Trends in veterinary sciences current aspects in veterinary morphophysiology biochemistry animal production food hygiene and clinical sciences

Bertotto 2 Effect of Altitude on Plasma Serotonin Levels in Horses.. An effect of sea-sonality was observed for all parameters, with generally higher values in summerand lower values in

Trends in

Veterinary

Sciences

Cristiano Boiti · Adriana Ferlazzo

Alberto Gaiti · Antonio Pugliese Editors

Current Aspects in Veterinary

Morphophysiology, Biochemistry,

Animal Production, Food Hygiene

and Clinical Sciences

Trends in Veterinary Sciences

Cristiano Boiti • Adriana Ferlazzo

Alberto Gaiti • Antonio Pugliese

Editors

Trends in Veterinary Sciences

Current Aspects in Veterinary

Morphophysiology, Biochemistry, Animal Production, Food Hygiene and Clinical Sciences

LXV Annual Meeting of The Italian Society for Veterinary Sciences Tropea-Drapia 2011 Selected Papers

123

Cristiano Boiti

Dipartimento di Scienze Biopatologiche

Università degli Studi di Perugia

ItalyAntonio PuglieseDipartimento Scienze veterinarieUniversità degli Studi di MessinaMessina

Italy

DOI 10.1007/978-3-642-36488-4

Springer Heidelberg New York Dordrecht London

Library of Congress Control Number: 2013937093

Ó Springer-Verlag Berlin Heidelberg 2013

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Part I Biology and Reproduction

1 Seasonal Effect on Hematological and Innate Immune

Parameters in Sea Bass (Dicentrarchus labrax) 3Francesco Pascoli, E Negrato, C Poltronieri,

G Radaelli and D Bertotto

2 Effect of Altitude on Plasma Serotonin Levels in Horses 9

G Bruschetta, P Di Pietro, M Miano,

C Cravana and A M Ferlazzo

3 Identification of Aquaporin 1 in Diplodus sargus 15

G Zanghì, S Campo, A D’Ascola, A Germanà

and A M Ferlazzo

4 Effect of Dephosphorylation on Donkey Milk Caseins 21

S Vincenzetti, A Vita, F M Carpi, D Micozzi and P Polidori

5 Distribution Pattern and Chemical Coding of Sympathetic

Trunk Ganglia Neurons Supplying the Boar

Urinary Bladder Trigone 27

F Gazza, M Botti, L Ragionieri, C Sorteni, D Russo,

P Clavenzani, R Chiocchetti, L Bo Minelli and R Panu

6 In Vivo Applications of Mesenchymal Stem Cells

and Platelet-Rich Plasma to Improve Tendon

Regeneration in Sheep 31

M Patruno, I Bronzini, L Maccatrozzo, A Perazzi, I Iacopetti,

G M De Benedictis, S Testoni, A Negro, F Mascarello

and T Martinello

v

7 Plasma Fatty Acid Profiles During the First Year in Dogs

with and without Hip Dysplasia: Preliminary Results 35

L Tidu, N Bacciu, G Rucco, S Nardi, M Santoro

and B Renaville

8 Signaling in Sperm Activation: A Common Strategy

for Different Organisms 41

I Saponaro, N Bernabò and M Mattioli

9 Tenogenic Differentiation of Ovine Amniotic Stem Cells

Co-Cultured with Tenocytes 45Valentina Curini, V Russo, O Di Giacinto, A Mauro, E Galiffa,

A Pomante and B Barboni

10 Cortisol Changes in Pregnant and Post-Partum Ewes:

Effects of Single or Twin Births 51

E Fazio, M Manera, S Mignacca, P Medica and A Ferlazzo

Part II Animal Pathology

11 Papillary and Chordoid Meningioma in the Dog:

Morphological Findings and Histological Grading 57

S Pavone and M T Mandara

12 Detection of Neutralizing Antibodies in Pigs Inoculated

with an Inactivated Vaccine Against Porcine

Circovirus Type 2 (PCV2) 63

S Petrini, M Paniccià, V Silenzi, F Ciuti, M Bresaola,

M Fortunati, G M De Mia, G Perugini and M Ferrari

13 Mycobacterium avium subsp paratuberculosis as an

Emergent Pathogen in Raw Ovine Milk Produced

in Central Italy 67

A R Attili, V Ngu Ngwa, L Pacifici, S Preziuso,

A Domesi and V Cuteri

14 Canine Filariosis in Sardinia: Epidemiological Findings

in the Ogliastra Region 73

A Scala, C Solinas, A P Pipia, G Sanna,

A Varcasia and G Tosciri

15 Comparison of Serum and Meat Juice for Detection

of Anti-Toxoplasma gondii Antibodies in Hunted

Wild Boars (Sus scrofa) 79

D Ranucci, F Veronesi, I Di Matteo, R Branciari,

D Miraglia, C Marini and D Piergili Fioretti

16 Eucoleus aerophilus (syn Capillaria aerophila) and Other

Trichinelloid Nematodes in Dogs from Liguria

(Northwest Italy) 85

F Macchioni, L Guardone, M C Prati and M Magi

17 Helminths in Sheep on Farms of the Basilicata Region

of Southern Italy 91

A Bosco, L Rinaldi, V Musella, D Pintus, M Santaniello,

M E Morgoglione, G Zacometti and G Cringoli

Part III Pharmacology and Clinical Science

18 Effects of Veterinary Drugs on Swimming Activity

in Two Freshwater Organisms 97

M Dalla Bona, V Di Leva and M De Liguoro

19 Interdisciplinary Evaluation of Toxicity in Ostreopsis Ovata:

Algal Biotoxins 103

A Ferrari, I Schiavetti, C Bolognesi, D Pavino and B Vivaldi

20 Aflatoxin M1Contamination and Antibacterial Residues

in Milk in Kosovo 109

G Gallina, A Rama, L Lucatello, C Benetti, D Bajraktari,

K Uka and C Montesissa

21 Heavy Metal Levels in Dog Liver and Kidney in Naples

(Campania, Italy) 115

F P Serpe, R Russo, R De Luna, S Florio,

M Esposito and L Severino

22 Ultrasonographic Assessment of Abdominal Lymph Nodes

in Normal Puppies: Preliminary Results 119

A La Pietra and M De Majo

23 Changes in the Metabolic Profile and Performance of Dairy

Cows Fed Two Dietary Crude Protein Concentrations 125

D Bernardini, S Segato, G Marchesini, A L Stefani,

G Gerardi and I Andrighetto

24 Impact of Physical Exercise on Release of Cardiac Troponins:

Evaluation in Healthy and Cardiopathic Dogs 129

M Pugliese, A Seminara, M De Majo, A La Pietra

and P P Niutta

25 Canine Erythrocyte Morphology: Observations

of a New Pattern, the ‘‘Quatrefoil’’ Erythrocyte 135George Lubas, Alessandra Gavazza, Biancaurora Gugliucci,

Anna Pasquini and Marianna Ricci

26 Pain Management in Companion Animals:

Medical–Legal Aspects 141

V Quartarone, A Fazio, G della Rocca, M Russo

and A Passantino

Part IV Food Inspection

27 Increase of TVBN and TMA-N in Skin and Gills

of Sparus aurata During Storage 149

A Giuffrida, F Giarratana, D Signorino, G Ziino

and A Panebianco

28 Actin Proteolysis in San Daniele Dry-Cured Ham 153

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

Part V Husbandry and Zootechnic

29 The Donkey Milk Food Chain: Quality and Certification 159Stefano Simonella, Cristina Panetta and Biagina Chiofalo

30 Effect of Different Rates of Postmortem pH Decline

on the Technological Quality of Calabrian Capocollo 165

L Nanni Costa, F Tassone, S Dall’Olio, S Carpino and V Russo

31 Preliminary Investigation of the Incidence of Obesity

in a Canine Population in the USA 171

G Biagi, I Cipollini, M Grandi, D Sarti and G Zaghini

32 Administration of Essential Oils Cinnamaldehyde, Eugenol,

and Capsicum to Beef Cattle: Effects on Health Status

and Growth Performance 177

R Compiani, C A Sgoifo Rossi, A Pizzi and V Dell’Orto

33 Extruded Linseed in the Diet of Grazing Goats:

Effects on Milk Conjugated Linoleic Acid 181Raffaella Tudisco, S Calabrò, M I Cutrignelli, M Grossi,

N Musco, V Piccolo and F Infascelli

Index 187

Part I

Biology and Reproduction

Chapter 1

Seasonal Effect on Hematological

and Innate Immune Parameters in Sea

Bass (Dicentrarchus labrax)

