RICERCA ITALIANA     

Research program

Quality, quality perception and product traceability in sheep meat production systems

University Co-ordinator

Università degli Studi di FIRENZE - SCIENZE ZOOTECNICHE - FIRENZE(FI)

Research Unit Leader

Alessandro GIORGETTI

Description

The RU of Florence will work in collaboration with the RUs of Udine (coordinator), Perugia, Campobasso and Catania studying local
and endangered breeds (Udine: Istriana; Campobasso: Merinizzata Italiana ; Perugia: Appenninica; Catania: Comisana;
Firenze: Pomarancina). Florence will attend to study the production of Pomarancina lamb, and the genetic characterization of all breeds considered by the other RUs.

In the phase 1, three type of lambs will be reared: semi-extensive, with prevalence of pasture end semi-intensive, with prevalence of indoor feeding; moreover for the semi-extensive typology will be considered the lambs of the two main periods of productions (fall and spring) for a total number of 36 lambs. The lambs will be reared following a common methodology developed by all RU's.
The lambs will be raised and fed according to a common methodology with the other RUs, also in the respect of the prerogatives of
the areas (Pomarance, Volterra, Castelnuovo Val di Cecina, Montecatini Val di Cecina, in province of Pisa). For this aim farms able to lodge these tests will be spotted.
During all the phases of rearing, will be carried out, as in the other RUs, the regular sampling of lambs feed intakes. These will
be subsequently sent to the UR engaged in the authentication of the meat. The methodologies of slaughtering, preparation and evaluation
of the carcass, reliefs, and procedures of sample conservation, the dissection of the main cut, will be suggested from the Scientific
Association of Animal Production (A.S.P.A., 1991. Metodologie relative alla macellazione degli animali di interesse zootecnico e
alla valutazione e dissezione della loro carcassa. ISMEA, Roma).

Phase 1 will be concluded with the mutual exchange of the samples
between all RUs. It will be done (according to the possibility to find the planned number of heads), 24 subjects for the semi-extensive typology of rearing and feeding of the mother, and 12 for the semi-intensive one. As the semi-extensive typology as concerning, 12 lambs will be produced in autumn and 12 in the spring. All the 36 heads will be homogenous for alive weight and state of fatness.
Water, milk, forages (grazed and not) and concentrate rations used for feeding the lambs and the mothers will be sampled, conserved and sent to the RU of Udine and Catania, engaged in the authentication of the meat. To the attainment of the previewed weight, lambs will be transported in a slaughtering centre and working of the endowed carcass, brand UE for the ovines. The carcasses, obtained using suggested methodology from the Scientific Association of Animal Production (A.S.P.A., 1991. op. cit.),after weightening and evaluation(Reg. the EEC n. 2137/1992 and n. 461/1993 and modifications and integrations) within an hour from slaughtering, will be maintained to room temperature (> 10°C) for 6 hours before cooling at 2°C.
After 24 hours from the slaughtering, every carcass will be weighted and measured (A.S.P.A., 1991. op. cit.).
Carcasses of every type will be photographed to obtain the standard indices. After the slaughtering, the metacarpial and metatarsial bones and perirenal fat will be collected from the right half-carcass of each experimental lamb (n =36). The bones will be frozen and under vacuum packed to be sent to the Campobasso RU for the evaluation of morphometric parameters (length, maximum diaphisary's diameter, weight and humidity) and metacarpial cartilage thickness detection. Samples of perirenal fat will be immediately frozen, under vacuum packed and wrapped in aluminum sheet, to be sent to the Catania RU, for the spectrophotometric analysis and terphens determination.

Therefore, in correspondence of the cross-section between muscles longissimus thoracis and lumborum, will be checked in triple sample: the pH (by pH meter, able to check temperature), the colour (L coordinates *, to * and b *, arrange CIELAB, using a portable colorimeter Minolta) and the water retention.
After the period of refrigeration, the right pelvic limb of 8 subjects (chosen between the most representative of each
typology and homogenous between them) will be cutted according to A.S.P.A. (1991, op. cit.), and dissected in order to estimate
the incidence of the fractions of lean, fat and bone. The parts will be packed under vacuum and conserved at -20°C. The meat samples
will be taken after 24 h from the slaughtering, under vacuum packed and frozen at -20°C, immediately or after 6 days of refrigeration
(based on the type analysis). The samples will be sent to the RUs of competence. To the RU of Florence the lambs' blood samples
will arrive before slaughtering for the genetic characterization. All the RU's will send samples of meat and food to the others, adopting the necessary measures to keep the chain of the cold, for the analysis.

