develop a microbial model for rapid fish shelf life predictions. Int J of Food Microbiol

60:171–184

Lougovois VP, Kyranas ER, Kyrana VR (2003) Comparison of selected methods of assessing

freshness quality and remaining storage life of iced gilthead sea bream (Sparus aurata). Food

Res Int 36:551–560

Mahmud MM, Hossain MA, Jahan I, Banerjee P, Rahaman MA (2007) Effect of delayed icing on

the quality characteristics of Bagda (Penaeus monodon fabricius, 1798). Int J Sustain Crop

Prod 2:24–30

Marrone R, Pennisi L, Colarusso G, Ghedini M, Ianieri A, Anastasio A (2007) Shelf-life di orate

(Sparus aurata) provenienti da allevamenti off-shore e confezionate in atmosfera protettiva.

Atti AIVI 17:194–198

Wood CM (1993) Ammonia and urea metabolism and excretion. In: Evans DH (ed) The

rhysiology of fishes. CRC Press, Florida, pp 379–425



Chapter 28



Actin Proteolysis in San Daniele

Dry-Cured Ham

M. L. Stecchini, A. Fabbro, M. Spaziani, E. Venir and G. Lippe



Abstract The aim of this work was to define the actin degradation pattern during

the production of dry-cured San Daniele ham, as a factor that could influence its

ripening and sensory characteristics. Biceps femoris muscle samples from San

Daniele hams were subjected to denaturing and reducing conditions and onedimensional sodium dodecyl sulphate–polyacrylamide gel electrophoresis followed

by immuno-detection analysis. The degradation of actin was not extensive and was

evident only after the salting stage, which could have labilized protein interactions in

the myofibrillar structure. This limited proteolysis may be associated with the

inaccessibility of the actin molecule to proteolytic enzymes.

Keywords Actin



Á Dry-cured ham Á Proteolysis



28.1 Introduction

The production of dry-cured ham requires a long processing time and is associated

with intense proteolytic activity on the myofibrillar as well as on the sarcoplasmic

proteins, resulting in their progressive degradation. Due to their low stability, the

cytosolic endopeptidases, calpains, poorly contribute to protein degradation.

In contrast, cathepsins significantly sustain proteolysis, but their activities gradually decrease throughout processing (Toldrà and Etherington 1988). A residual

activity of only 5–10 % for cathepsins B, H, and L has been reported at 15 months

of processing (Toldrà et al. 1993). The initial breakdown of muscle proteins by

endopeptidases is followed by the action of exopeptidases, giving rise to small

peptides and free amino acids. These final products can contribute directly or

M. L.Stecchini (&) Á A. Fabbro Á M. Spaziani Á E. Venir Á G. Lippe

Department of Food Science, University of Udine, Udine, Italy

e-mail: mara.stecchinini@uniud.it



C. Boiti et al. (eds.), Trends in Veterinary Sciences,

DOI: 10.1007/978-3-642-36488-4_28, Ó Springer-Verlag Berlin Heidelberg 2013



153



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M. L. Stecchini et al.



indirectly, as precursors of other compounds (keto acids, amines, aldehydes,

methyl ketones, etc.), to flavor development in dry-cured ham (Hinrichsen and

Pedersen 1994). On the other hand, excessive proteolysis could be responsible for

the appearance of defects (softness and bitter flavor) and might be avoided by

removing raw materials with high proteolytic activity, as suggested for Parma ham

(Schivazappa et al. 2002). Structural modifications also associated with proteolysis

are essential in determining the typical texture of dry-cured ham and contributing

to flavor perception (Larrea et al. 2007).

A number of studies have been published on the use of nonprotein nitrogen

fraction as an indicator of dry-cured ham proteolysis. More recently, proteomics

has been used to study the evolution of myofibrillar and sarcoplasmic protein

hydrolysis (Di Luccia et al. 2005; Picariello et al. 2006). This latter approach is

useful for identifying molecular markers to predict and discriminate quality

characteristics. In a recent proteomic study on Slovenian dry-cured hams (Kraški

pršut) (Škrlep et al. 2011), the sensory defects found were related to the degradation patterns of proteins, including actin.