Francesco Pascoli, E Negrato, C Poltronieri, G Radaelli

and D Bertotto

Abstract The temperate aquatic environment is affected by two primary seasonalcomponents, temperature and photoperiod Many organisms respond to seasonalchange physiologically, behaviorally or both The aim of this study was toinvestigate the effect of seasonality on cortisol, hematological, and innate immuneparameters in European sea bass (Dicentrarchus labrax) reared under traditionalsemi-intensive aquaculture Sea bass were reared in an outdoor pond.Serum cortisol, hematocrit, leucocrit, serum lysozyme activity, and totalglutathione (GSH) were monitored bimonthly for 14 months An effect of sea-sonality was observed for all parameters, with generally higher values in summerand lower values in winter These results could improve the understanding of theinfluence of seasonal cues on the immune system and the stress response in fish, tooptimize husbandry practices

Keywords Fish
Innate immunity
Cortisol
Hematology

1.1 Introduction

In the literature, there are numerous studies on the influence of seasonality on fishphysiology The temperate aquatic environment is influenced throughout the year

by two main seasonal cues, temperature and photoperiod (Morgan et al 2008)

In fish, seasonality coordinates reproduction, affects body weight and

F Pascoli ( &)
E Negrato
C Poltronieri
G Radaelli
D Bertotto

Dipartimento di Biomedicina Comparata e Alimentazione,

Università degli Studi di Padova, Legnaro (PD), Italy

e-mail: francesco.pascoli@unipd.it

D Bertotto

e-mail: daniela.bertotto@unip.it

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

DOI: 10.1007/978-3-642-36488-4_1, Ó Springer-Verlag Berlin Heidelberg 2013

3

physiological status, regulates food intake and locomotion, and is thought also tocoordinate the immune response (Bowden et al.2007) In general, physiologicalparameters are reduced in winter and raised in summer (Bowden et al.2007).The purpose of this study was to investigate the effects of seasonality ongrowth, cortisol, immunological, and hematological parameters in sea bass rearedaccording to conventional semi-intensive method over a period of 14 months.

1.2 Materials and Methods

Juvenile sea bass (Dicentrarchus labrax) were reared in an outdoor tank from May

2009 to July 2010 and monitored every 2 months (initial weight 69 g; final weight

350 g; stocking density 2–12 kg/m3) At each sampling, 20 animals were caughtand measured (total and standard length and weight) to observe growth andcondition factor (K) Blood samples were collected from the caudal vein.Serum cortisol analysis was carried out by radioimmunoassay (RIA), as described

by Simontacchi et al (2008) Hematocrit and leucocrit were obtained by centrifugation of whole blood (12,500 rpm for 5 min) Serum lysozyme activitywas measured by a turbidimetric assay, as described by Parry et al (1965).Total glutathione (GSH) was determined by an enzymatic assay adapted tomicrotiter plate (Baker et al.,1990)

micro-1.3 Results

Weight increased from 69.1 ± 3.0 g to 345.5 ± 13.6 g after 14 months.During this period, the condition factor worsened from 1.02 ± 0.03 to1.21 ± 0.01, with the lowest values in December 2009, January 2010, and March

2010 (0.94 ± 0.02, 0.94 ± 0.02, and 0.95 ± 0.02, respectively) and a significantincrease in May and July 2010 (1.22 ± 0.02 and 1.21 ± 0.01, respectively).Serum cortisol was significantly higher in May 2009, May 2010, and July 2010compared to the other months (p \ 0.05; Fig.1.1) The lowest levels wererecorded in October and December 2009, and January and March 2010 (p \ 0.05).The hematocrit was significantly lower in January and March 2010 than theother samples (p \ 0.05; Fig.1.2) The leucocrit was significantly lower inDecember 2009, January 2010, and March 2010 compared to the other months(p \ 0.05; Fig.1.3) The highest value was recorded in October (p \ 0.01).Serum lysozyme activity increased from May to October 2009, then decreaseduntil January 2010 and increased again after that point (Fig.1.4) The lowest valueswere recorded in January (p \ 0.01) Higher values were found in July 2010 than inMay, July, and December 2009 and March 2010, but these were not significantlydifferent from those in October 2009 and May 2010

The GSH decreased from July to December 2009 and then increased until July

2010 (Fig.1.5) The lowest values were found in October and December 2009 andJanuary and March 2010, and the highest were in July 2010 (p \ 0.05)

Fig 1.1 Variations in serum cortisol of sea bass over a 14-month period (mean ± SE) Different letters indicate significant differences (p \ 0.05)

Fig 1.2 Variations in hematocrit of sea bass over a 14-month period (mean ± SE) Different letters indicate significant differences (p \ 0.05)

1 Seasonal Effect on Hematological and Innate Immune Parameters 5

Fig 1.3 Variations in leucocrit of sea bass over a 14-month period (mean ± SE) Different letters indicate significant differences (p \ 0.05)

Fig 1.4 Variations in serum lysozyme activity of sea bass over a 14-month period (mean ± SE) Different letters indicate significant differences (p \ 0.05)

1.4 Discussion

In this study, we investigated the effect of seasonality on some hematological andstress parameters in sea bass reared under conventional semi-intensive aquaculture.The effect of seasonality was observed for all parameters investigated The growthand condition factors showed a significant reduction during the coldest months(October to March), due to a lower water temperature, which affects food intake,and consequently diet and metabolism (Pastoureaud1991)

Serum cortisol levels showed a seasonal pattern, with lower values during thecoldest months and higher values in the warmer months, consistent with thefluctuations of temperature and photoperiod as already reported by Planas et al.(1990)

The lowest hematocrit values were recorded in January and March, in lation with the lowest temperatures, which increase the solubility of oxygen in thewater and reduce metabolism, requiring a lower number of red blood cells to carryoxygen around the body (Stolen et al 1984) The highest leucocrit values wererecorded in October 2009, whereas the lowest were during the winter months.Seasonal changes in hematological and immunological parameters have beenfound in other species It is hypothesized that shorter daylight hours can inducechanges in the immune system to prepare for winter In our study, the highestWBC values, found in October, may also be related to a viral encephalitis thatoccurred in August 2010 and that has caused the death of 8.31 % of the fish

corre-Fig 1.5 Variations in glutathione (GSH) of sea bass over a 12-month period (mean ± SE) Evaluation of GSH in May 2009 was not possible because of a lack of serum Different letters indicate significant differences (p \ 0.05)

1 Seasonal Effect on Hematological and Innate Immune Parameters 7

Serum lysozyme activity showed a seasonal trend related to temperature andphotoperiod, with lower values during the colder months, consistent with otherstudies in the literature.

The observed levels of GSH suggest a relationship with seasonality, as reported

in previous studies In mammals, fasting and inflammatory processes can affect thelevels of GSH, causing a decrease in plasma levels (Malmezat et al 2000).Consequently, the low levels found in this study during the colder months may berelated to winter starvation and to the viral encephalitis that occurred in Augustand September

In conclusion, all parameters exhibited a seasonal pattern, similar to studiesperformed in other species These results may contribute to a better understanding

of the mechanisms that regulate the influence of seasonal components on theimmune and stress responses in fish, leading to optimized husbandry practices

References

Baker MA, Cerniglia GJ, Zaman A (1990) Microtiter plate assay for the measurement of glutathione and glutathione disulfide in large number of biological samples Anal Biochem 190:360–365

Bowden TJ, Thompson KD, Morgan AL, Gratacap RML, Nikoskelainen S (2007) Seasonal variation and the immune response: a fish perspective Fish Shellfish Immunol 22:695–706 Malmezat T, Breuille D, Capitan P, Minard PP, Obled C (2000) Glutathione turnover is increased during the acute phase of sepsis in rats J Nutr 130:1239–1246

Morgan AL, Thompson KD, Auchinachie NA, Migaud H (2008) The effect of seasonality on normal haematological and innate immune parameters of rainbow trout Oncorhynchus mykiss

L Fish Shellfish Immunol 25:791–799

Parry RM, Chandan RC, Shahani RM (1965) A rapid sensitive assay of muramidase Proc Soc Exp Biol Med 119:384–386

Pastoureaud A (1991) Influence of starvation at low temperatures on utilization of energy reserves, appetite recovery and growth character in sea bass, Dicentrarchus labrax Aquaculture 99:167–178

Planas J, Gutierrez J, Fernandez J, Carrillo M, Canals P (1990) Annual and daily variations of serum cortisol in sea bass Dicentrarchus labrax L Aquaculture 91:171–178

Simontacchi C, Poltronieri C, Carraro C, Bertotto D, Xiccato G, Trocino A, Radaelli G (2008) Alternative stress indicators in seabass Dicentrarchus labrax L J Fish Biol 72:747–752 Stolen JS, Gahn T, Kasper V, Nagle JJ (1984) The effect of environmental temperature on the immune response of a marine teleost (Faralichrhys denrufus) Dev Comp Immunol 8:89–98

To evaluate animal welfare linked to physiological status, cortisol levels, bloodcell count and hematochemical variables (glucose, total proteins, albumin, creat-inine, urea, aspartate transaminase, creatine kinase, lactate dehydrogenase, tri-glycerides and total cholesterol) were measured Comparison of mean plasmalevels of 5-HT, tryptophan, and cortisol in horses coming from different altitudeswere not significantly different Plasma 5-HT levels were affected by altitude in agender-dependent way, showing an opposing trend between mares and geldings,with the highest levels in plasma of geldings coming from farms at sea level Both5-HT and tryptophan were higher in mares than in geldings from 600 m above sealevel Plasma cortisol levels, which were significantly higher in mares than ingeldings at sea level, were more affected by gender than altitude.

