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2…Limits of Treatment of Pain in Animals
Pain Management in Companion Animals
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
V. Quartarone et al.
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
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.
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
Pain Management in Companion Animals
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
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
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.
Keywords Gilthead seabream
Total volatile basic nitrogen (TVBN)
Trimethylamine nitrogen (TMA-N) Skin Gills Shelf life
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
C. Boiti et al. (eds.), Trends in Veterinary Sciences,
DOI: 10.1007/978-3-642-36488-4_27, Ó Springer-Verlag Berlin Heidelberg 2013
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.
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
Increase of TVBN and TMA-N in Skin and Gills of Sparus aurata During Storage
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
A. Giuffrida et al.
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.
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
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
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
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
Actin Proteolysis in San Daniele
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.
Á Dry-cured ham Á Proteolysis
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
C. Boiti et al. (eds.), Trends in Veterinary Sciences,
DOI: 10.1007/978-3-642-36488-4_28, Ó Springer-Verlag Berlin Heidelberg 2013
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
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.
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
Actin Proteolysis in San Daniele Dry-Cured Ham
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.
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’’.
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
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