This study aimed to investigate the degradation of actin during processing of

San Daniele dry-cured ham, providing a characterization of the process with

respect to myofibrillar protein fragmentation, which could influence the final

sensory characteristics.



28.2 Materials and Methods

Hams were obtained from Large White 9 Landrace pigs, suitable for the protected

designation of origin (PDO) Prosciutto di San Daniele. The internal Biceps femoris

muscles, characterized by low salt penetration, were excised from the hams and

analyzed at crucial steps of the ham-curing process (T1 = out of salting,

14–18 days; T2 = introduction to resting, 35 days; T3 = after washing and drying, 117 days; T4 = after greasing, 211–221 days; T5 = end of curing, 413 days).

The protein extraction was carried out in a sodium dodecyl sulfate (SDS)

solution containing 100 mM dithiothreitol (DTT) to optimize protein recovery.

Extracts were analyzed using one-dimensional denaturing electrophoresis (SDSPAGE) for the separation of proteins between 10 and 100 kDa. Western blotting

was then run with anti-actin antibody (Sigma-Aldrich) and immunoreactive bands

were detected by chemiluminescence.



28.3 Results

In all samples, the presence of intact actin with an apparent molecular weight of

42 kDa was observed. Dry-cured ham processing gave rise to a limited degradation of this myofibrillar protein. From the drying phase, fragments around 29 and



28



Actin Proteolysis in San Daniele Dry-Cured Ham



155



22 kDa were detected using anti-actin antibody. After 210–220 days, a new

fragment of 38 kDa appeared and remained intact to the end of the curing, along

with the 29- and 22-kDa fragments.



28.4 Discussion

Despite the number of papers published on dry-cured ham proteolysis, the research

has only recently focused on actin proteolysis. The presence of intact actin was

restricted to the first 6 months of the Italian dry-cured ham process. Actin then

seemed to disappear after 10 months of processing (Di Luccia et al. 2005). Such a

complete hydrolysis of actin was not observed in Spanish dry-cured hams (Teruel),

where actin appeared to decrease less in the Semimembranosus muscle than in the

Biceps femoris (Larrea et al. 2006). Small (1502–1971 Da) peptide fragments of

actin have been identified at the end of Serrano dry-cured ham processing, supporting the relevance of action of cathepsin D (Sentandreu et al. 2007). Cathepsin

D may remain active during a large part of the processing period, although a

reduction is expected with increasing NaCl concentrations (Sarraga et al. 1993).

We hypothesize that actin degradation in San Daniele ham, which seems to be

similar to the degradation of myosin previously reported (Spaziani et al. 2009),

may occur because of weaker myofibrillar-protein electrostatic interactions

induced by modifications of ionic strength due to salt penetration during processing. This would explain the lag between the salting step and the appearance of

proteolytic fragments, which are probably generated by cathepsin activities.

Acknowledgments This research was supported by a grant from ‘‘Legge regionale L.R. 26/2005

articolo 23: Innovazione e Ottimizzazione nella Filiera del Prosciutto Crudo Tipico’’.



References

Di Luccia A, Picariello G, Cacace G, Scaloni A, Faccia M, Liuzzi V, Alviti G, Spagna Musso S

(2005) Proteomic analysis of water soluble and myofibrillar protein changes occurring in drycured hams. Meat Sci 69:479–491

Hinrichsen LL, Pedersen SB (1994) Relationship among flavor, volatile compounds, chemical

changes and microflora in Italian-type dry-cured ham during processing. J Sci Food Agric

43:2932–2940

Larrea V, Hernando I, Quiles A, Lluch MA, Pérez-Munuera I (2006) Changes in proteins during

Teruel dry-cured ham processing. Meat Sci 74:586–593

Larrea V, Pérez-Munuera I, Hernando I, Quiles A, Llorca E, Lluch MA (2007) Microstructural

changes in Teruel dry-cured ham during processing. Meat Sci 76:574–582

Picariello G, De Martino A, Mamone G, Ferranti P, Addeo F, Faccia M, Spagna Musso S, Di

Luccia A (2006) Proteomic study of muscle sarcoplasmic proteins using AUT-PAGE/SDSPAGE as two-dimensional gel electrophoresis. J Chromatogr B 833:101–108



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