Keywords Horse
Plasma serotonin
Tryptophan
Biochemical variablesAbbreviations

5-HT Serotonin

Try Tryptophan

G Bruschetta ( &)
P Di Pietro
M Miano
A M Ferlazzo

Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali,

Sezione di Biochimica, University of Messina, Messina, Italy

e-mail: gbruschetta@unime.it

C Cravana

Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali,

Sezione di Fisiologia, University of Messina, Messina, Italy

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

DOI: 10.1007/978-3-642-36488-4_2, Ó Springer-Verlag Berlin Heidelberg 2013

9

2.1 Introduction

Serotonin, or 5-hydroxytryptamine (5-HT), is a neurotransmitter producedindependently in the central nervous system and peripheral tissues from twodistinct isoforms of the enzyme tryptophan hydroxylase (TPH-1 and TPH-2),which catalyses the rate limiting step of the synthesis process Its many biologicalfunctions include pulmonary arterial smooth muscle cell proliferation, smoothmuscle bronchial vasoconstriction and local microthrombosis (MacLean et al

2000) Indeed, high circulating 5-HT levels were associated with the onset ofpulmonary arterial hypertension (PAH) (Hervé et al 1995) Blood 5-HT levelswere increased in hypoxic conditions, even in mice (Callebert et al.2006) 5-HT issynthesised from the amino acid tryptophan (Try) in the brain, in mast cells and inintestinal enterochromaffin cells, where the gene of the classic isoenzyme TPH-1,which controls the peripheral 5-HT production, is mostly expressed (Walther andBader 2003) Both TPH-1 and peripheral 5-HT play an essential role in thedevelopment of hypoxia-induced increased pulmonary pressure and pulmonaryvascular remodelling (Morecroft et al.2007) Platelets do not synthesise 5-HT, butthey are its major site of storage and transport in the peripheral blood (Andres et al

1993) The 5-HT uptake within them keeps plasma 5-HT concentrations low

In the horse, plasma 5-HT values are reported to be higher than in humans (Baileyand Elliott1998; Di Pietro et al.2010; Lebelt et al.1998), and often an increase ofcirculating 5-HT is linked to common pathological conditions such as laminitis(Bailey et al.2009) Considering the clinical importance of this variable, the aim

of this study was to evaluate, in horses of different breeds, the effect of horsefarm altitude on plasma levels of 5-HT and Try, its precursor amino acid.Moreover, some haematochemical and hormonal parameters were determined tosignal the animal welfare status and/or the presence of any stress conditions

2.2 Materials and Methods

Twenty clinically healthy horses (9 geldings and 11 mares) of various breeds (SanFratellana, Sella Italiana and crossbred) were used Mean age was 10 ± 6 years.Horses came from farms and riding schools in Messina, Catania and theirnearby environs at sea level or at an altitude of 600 m above sea level (asl).All horses housed in Messina, in individual boxes, were fed with fresh forages,concentrates and water Blood samples were collected from the jugular vein intoEDTA tubes at 8:30 a.m., at approximately 25°C, in the month of May Blood cellcount was performed, and after centrifugation at 2,000 g, the following haemato-chemical parameters were detected: glucose (Glu), total proteins (TP), albumin(ALB), creatinine (Crea), urea, aspartate transaminase (AST), creatine kinase(CPK), lactate dehydrogenase (LDH), triglycerides (TG), total cholesterol (TCho),

by spectrophotometry and serum cortisol levels (in duplicate, by ELISA kit [Radim,

Pomezia, Italy]) The platelet poor plasma (PPP) fraction was obtained bycentrifugation at 4,500 g Equal volumes of plasma, N-methylserotonin (internalstandard) and protein precipitation reagent were vigorously vortex mixed, incubated

at 4°C and centrifuged at 4,500 g The obtained supernatant was used forthe detection of plasma serotonin and tryptophan by reverse phase HPLC (Waters1,525 binary HPLC pump) with electrochemical detector (ESA) Statistical analysiswas done by Student’s unpaired t test and Pearson’s correlation and linearregression tests

2.3 Results

The blood cell count and haematochemical parameters were in the physiologicalrange for all animals (Kaneko1989; Ubaldi et al.1982) and indicated an overallstate of wellness Preliminary data (Table2.1) did not show any significantdifferences of plasma 5-HT, tryptophan and cortisol levels with altitude change,nor significant correlations among them However, a difference in plasma 5-HTlevels based on altitude was observed when the horses were divided by gender(Table2.2) Plasma tryptophan levels (Table2.2) were similar in mares andgeldings at sea level, but slightly higher in mares at 600 m asl, with a trendcomparable to 5-HT In mares, higher concentrations of cortisol were observed inboth groups (Table2.2), which were significant at sea level and at 600 m asl

2.4 Discussion

Mares seem to respond to higher altitude with greater 5-HT values compared tothose housed at sea level (Table2.2), consistent with observations in cows(Bruschetta et al.2010) and rats (Awabdy et al.2003) At higher altitude, plateletfunction could be downregulated, producing a decrease of platelet serotoninuptake and increased circulating serotonin levels On the contrary, in geldings, therewas a significant increase in 5-HT at sea level Moreover, a gender-dependent effect

on plasma 5-HT levels was detected in horses at sea level, which was slight inhorses at 600 m asl An analysis using a larger sample size could support theseresults and clarify the existence of an interaction among different variables that mayhave opposite effects on plasma 5-HT levels Further, significant findings couldcome from the comparison of horses coming from farms or riding schools at sealevel with horses farmed at altitudes higher than 1,000 m asl

Table 2.1 Plasma levels of 5-HT, Try and cortisol (Mean ± S.D.) in horses at different altitudes

2 Effect of Altitude on Plasma Serotonin Levels in Horses 11

The major changes in tryptophan values were observed in mares These datacould suggest the existence of sexual dimorphism, which could be deeper investi-gated using a greater number of horses The higher concentration of cortisolobserved in mares in both groups (Table2.2) confirms the existence of a regulation

of the hypothalamic–pituitary–adrenal axis of horses in a gender-dependentmanner, as already observed in humans (Stroud et al.2011) and rats (Viau et al

2005) With a larger sample size, this could be of interest for geldings farmed at

Awabdy D, Bryan-Lluka LJ, Wanstall JC (2003) 5-hydroxytryptamine and platelets: uptake and aggregation in hypoxic pulmonary hypertensive rats Eur J Pharmacol 459:1–7

Bailey SR, Elliott J (1998) Plasma 5-hydroxytryptamine constricts equine digital blood vessels

in vitro: implications for pathogenesis of acute laminitis Equine Vet J 30:124–130 Bailey SR, Adair HS, Reinemeyer CR, Morgan SJ, Brooks AC, Longhofer SL, Elliott J (2009) Plasma concentrations of endotoxin and platelet activation in the developmental stage of oligofructose-induced laminitis Vet Immunol Immunopathol 129:167–173

Bruschetta G, Di Pietro P, Sanzarello L, Giacoppo E, Ferlazzo AM (2010) Plasma serotonin levels in Italian Fresian dairy cows Vet Res Commun 34(Suppl.1):S17–S20

Callebert J, Esteve JM, Hervé P, Peoc’h K, Tournois C, Drouet L, Launay JM, Maroteaux L (2006) Evidence for a control of plasma serotonin levels by 5-hydroxytryptamine(2B) receptors in mice J Pharmacol Exp Ther 317:724–731

Di Pietro P, Bruschetta G, Sanzarello L, Ferlazzo AM, Medica P (2010) Horse platelet poor plasma serotonin levels after trekking Proc It Soc Vet Sci 64:3–5

Hervé P, Launay JM, Scrobohaci ML, Brenot F, Simonneau G, Petitpretz P, Poubeau P, Cerrina J, Duroux P, Drouet L (1995) Increased plasma serotonin in primary pulmonary hypertension.