Phase 2 will be dedicated to chemical-bromatological, physical-sensorial analyses of samples of meat and feed in order to obtain:

A) characterization of the meat produced from all typology of lamb;
B) verifying of the authenticity of the geographical origin and of the lambs feeding;
C) genetical characterization of the breeds.

Every RU will carry out analyses on own breed, with the exception of the consumer test, that will be carry out exclusively on the samples of meat from the own typology, under the coordination of the RU of Udine, and according to the central location test methodology.

The RU of Florence will test blood and hair samples from every RU (40-50 samples for each breed) to perform the genetic characterization.
The samples should be collected from ewes and rams pertaing to at least 5 heards for each breed. We suggest the utilisation of 6/8 ewes and 1/2 rams (avoiding sibs and half-sib)in each heard.
To obtain DNA from the blood samples will be used dedicated commercial DNA extraction's kit ; To obtain DNA from hair samples will be used corrected Huff '93 metodologies.
In order to reveal the existing level of polimorfism within and between the studied breeds we will amplify 15 microsatellite loci.
These microsatellite are the one choosed by ISAG to evaluate paternity test, and are divided in two multiplexable panels.
Multiplex 1: CSRD0247 - HSC – INRA0063 – MAF0214 – OarAE0129 – OarCP0049 – OarFCB0011 – OarFCB0304; Multiplex 2:
D5S2 - INRA0005 – INRA0023 – MAF0065 – McM0527 – OarFCB0020 – SPS0113 (proposed by Laboratorio Gruppi Sanguigni,
LGS Cremona).
MICROSATELLITES CHARACTERISTICS
Locus Primer sequences Dye
CSRD0247 GGA CTT GCC AGA ACT CTG CAA T HEX
CAC TGT GGT TTG TAT TAG TCA GG
HSC CTG CCA ATG CAG AGA CAC AAG A 6'FAM
GTC TGT CTC CTG TCT TGT CAT C
INRA0063 GAC CAC AAA GGG ATT TGC ACA AGC 6'FAM
AAA CCA CAG AAA TGC TTG GAA G
MAF0214 AAT GCA GGA GAT CTG AGG CAG GGA CG TET
GGG TGA TCT TAG GGA GGT TTT GGA GG
OarAE0129 AAT CCA GTG TGT GAA AGA CTA ATC CAG TET
GTA GAT CAA GAT ATA GAA TAT TTT TCA ACA CC
OarCP0049 CAG ACA CGG CTT AGC AAC TAA ACG C HEX
GTG GGG ATG AAT ATT CCT TCA TAA GG
OarFCB0011 GCA AGC AGG TTC TTT ACC ACT AGC ACC 6'FAM
GTG GGG ATG AAT ATT CCT TCA TAA GG
OarFCB0304 CCC TAG GAG CTT TCA ATA AAG AAT CGG HEX
CGC TGC TGT CAA CTG GGT CAG GG
Multiplex 2
Locus Primer sequences Dye
D5S2 TAC TCG TAG GGC AGG CTG CCT G 6'FAM
GAG ACC TCA GGG TTG GTG ATC AG
INRA0005 GTC CAT TGC CTC AAA TCA ATT C HEX
AAA CCA CTT GAC TAC TCC CCA A
INRA0023 GAG TAG AGC TAC AAG ATA AAC TTC TET
TAA CTA CAG GGT GTT AGA TGA ACT C
MAF0065 AAA GGC CAG AGT ATG CAA TTA GGA G TET
CCA CTC CTC CTG AGA ATA TAA CAT G
McM0527 GTC CAT TGC CTC AAA TCA ATT C HEX
AAA CCA CTT GAC TAC TCC CCA A
OarFCB0020 GGA AAA CCC CCA TAT ATA CCT ATA C 6'FAM
AAA TGT GTT TAA GAT TCC ATA CAT GTG
SPS0113 AAA GTG ACA CAA CAG CTT CTC CAG 6'FAM AAC GAG TGT CCT AGT TTG GCT GTG
PCR CONDITIONS
Multiplex 1
DNA 100 ng
Buffer 10X 1 microliter
MgCl2 (25 mM) 1,2 microliter
DNTPs (10 mM) 0,4 microliterl
Taq Gold (5U/ microliter) 0,4 microliter
Primers (10 microM):
CSRD247 0,4 + 0,4 microliter
HSC 0,3 + 0,3 microliter
INRA0063 0,2 + 0,2 microliter
MAF0214 0,5 + 0,5 microliter
OarAE0129 0,2 + 0,2 microliter
OarCP0049 0,05 + 0,05 microliter
OarFCB0011 0,15 + 0,15 microliter
OarFCB0304 0,3 + 0,3 microliter
In a total volume of 10 microliter
In a total volume of 10 microliter
AMPLIFICATION PROGRAMME
95°C x 12' 31 x (95°C x 20" - 63°C x 1' - 72°C x 1'