Gender Geldings (n 5) Mares (n 7) Geldings (n 4) Mares (n 4) 5-HT (ng/ml) 64.4 ± 6.9 49.0 ± 13.1a 44.3 ± 10.0a 55.5 ± 12.6 Try (lg/ml) 7.52 ± 1.47 7.55 ± 1.42 7.09 ± 1.44 8.80 ± 1.84 Cortisol (ng/ml) 71.0 ± 13.8 95.4 ± 19.8a 84.0 ± 12.2 99.3 ± 14.5a

a vs geldings at sea level: p \ 0.05

Lebelt D, Zanella AJ, Unshelm J (1998) Physiological correlates associated with cribbing behaviour in horses: changes in thermal threshold, heart rate, plasma beta-endorphin and serotonin Equine Vet J Suppl 27:21–27

MacLean MR, Hervé P, Eddahibi S, Adnot S (2000) 5-hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension Br J Pharmacol 131:161–168

Morecroft I, Dempsie Y, Bader M, Walther DJ, Kotnik K, Loughlin L, Nilsen M, MacLean MR (2007) Effect of tryptophan hydroxylase 1 deficiency on the development of hypoxia-induced pulmonary hypertension Hypertension 49:232–236

Stroud LR, Papandonatos GD, Williamson DE, Dahl RE (2011) Sex differences in cortisol response to corticotropin releasing hormone challenge over puberty: Pittsburgh Pediatric Neurobehavioral Studies Psychoneuroendocrinology 36:1226–1238

Ubaldi A, Corbella E, Montanari P (1982) Chapter 7 In: Ubaldi A, Corbella E, Montanari P (eds) Diagnostica chimico-clinica veterinaria Casa editrice Ambrosiana, Milano, p 152

Viau V, Bingham B, Davis J, Lee P, Wong M (2005) Gender and puberty interact on the induced activation of parvocellular neurosecretory neurons and corticotrophin-releasing hormone messenger ribonucleic acid expression in the rat Endocrinology 146:137–146 Walther DJ, Bader M (2003) A unique central tryptophan hydroxylase isoform Biochem Pharmacol 66:1673–1680

stress-2 Effect of Altitude on Plasma Serotonin Levels in Horses 13

Chapter 3

Identification of Aquaporin 1 in Diplodus

sargus

G Zanghì, S Campo, A D’Ascola, A Germanà and A M Ferlazzo

Abstract Aquaporin 1 (AQP-1) is the first member of the aquaporin family, whichincludes seven homologs in teleosts, involved in the selective transport of water,small neutral molecules, and ions AQPs contain six transmembrane helices, fiveconnecting loops, and the amino and carboxyl ends protrude into the cytoplasm.AQPs are important for osmoregulation in fish gills (independent of salinity),kidneys, and intestine Here, the nucleotide and amino acid sequences of AQP-1 inDiplodus sargus were characterized, and a phylogenetic tree was built to study itsevolution Results showed that AQP-1 mRNA is 1,325 nucleotides in length, andthe deduced protein contains two canonical Asn-Pro-Ala (NPA) consensus motifsand all features for water transport D sargus AQP-1 is located in a homophyleticbranch with Sparus aurata only, inside of a sub-tree in a paraphyletic position withCentropristis striata and Osmerus mordax Although the structure of the AQPprotein in different species is conserved, results showed that the S aurata and

D sargus AQP-1 proteins have evolved similarly

Keywords D sargus
Aquaporin 1
Biochemistry
Nucleotide and amino acidsequences

G Zanghì ( &)
A M Ferlazzo

Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali,

Sezione di Biochimica, Università degli Studi di Messina, Messina, Italy

e-mail: gzanghi@unime.it

S Campo
A D’Ascola

Dipartimento di Scienze Biochimiche, Fisiologiche e Della Nutrizione,

Sezione di Chimica, Università degli Studi di Messina, Messina, Italy

A Germanà

Dipartimento di Morfologia, Biochimica, Fisiologia e Produzioni Animali,

Sezione di Morfologia, Università degli Studi di Messina, Messina, Italy

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

DOI: 10.1007/978-3-642-36488-4_3, Ó Springer-Verlag Berlin Heidelberg 2013

15

3.1 Introduction

The survival of fish in the water environment is strictly associated with the correctfunctional properties of some critical structures, such as ionic channels, which playkey roles in electrochemical and osmotic equilibria Aquaporins (AQPs) facilitatethe very fast flux of water molecules to the inside and outside of cells of specifictissues (e.g., proximal tubules, erythrocytes, and membranes of vegetal cellvacuoles) Their presence is necessary for transport of water, which, as a polarmolecule, negligibly diffuses through biological membranes Currently, 13 iso-forms of AQPs have been identified in mammals, variously distributed to variousorgans (Ablimit et al.2006; Lee et al.1997; Lopez et al.2007), and 17 AQPs havebeen identified in both fresh and marine teleosts (Cerdà and Finn2010), particularly

in pufferfish and zebrafish (Tingaud-Sequeira et al.2010) The duplicate isoforms

of aquaporin 1 (AQP-1) are involved in intestinal water absorption in marine fish(Tingaud-Sequeira et al.2008) AQPs comprise a superfamily of intrinsic proteins,with a molecular weight between 36 and 78 kDa, located inside the membrane lipidbilayer, allowing the bidirectional transport of water (Agre2006) Two families ofAQPs were identified (Gonen and Walz2006): specific aquaporins, allowing onlytransport of water, and aquaglyceroporins, allowing transport of water, glycerol,and other neutral molecules (Agre et al.2002; Litman et al.2009) Both proteinscontain transmembrane domains that cross the phospholipid layer (Fu and Lu

2007) AQPs are ethero- and homotetramers Every protein subunit contains its ownpore for transport of one water molecule at a time Each subunit is characterized byone of six transmembrane domains that cross the lipid bilayer and faces each other:the first three domains (a, b, c) oppose the other three (a1, b1, c1) In the channelpore, the N- and C-terminal ends of the polypeptide chains are oriented toward theintracellular environment Moreover, the AQP structure contains extracellularloops (A, C, E) and intracellular loops (B, D, F) (Jung et al.1994) The aqueouschannel pore is formed by loops B and E, folded to create the central pore.The loops contain particular Asn-pro-ala (NPA) motifs, allowing an ‘‘hourglass’’structure (Agre et al.1999) AQP-1 is the first member of the AQP family, whichincludes seven homologous proteins in teleosts, that is particularly important insome osmoregulatory fish organs, especially gills, where it is expressed in greatamounts, in addition to the kidney and gut (Giffard-Mena et al 2007).Among marine teleosts of the perciform family, Diplodus sargus (Bargelloni et al

2005) is found in the Mediterranean Sea, Black Sea, and Eastern Atlantic Ocean.Young specimens live on the surface, and lonely adults live on rocky seabeds atdepths of 20–30 m and are 35–45 cm in length D sargus reproduces at thebeginning of autumn, and its fries remain for some time in brackish waters, thenadapt to the changing water salinity In order to understand the molecular mech-anisms allowing this species to adapt in an extremely good way to different envi-ronmental conditions, the aim of this study was to characterize AQP-1 nucleotide

and amino acid sequences by constructing a phylogenetic tree to evaluate itsevolution The molecular study of this perciform family fish is very interesting,from both a scientific point of view and its commercial consequences.

3.2 Materials and Methods

The research was carried out on 10 specimens of D sargus, 250 g body weight,farmed for 10 days under normal salinity conditions in an aquarium containing 300 Lfresh seawater and gassed with an integrated air pump oxygenator After anesthesiawith MS-222 (40 mg/L ethyl-m-aminobenzoate methanesulfonate), fish were sac-rificed for gill sampling (50–100 mg) After homogenization and total mRNAextraction using the Trizol Reagent kit (Invitrogen, USA) followed by RT-PCR, theAQP-1 nucleotide sequence was identified using the rapid amplification of cDNAends (RACE) technique To build the phylogenetic tree, AQP-1 was aligned usingClustalW software with AQPs 1 and 3 of 33 temperate and Antarctic teleosts,

a Renibacterium, and other higher vertebrates, representing the outgroup The treewas calculated by the Neighbor-Joining method with a 1,000 repetitions Bootstrapusing the MEGA 5.05 program, whereas the calculus matrix was experimentallyevaluated using ProTest software Phylogenetic trees were drawn to scale, whereasevolutionary distances were calculated using the Poisson correction method.The phylogenetic tree, so drawn, showed a good resolution, given that only one knothad a value lower than 0.5

3.3 Results

In Fig.3.1, the nucleotide sequence of D sargus AQP-1 is reported The mRNA is1,325 nucleotides in length; its start codon is located at position 116, the stop codon

is located at position 901, and the polyadenylation signals are located at positions

957 and 1,303 In Fig.3.2, the amino acid sequence of the deduced protein, which is

261 amino acid residues in length and highly homologous to those of other studiedfish species is shown The protein contains two particular NPA motifs and allessential features for water transport The two NPA domains can be observed atpositions 70 and 184, and contain loops B and E, respectively In addition, it ispossible to see amino acid residues of the other four loops The phylogenetic treecan be divided into four main sub-trees The first contains AQPs of outgroupspecies (chicken, Xenopus, and human) Reptile AQP, Vitis vinifer, lies on a distinctbranch On the first sub-tree, in an unusual way, AQPs of two teleosts are included.The fourth sub-tree contains the majority of AQP-3, while the third sub-treecontains the AQP-1 of some teleosts and human AQP-2 Finally, AQP-1 of almostall analyzed teleosts is included in the second sub-tree, which contains differentclusters, one formed only from the D sargus and Sparus aurata AQPs that

obviously are monophyletic, but are in a paraphyletic position with AQP-1 ofCentropristis striata and Osmerus mordax The length of the branches of thiscluster also shows that D sargus and S aurata AQPs have undergone a very recentand common diversification.

3.4 Discussion

In conclusion, D sargus mRNA and its deduced protein have been identified.The drawn phylogenetic tree shows that the evolutionary diversification of thisgene is similar to that of S aurata This study will be strengthened by thecharacterization and identification of AQP-3 in the same species; AQP-3, in whichtwo duplicated genetic isoforms have been found in zebrafish (Danio rerio), plays

a fundamental role in physiological osmoregulatory processes in different tean organs (Cutler et al.2007)

teleos-Fig 3.1 Nucleotide sequence of the AQP-1 gene (GenBank provisory accession number = JN210582)

Fig 3.2 Amino acid

sequence of AQP-1 and

consensus loops

Acknowledgments This research work was carried out by PRA 2007 funds at the University of Messina.

References

Ablimit A, Matsuzaki T, Tajika Y, Aoki T, Hagiwara H, Takata K (2006) Immunolocalization of water channel aquaporins in the nasal olfactory mucosa Arch Histol Cytol 69:1–12 Agre P (2006) The aquaporin water channels Proc Am Thorac Soc 3:5–13

Agre P, Mathai JC, Smith BL, Preston GM (1999) Functional analyses of aquaporin water channel proteins Methods Enzymol 294:550–572

Agre P, King LS, Yasui M, Guggino WB, Ottersen OP, Fujiyoshi Y, Engel A, Nielsen S (2002) Aquaporin water channels—from atomic structure to clinical medicine J Physiol 542:3–16 Bargelloni L, Alarcon JA, Alvarez MC, Penzo E, Magoulas A, Palma J, Patarnello T (2005) The Atlantic-Mediterranean transition: discordant genetic patterns in two seabream species, Diplodus puntazzo (Cetti) and Diplodus sargus Diplodus (L.) Mol Phylogenet Evol 36:523–535

Cerdà J, Finn RN (2010) Piscine aquaporins: an overview of recent advances J Exp Zool A Ecol Genet Physiol 313:623–650

Cutler CP, Martinez AS, Cramb G (2007) The role of aquaporin 3 in teleost fish Comp Biochem Physiol A Integr Physiol 148:82–91

Fu D, Lu M (2007) The structural basis of water permeation and proton exclusion in aquaporins Mol Membr Biol 24:366–374

Giffard-Mena I, Boulo V, Aujoulat F, Fowden H, Castille R, Charmantier G, Cramb G (2007) Aquaporin molecular characterization in the sea-bass (Dicentrarchus labrax): the effect of salinity on AQP1 and AQP3 expression Comp Biochem Physiol A Integr Physiol 148:430–444

Gonen T, Walz T (2006) The structure of aquaporins Q Rev Biophys 39:361–396

Jung JS, Preston GM, Smith BL, Guggino WB, Agre P (1994) Molecular structure of the water channel through aquaporin CHIP The hourglass model J Biol Chem 269:14648–14654 Lee MD, King LS, Agre P (1997) The aquaporin family of water channel proteins in clinical medicine Medicine (Baltimore) 76:141–156

Litman T, Søgaard R, Zeuthen T (2009) Ammonia and urea permeability of mammalian aquaporins In: Beitz E (ed) Aquaporins Handbook Of Experimental Pharmacology Springer, Berlin, pp 327–358

Lopez IA, Ishiyama G, Lee M, Baloh RW, Ishiyama A (2007) Immunohistochemical localization

of aquaporins in the human inner ear Cell Tissue Res 328:453–460

Tingaud-Sequeira A, Chauvigné F, Fabra M, Lozano J, Raldúa D, Cerdà J (2008) Structural and functional divergence of two fish aquaporin-1 water channels following teleost-specific gene duplication BMC Evol Biol 8:259

Tingaud-Sequeira A, Calusinska M, Finn RN, Chauvigné F, Lozano J, Cerdà J (2010) The Zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals Biomed Central BMC Evol Biol 10:38

Chapter 4

Effect of Dephosphorylation on Donkey

Milk Caseins

S Vincenzetti, A Vita, F M Carpi, D Micozzi and P Polidori

Abstract Donkey milk caseins display great microheterogeneity bytwo-dimensional electrophoresis (2-DE), probably because of a variable degree ofphosphorylation and/or alternative splicing phenomena In this work, we haveinvestigated the complexity of the donkey milk caseins: The whole casein fractionwas subjected to treatment with calf intestinal alkaline phosphatase to achievedephosphorylation The obtained apo forms were analyzed by 2-DE and comparedwith whole donkey caseins As a result, donkey milk b-casein and its splicingvariants are present as different phosphorylated isoforms, whereas aS1-caseindisplays several isoforms that are derived from both phosphorylation andglycosylation phenomena

Keywords Alpha-casein
Beta-casein
Dephosphorylation
Donkey milk

Two-dimensional electrophoresis

4.1 Introduction

Caseins are a family of acid phosphoproteins (aS1-, aS2-, b-, and j-caseins) that aresynthesized in the mammary gland in response to hormonal stimuli and formmicellae in milk In the mare’s milk, the primary structures of the aS1- and

S Vincenzetti ( &)
A Vita

Scuola di Scienze Mediche Veterinarie, Università di Camerino, Camerino (MC), Italy e-mail: silvia.vincenzetti@unicam.it

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

DOI: 10.1007/978-3-642-36488-4_4, Ó Springer-Verlag Berlin Heidelberg 2013

21

b-caseins show several polymorphic patterns due to alternative splicing nomena; furthermore, there are some other isoforms of caseins due to post-translational modifications such as phosphorylation and glycosylation (Miclo et al.

phe-2007) The phosphate group of casein may affect many of its features, such as theirdigestion and the bioavailability of divalent cations It is known that in cow’s milkprotein allergy, caseins are the proteins widely responsible for this allergic phe-nomenon In particular, some authors have shown that the aS1-casein in itsphosphorylated form plays an important role in the allergenicity of milk and that ingeneral, the aS- and b-caseins that possess serine-phosphorylated residues can beconsidered immunoreactive and resistant to digestion (Tezcucano et al.2007) Inthis work, we investigated the complexity of donkey milk caseins pattern by two-dimensional electrophoresis (2-DE) analyses For this purpose, the total caseinsobtained by isoelectric precipitation were dephosphorylated by alkaline phos-phatase, showing that donkey’s milk b-casein and its splice variants are largelypresent as phosphorylated isoforms, while donkey’s milk aS1-casein shows dif-ferent isoforms that may result from both phosphorylation and glycosylationphenomena

4.2 Materials and Methods

Donkey’s milk was collected from 10 Martina Franca breed asses at an diate stage of lactation The milk was skimmed by centrifugation at 3,000 9 g for

interme-30 min, at a temperature of 15°C The casein fraction was obtained from skimmedmilk by acidic precipitation at pH 4.6 with 10 % acetic acid, followed bycentrifugation at 3,000 g for 10 min The casein pellet was then resuspended in

50 mM Tris/HCl, pH 7.5, 1 mM DTT, and 8 M urea The 2-DE was performed asfollows: 100 lg casein was treated with the 2D-Clean-Up kit (GE Healthcare) andresuspended in rehydration buffer (8 M urea, 2 % CHAPS, 65 mM DTT, 0.001 %bromophenol blue, and 0.5 % IPG buffer, pH 4–7) Isoelectric focusing was per-formed on an immobilized gradient at pH 4–7 (Immobiline DryStrip gel, 18 cm,