Multiplex 2
DNA 100 ng
Buffer 10X 1 microliter
MgCl2 (25 mM) 1,2 microliter
DNTPs (10 mM) 0,4 microliter
Taq Gold (5U/ microliter) 0,4 microliter
Primers (10 microM):
D5S2 0,2 + 0,2 microliter
INRA0005 0,3 + 0,3 microliter
INRA0023 1,0 + 1,0 microliter
MAF0065 0,4 + 0,4 microliter
McM0527 0,2 + 0,2 microliter
OarFCB0020 0,4 + 0,4 microliter
SPS0113 0,1 + 0,1 microliter
In a total volume of 10 microliter
Amplification programme
95°C x 10' 31 x (94°C x 30" - 55°C x 30" - 72°C x 1'
Microsatellite characteristic
Locus Exp Size
MULTIPLEX 2
CSRD0247 209 - 261
HSC 267 - 301
INRA0063 169 - 207
MAF0214 181 - 265
OarAE0129 135 - 165
OarCP0049 82 - 140
OarFCB0011 122 - 148
OarFCB0304 148 - 190
MULTIPLEX 2
D5S2 190 - 210
INRA0005 120 - 180
INRA0023 201 - 219
MAF0065 121 - 159
McM0527 165 - 179
OarFCB0020 92 - 118
SPS0113 130 – 158
Finally the amplified regions will be analyse by a capillary sequencer to obtain the exact allelic number and dimension.
The data derived from the sizing will treat as follows: in order to obtain information on population's structure we'll perform an
analisys of molecular variance (AMOVA); this type of analisys estimates the level of between- and within population genetic variation.
Tests for significant genic differentiation among population pairs will be performed using exact tests for genetic heterogeneity and
allele frequencies for all populations. Wright's F-statistics (FST) will subsequently estimated in order to measure the extent of
genetic differentiation between populations using the classical estimator "teta" of Weir & Cockerham (1984).
Indices of genetic similarity/dissimilarity between populations will be calculated; then the following genetic distances will be evaluated:
Sanghvi's distance (1953).
Cavalli Sforza e Edwards's distance (1967)
Nei Standard's distance (1972)
Nei's Minimum distance (1973).
Nei s Da distance (revisionata 1983).
Reynolds's distance (1983)
Golstein's distance (1995)
The genetic distances are a powerful to draw phylogenetic trees to describe both the current population structure and the evolutionary history of sub populations. The most suitable methods to design trees are neighbour-joining and maximum parsimony.
In this study, individual assignment based on microsatellite data will be use to indirectly identify the level of genic flux between
studied populations. For this type of test will be used methods based on the likelihood like frequency method and Bayesian one, and
methods based on the genetic distance. Finally, methods based on the belonging probability will be used. In order to complete this kind of
analisys we'll perform the extinction probability test.
Furthermore the total contribution of genetic diversity to a set of breeds will be analysed.
This methodology employes the allelic richness as discriminating measure, in place of the most common indices such as heterozigosity or number of polymorfic loci.
This means that this indicator takes in consideration the unicity of the population studied, in terms of allelic patrimony, stressing the
level of biodiversity that every breed brings within a particular set of other populations.
The calculation of this estimator involves the following parameters:
Diversity within population (Hs)
Total Diversity (Ht).
Coefficient of genic differentiation (Gst).
Divergences from the other populations (DHs, DHt, DGst).
Contribution to the total allelic richness (Crt).