GE Healthcare) The IPG strip was rehydrated for 12 h at 15°C to avoid idation (at the level of a residue of Asn), which may occur when the isoelectricfocusing is carried out at temperatures above 20°C (Matéos et al 2009).The electrophoretic conditions were 50 A/strip, 1 h at 500 V, 1 h at 1,000 V, and

deam-4 h at 8,000 V The second dimension consisted of 13 % SDS-PAGE

After electrophoresis, proteins were stained using Coomassie blue or blotted topolyvinylidene difluoride membranes to analyze each spot by N-terminalsequencing Spot analysis was carried out using PDQuest software (Version 7.1.1;Bio-Rad Laboratories) for spot-intensity calibration, as well as calculation ofmolecular mass and isoelectric point (pI) In the experiment of casein dephos-phorylation, the casein fraction was resuspended in 10 ml 0.4 % ammoniumbicarbonate, pH 8.5, containing 0.5 mM MgCl2 The solution was heated to 80°Cfor 5 min to disperse the caseins and then was lyophilized in 5.3-mg aliquots

Lyophilized caseins were resuspended in 50 mM Tris/HCl, 10 mM MgCl2, and

1 mM DTT, pH 7.5, and treated with alkaline phosphatase (20 units) for 3 h at

37°C After incubation, the dephosphorylated casein samples were frozen andfreeze-dried Subsequently, dephosphorylated caseins were subjected to 2-DE asdescribed above The control consisted of caseins treated in the same way as thosesubjected to dephosphorylation but incubated in the absence of alkalinephosphatase

4.3 Results

Figure4.1shows the electrophoretic pattern of the phosphorylated casein fraction(control, Fig.4.1a) compared with dephosphorylated caseins (Fig 4.1b) A shift ofdephosphorylated caseins toward more basic pH values with respect to the control

is evident, because of the removal of the negative charge of the phosphate groups.The phosphorylated and dephosphorylated caseins were analyzed by PDQuestsoftware and N-terminal sequencing, and the results are summarized in Table4.1.Spots A–H of the phosphorylated casein fraction are b-caseins with molecularmasses (Mr) ranging from 31.15 to 33.80 kDa and pIs ranging from 4.60 to 4.90.Spots I–N are aS1-caseins with lower molecular weights with respect to theb-caseins, ranging from 27.40 to 31.20 kDa and more basic pI values, rangingfrom 4.90 to 5.40 (Fig.4.1a and Table4.1) Spots A1 and B1 observed in thedephosphorylated casein fraction (Fig.4.1b and Table4.1) are two b-caseins withvery similar molecular masses (33.50 and 32.87 kDa, respectively) but different pIvalues (5.70 and 5.90, respectively) Spots C1 and I1 are two b-caseins but withslightly lower molecular masses while spots E1, F1, G1, and H1 are aS1-caseinswith different pI and molecular mass values Under our experimental conditions,

we have not observed aS2- and j-caseins, which might be present in trace amounts

in donkey’s milk, as reported by other authors (Bertino et al.2010)

Fig 4.1 Casein dephosphorylation in donkey’s milk: a phosphorylated caseins (control);

b dephosphorylated caseins

4 Effect of Dephosphorylation on Donkey Milk Caseins 23

4.4 Discussion

The phosphorylated casein fraction shows, after 2-DE analysis, the presence ofabout 14 major spots with molecular masses ranging between 27.24 and33.74 kDa and pI values between 4.60 and 5.40 (Fig.4.1a and Table4.1).N-terminal analysis showed the presence of as1- and b-caseins, suggesting that theheterogeneity observed in the casein fraction could be due to different degrees ofphosphorylation or glycosylation (Bertino et al 2010; Chianese et al 2010).Through the NetPhos 2.0 and Netglycate 1.0 software (available on the website

www.expasy.ch), it is possible to predict phosphorylation and glycosylation onas1- and b-caseins In donkey milk b-casein, there are 11 serine residues and onethreonine residue that can be potentially phosphorylated, and there is one lysineresidue that can be potentially glycosylated, whereas in as1-casein, there 10, 2, and

1 potentially phosphorylated sites on serine, threonine, and tyrosine residues,respectively Furthermore, there are seven potential lysine residues glycosylated.Experiments on casein dephosphorylation have shown that the heterogeneityobserved in the donkey’s milk casein fraction is essentially due to post-transla-tional modifications, in particular, to phosphorylation In fact, comparing the 2-DE

Table 4.1 Characterization of phosphorylated and dephosphorylated caseins by N-terminal sequencing Spot analysis was carried out by PDQuest software Version 7.1.1 (Bio-Rad Laboratories)

patterns obtained using the phosphorylated and dephosphorylated caseins, it can beassumed that spots A, B, C, and D (Fig.4.1a) assigned the phosphorylatedb-caseins, are modified after dephosphorylation of spots A1 and B1 (Fig.4.1b),where they show similar molecular weights but different pI values Therefore,spots A1 and B1 could be two isoforms that may be derived from a geneticpolymorphism or from the glycosylation of a lysine residue, according to theprediction of glycosylation sites on b-casein Presumably, spots C1 and I1(Fig.4.1b, Table4.1), characterized by lower molecular masses (31.90 and32.30 kDa, respectively) than the other two b-casein spots (A1 and B1), may bethe dephosphorylated splice variants of b-casein In fact, Matéos and collaborators(2010) showed the presence of b-casein D5 (30 kDa) characterized as a splicevariant of exon 5, in mare’s milk Therefore, in conclusion, spots A, B, C, and D ofdonkey’s milk b-casein (Fig.4.1a) could correspond to different isoforms withdifferent degrees of phosphorylation, while spots E, F, G, and H may be differentphosphorylated isoforms of splice variants Finally, the prediction of glycosylationsites on donkey’s milk as1-caseins highlights the potential for glycosylation ofseven lysine residues that may be responsible for the presence of different spots(D1, E1, F1, G1, and H1) observed in donkey’s milk as1-casein even afterdephosphorylation.

Acknowledgments This work has been supported by the Italian Ministry of Agriculture; Principal Investigator: Prof Polidori Paolo.

References

Bertino E, Gastaldi D, Monti G, Baro C, Fortunato D, Perono Garoffo L, Coscia A, Fabris C, Mussap M, Conti A (2010) Detailed proteomic analysis on DM: insight into its hypoallergenicity Front Biosci 2:526–536

Chianese L, Calabrese MG, Ferranti P, Mauriello R, Garro G, De Simone C, Quarto M, Addeo F, Cosenza G, Ramunno L (2010) Proteomic characterization of donkey milk ‘‘caseome’’.

J Chromatogr A 1217:4834–4840

Matéos A, Girardet JM, Mollé D, Dary A, Miclo L, Gaillard JL (2009) Two-dimensional cartography of equine beta-casein variants achieved by isolation of phosphorylation isoforms and control of the deamidation phenomenon J Dairy Sci 92:2389–2399

Matéos A, Girardet JM, Mollé D, Corbier C, Gaillard JL, Miclo L (2010) Identification of phosphorylation sites of equine beta-casein isoforms Rapid Commun Mass Spectrom 24:1533–1542

Miclo L, Girardet JM, Egito AS, Mollé D, Martin P, Gaillard JL (2007) The primary structure of

a low-Mr multiphosphorylated variant of beta-casein in equine milk Proteomics 7:1327–1335 Tezcucano Molina AC, Alli I, Konishi Y, Kermasha S (2007) Effect of dephosphorylation on bovine casein Food Chem 101:1263–1271

4 Effect of Dephosphorylation on Donkey Milk Caseins 25

Chapter 5

Distribution Pattern and Chemical Coding

of Sympathetic Trunk Ganglia Neurons

Supplying the Boar Urinary Bladder

Trigone

F Gazza, M Botti, L Ragionieri, C Sorteni, D Russo, P Clavenzani,

R Chiocchetti, L Bo Minelli and R Panu

Abstract Sympathetic trunk ganglia (STG) neurons projecting to the urinarybladder trigone of the boar were studied by coupling retrograde tracing Fast Blue(FB) and double-labeling immunofluorescence methods FB-positive neurons werelocalized in the L1-S3 STG Immunohistochemical staining revealed the catechol-aminergic (tyrosine hydroxylase-/dopamine beta hydroxylase immunoreactivity)phenotype of the majority of FB-positive neurons, which also preferentiallyexpressed neuropeptide Y In addition, some of the FB-positive dopaminergicperikarya were immunoreactive for calcitonin gene-related peptide, substance P,vasoactive intestinal peptide, vesicular acetylcholine-transporter, neuronal nitricoxide synthase, somatostatin, and leu-enkephalin Functional hypotheses have beenformulated

Keywords Boar
Autonomic neurons
Retrograde neuronal tracer

Immunohistochemistry

5.1 Introduction

The urethral bladder trigone (UBT) is a limited area of the urinary bladder wherethe majority of the bladder’s vessels and nervous fibers enter (Birder et al.2010)and where intramural neurons are more concentrated (Andersson2002; Pidsudko

2004) The UBT has remarkable clinical importance, because it is a site for vesical

F Gazza
M Botti
L Ragionieri
L Bo Minelli (&)
R Panu

Dipartimento di Scienze Medico-Veterinarie, Università degli Studi di Parma, Parma, Italy e-mail: luisa.bo@unipr.it

C Sorteni
D Russo
P Clavenzani
R Chiocchetti

Dipartimento di Scienze Mediche-Veterinarie, Università degli Studi di Bologna,

Bologna, Italy

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

DOI: 10.1007/978-3-642-36488-4_5, Ó Springer-Verlag Berlin Heidelberg 2013

27

cancer origin and proliferation in both human (Shokeir 2004) and veterinarymedicine (Saulnier-Troff et al.2008; Sakai2011; Wilson et al.2007) Therefore, athorough knowledge of the innervation of this area could have important clinicalconsequences The aim of this study was to identify and neurochemicallycharacterize the sympathetic trunk neurons involved in the innervation of the boarUBT, combining retrograde neuronal transport Fast Blue (FB) with adouble-labeling immunofluorescence method.

5.2 Materials and Methods

The study was carried out on three 40 kg boars Under general anesthesia, theyunderwent laparatomy via the ventral midline from the umbilical to pubic regions.The urinary bladder was deflected caudally to expose the UBT 50 ll of 2 %aqueous solution of FB was injected in 5 random sites (10 ll per site) in thebladder wall of the trigone with a glass Hamilton microsyringe After an appro-priate survival time (15 days), the animals were deeply anaesthetized and sub-jected to euthanasia by i.v administration of embutramide, mebenzonium iodide,and tetracaine hydrochloride (Tanax, Intervet, and Italia; 0.3 ml/kg) Bilateralsympathetic trunk ganglia (STG) from T1 to Co1 were collected Double immu-nolabeling reactions were carried out on 16 lm thick cryosections of thelumbo-sacral ganglia in which FB positive (FB+) neurons were present, to high-light the eventual co-existence of tyrosine hydroxylase (TH) with dopamine bhydroxylase (DbH), vesicular choline acetyl transferase (VChAT), neuronal nitricoxide synthase (n-NOS), calcitonin gene-related peptide (CGRP), leu-enkephaline(LENK), neuropeptide Y (NPY), substance P (SP), vasoactive intestinalpolypeptide (VIP), and somatostatin (SOM)

5.3 Results

There were 1845 ± 259 (mean ± S.E.M., n = 3) FB+ neurons distributed in theL1-S3 STG The vast majority of them (87 ± 5 % of counted FB+ neurons) werecounted in the L7-S1 ganglia, primarily in the left ganglion Morphologically, theFB+ cells were multipolar with an ellipsoidal shape and a major axis of

30 ± 5 lm (200 cells measured for each animal), generally parallel to thelongitudinal axis of the ganglion The labeled cells were isolated or clustered intosmall groups of two or three neurons scattered in the ganglion In the most positiveganglia, the labeled cells were localized almost exclusively along one side of theganglion

Immunohistochemistry showed that 66 ± 10 % of FB+ neurons showedimmunoreactivity (IR) for TH and that 92 ± 7 % of these cells co-expressed DbH,revealing the catecholaminergic nature of these neurons

We observed that 59 ± 8 % of FB+ neurons showed IR to NPY, whereas thepercentage of FB+ neurons that co-expressed TH- and NPY-IR was 52 ± 7 %.The neurons projecting to the UBT showed positivity also for the other markerstested, in the following percentages: CGRP- (24 ± 3 %), SP- (23 ± 2 %),VIP- (19 ± 2 %), nNOS- (15 ± 2 %), VAChT- (15 ± 2 %), LENK- (14 ± 7 %),and SOM-IR (12 ± 3 %).

Moreover, the FB+/TH-IR neurons co-expressed CGRP- (20 ± 2 %),SP- (19 ± 5 %), VIP- (15 ± 2 %), VAChT- (15 ± 2 %), nNOS- (12 ± 3 %),SOM- (11 ± 4 %), and LENK-IR (10 ± 3 %)

VAChT-, CGRP-, LENK-IR various fibers, and in minor quantities, nNOS-IRnerve terminals were observed around the FB+ neurons

5.4 Discussion

The combined use of FB and double immunofluorescence methods carried out onneurons of the STG innervating the boar UBT documented that the contribution ofthese neurons was quantitatively and qualitatively diversified The distribution ofthe FB+ neurons along one side of the ganglia mainly involved in the innervation

of the UBT is particular In the past, a somatotopic organization has beendocumented in the caudoventral portions of the STG for the neurons projecting tothe pig colon (Skobowiat et al.2010)

This localization at the periphery of the ganglia, the small dimensions, thecatecholaminergic character, and/or the positivity to NPY or, less frequently toSOM, allow us to hypothesize that the majority of the UBT projecting neuronshave a vasoconstrictor activity (Majewski1999)

The adrenergic innervation could also be destined to the musculature of thetrigone, where in certain species, the high density of the adrenergic fibers has beenproven (Janig and McLachlan1987; Lakomy et al.1989,1990) This could be related

to the change of function of the caudal part of the urinary bladder from ‘‘storage’’ to

References

Andersson KE (2002) Bladder activation: afferent mechanisms Urology 59(5A):43–50 Birder L, de Groat WC, Mills I, Morrison J, Thor K, Drake M (2010) Neural control of the lower urinary tract: peripheral and spinal mechanisms Neurourol Urodyn 29:128–139

Gibbins IL, Morris JL (2000) Pathway specific expression of neuropeptides and autonomic control of the vasculature Regul Pept 93(1–3):93–107

Jänig W, McLachlan EM (1987) Organization of lumbar spinal outflow to distal colon and pelvic organs Physiol Rev 67(4):1332–1404

Lakomy M, Kaleczyc J, Wasowicz K (1989) Adrenergic innervation of the ureters, urinary bladder, and urethra in pigs Gegenbaurs Morphol Jahrb 135(2):347–355

Lakomy M, Wasowicz K, Kaleczyc J, Chmielewski S (1990) AChE-positive innervation of the ureters, urinary bladder, and urethra in pigs Z Mikrosk Anat Forsch 104(2):316–326 Majewski M (1999) Synaptogenesis and structure of the autonomic ganglia Folia Morphol (Warsz) 58(3 Suppl 2):65–99 (Review)

Pidsudko Z (2004) Distribution and chemical coding of neurons in intramural ganglia of the porcine urinary bladder trigone Folia Histochem Cytobiol 42:3–11

Sakai H, Yonemaru K, Takeda M, Niimi K, Murakami M, Hirata A, Yanai T (2011) Ganglioneuroma in the urinary bladder of a dog J Vet Med Sci 73(6):801–803

Saulnier-Troff FG, Busoni V, Hamaide A (2008) A technique for resection of invasive tumors involving the trigone area of the bladder in dogs: preliminary results in two dogs Vet Surg 37:427–437

Shokeir AA (2004) Squamous cell carcinoma of the bladder: pathology, diagnosis and treatment BJU Int 93:216–220

Skobowiat C, Calka J, Wasowicz K, Majewski M (2010) Distribution pattern and chemical coding of neurons of the sympathetic chain ganglia supplying the descending colon in the pig Acta Vet Hung 58(2):189–198

Wilson HM, Chun R, Larson VS, Kurzman ID, Vail DM (2007) Clinical signs, treatments, and outcome in cats with transitional cell carcinoma of the urinary bladder: 20 cases (1990–2004).

J Am Vet Med Assoc 231:101–106

Chapter 6

In Vivo Applications of Mesenchymal

Stem Cells and Platelet-Rich Plasma

to Improve Tendon Regeneration in Sheep

M Patruno, I Bronzini, L Maccatrozzo, A Perazzi, I Iacopetti,

G M De Benedictis, S Testoni, A Negro, F Mascarello

and T Martinello

Abstract The ‘‘restitutio ad integrum’’ pursue in the treatment of tenodesmiclesions might represents a tangible target thanks to the increased number of novelcellular-based therapies In this work, we evaluated the efficacy of the application

of platelet-rich plasma (PRP), mesenchymal stem cells (MSCs), and PRP ? MSCs

to experimentally injured sheep deep digital flexor tendon (DM n° 97/2010-B).Our results indicate that the in vivo integration of injected MSCs was successful asverified by the presence of green fluorescent protein GFP-positive cells.Tissue architecture and the tendon linear fiber pattern were significantly improved

on histologic sections, especially after the use of MSCs, and the clinic evaluationwas also satisfactory after the use of PRP

Keywords Adult stem cells
Platelet-rich plasma
Tendon regeneration

6.1 Introduction

In veterinary medicine, the tenodesmic pathologies are clinically relevant because:(1) they occur very often; (2) it is difficult to obtain a complete functionalrecovery; and (3) they cause long periods of inactivity The commonly usedtherapeutical protocols so far does not involve achieving a ‘‘restitutio ad

M Patruno ( &)
I Bronzini
L Maccatrozzo
F Mascarello
T Martinello

Department of Comparative Biomedicine and Food Science, University of Padova,

Legnaro, Italy

e-mail: marco.pat@unipd.it

A Perazzi
I Iacopetti
G M De Benedictis
S Testoni

Department of Department of Animal Medicine, Production and Health,

University of Padova, Legnaro, Italy

A Negro

Department of Biomedical Science, University of Padova, Padua, Italy

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

DOI: 10.1007/978-3-642-36488-4_6, Ó Springer-Verlag Berlin Heidelberg 2013

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integrum’’, and therefore the research community continues to look for newtherapies that are able to increase the regeneration of damaged tissue In particular,the advantages of using mesenchymal stem cells (MSCs) isolated from adulttissues (Martinello et al.2010) and biomaterials such as platelet-rich plasma (PRP)are being investigated The therapeutical potential based on the regenerativecapability of stem cells is a real possibility, even in veterinary medicine (Ferrari

et al.2007; Crovace et al 2010; Watts et al.2011), and the beneficial effects ofgrowth factors found in PRP are already known (Cenni et al.2010) Moreover, theassociation of MSCs and PRP likely will allow for improved in vivo growth ofimplanted cells; therefore, their combined use represents an intriguing option inthe treatment of tenodesmic pathologies in the veterinary orthopedic fields.Our research aims to study the efficacy of MSCs, together with the use of PRP,implanted in experimentally injured tendons of sheep

6.2 Materials and Methods

All protocols used in this research involving live animals were approved by theUniversity Ethics Committee for Animal Experimentation and by the ItalianMinistry of Health on 17 May 2010 (DM n° 97/2010-B) The project was dividedinto three phases: (1) bilateral induction of experimental tendon lesions in threegroups of sheep; (2) monolateral application of PRP in one group, MSCs in thesecond group, and both PRP ? MSCs in the third group, with a clinical follow-upfor all groups; and (3) post mortem histological analysis of tendons For eachsheep, the left hind limb received one of the three treatments, and the right hindlimb was injected with the same volume of saline solution phosphate-bufferedsaline (PBS) and served as the internal control (placebo)

Nine female Bergamasca sheep homogeneous for size and age were used in thisstudy Parasitological and biochemistry exams were carried out to ensure the goodhealth of the subjects Sheep were sedated by intravenous administration of0.2 mg/kg Metadone (Eptadone) and 10 lg/kg Medetomidine (SedatorÒ) andpositioned in lateral recumbency Into each deep digital flexor tendon (DDFT),

500 IU filter sterilized bacterial Collagenase type 1A (C-9891, Sigma, Milan,Italy) was injected bilaterally (left and right hind legs) under ultrasonographicguidance The injection was performed using a 23 gage needle positioned 15 cm atthe proximal–distal direction from the calcaneal bone Seven days after creation ofthe lesions, the animals were treated with one of the three methods describedabove

1 ml of PRP (972 9 106platelets) was prepared following standard proceduresand inoculated as a liquid, taking advantage of the intrinsic coagulation factors thatinduce its gelification in situ once injected One hundred milliliters of peripheralblood were collected from the jugular vein of sheep 8 weeks before treatment Toisolate MSCs, a Ficoll-paque solution (Amersham Biosciences) was usedaccording to a protocol described previously for horse but adaptable for sheep

(Martinello et al 2010) Isolated cells were also transfected with a plasmidexpressing the green fluorescent protein (GFP) A total of 15 9 106MSCs wereused for each inoculation in 1 ml hyaluronic acid (Hyalgan, Fidia, Padova, Italy)solution as a vector solution for the second group of sheep and with 1 ml PRP forthe third group The efficacy of each treatment was monitored through clinical andultrasound examinations conducted regularly until sacrifice of the animal 1 monthafter treatment Histological and immunohistochemical analyses were carried out

to evaluate different parameters involved in tendon regeneration: the alignment ofthe collagen fibers, the amount, and morphology of new cells, the grade ofvascularization, the presence of inflammatory elements, and the expression ofcollagen types I and III together with matrix fibrillar proteins

6.3 Results

The clinical and ultrasound examinations revealed that in all subjects, a localreaction to the collagenase injection was evident as well as the formation of aperitendinous edema associated with loss of ecogenity of the tissue The clinicalfollow-up results were similar in all three groups of treated animals, and the treatedlimbs showed a quicker reduction of clinical signs due to the inflammatoryresponse respect to control limbs The ecographic controls at 7, 14, 21, and

28 days from the treatment indicated a slow but progressive filling of the anechoicarea in the treated tendons

Regarding the histological profile, we observed complete disorganization of thecontrol tendons (untreated lesions, placebo), whereas in the subjects treated withPRP, better tissue organization was seen, although tenoblasts appeared still round

in shape By contrast, in subjects treated with MSCs, better alignment of collagenfibers was observed, tenoblasts appeared similar to proper tenocytes, and a higherexpression of collagen I was detected The expression of collagen III was observed

in the placebo and weakly in tendons treated with PRP but not in tendons treatedwith MSCs The expression of matrix proteins cartilage oligomeric matrix protein(COMP) and biglycan was increased in all treated tendons The presence andviability of injected cells was confirmed using an antibody against GFP in tendonstreated with MSCs alone and PRP ? MSCs

6.4 Discussion

Our data allow for speculation about the ability of adult stem cells to improveregenerative potential of tendon, because treatment with these cells stimulatedmechanisms that resulted in histological features similar to native tendons It isalways very difficult to evaluate the quality of matrix produced using clinical andultrasound investigations, and our results did not show significant differences in

6 In Vivo Applications of Mesenchymal Stem Cells and Platelet-Rich Plasma 33

outcomes after the use different treatments (PRP, MSCs, and PRP ? MSCs).Histological examinations indicated that all treatments reduced the vascularizationand inflammatory response Treatment with PRP improved the general organiza-tion of injured tendons However, the use of MSCs seemed to increase the quality

of the repaired tendons according to histological features and because of a lack ofexpression of collagen III; the latter is a collagen type that is present in naturalhealing tendons but does not allow full recovery of the mechanical characteristics

of tendons Moreover, only tendons treated with MSCs exhibited an elongatedmorphology that is similar to that of quiescent tenocytes

Ferrari M, Corradi A, Lazzaretti M, De’Cillà M, Losi CG, Villa R, Lanfranchi A (2007) Adult stem cells: perspectives for therapeutic applications Vet Res Commun 31(Suppl 1):1–8 Martinello T, Bronzini I, Maccatrozzo L, Iacopetti I, Sampaolesi M, Mascarello F, Patruno M (2010) Cryopreservation does not affect the stem characteristics of multipotent cells isolated from equine peripheral blood Tissue Eng Part C 16:771–781

Watts AE, Yeager AE, Kopyov OV, Nixon AJ (2011) Fetal derived embryonic-like stem cells improve healing in a large animal flexor tendonitis model Stem Cell Res Ther 2:4 doi: 10.1186/scrt45

Chapter 7

Plasma Fatty Acid Profiles During

the First Year in Dogs

with and without Hip Dysplasia:

Preliminary Results

L Tidu, N Bacciu, G Rucco, S Nardi, M Santoro and B Renaville

Abstract At the Military Veterinary Center of Grossetto, where operative dogsfrom the Italian Army are raised and trained, more than 70 % of the discharges forunfitness are due to articular pathologies like hip and elbow dysplasia The aim ofthis study was to investigate fatty acid metabolism of dogs during the growthphase, and its modulation by a fish-based diet Only 2 out of the 32 subjects wereaffected by articular pathologies during the study Still, both subjects had lowerlevels of arachidonic acid and higher levels of docosahexaenoic acid Moreover,

we observed that the ratio of eicosapentaenoic to docosahexaenoic acids, anindicator of delta-6 desaturase activity, drops dramatically during the first year inthe German shepherd

Keywords Dog
Dysplasia
Fatty acids

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

DOI: 10.1007/978-3-642-36488-4_7, Ó Springer-Verlag Berlin Heidelberg 2013

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