Animal, page 1 of 10 © The Animal Consortium 2019

doi:10.1017/S1751731119000405
animal

Meat quality traits and the expression of tenderness-related

genes in the loins of young goats at different ages
E. Saccà†, M. Corazzin, S. Bovolenta and E. Piasentier
Department of Agricultural and Environmental Sciences, University of Udine, Via Sondrio, 2/A, 33100 Udine, Italy

(Received 30 May 2018; Accepted 7 February 2019)

Goat meat is considered healthy because of its low fat content, but it is often rather tough. Tenderness is the most important
attribute of quality during meat consumption and there is scarce information about the expression of genes involved in the meat
tenderization process in goats. The aim of this trial was to assess certain meat quality traits and the expression, at the messenger
RNA (mRNA) and protein levels, of specific genes involved in the tenderization process of the longissimus lumborum (LL) in young
male goats (Capra hircus) at different ages. Samples of LL were collected at slaughter from 32 Alpine goats that were divided into
three categories: 9 suckling kids (Sk) at 5.4 ± 0.15 weeks of age, 16 chevons (Ch) at 17.1 ± 0.55 weeks of age and 7 post-puberal
goats (Pu) at 34.3 ± 2.5 weeks of age. Animal and carcass variables (live weight gain, live weight, carcass weight and fat deposits)
and quality traits of meat (lipid content, ultimate pH, color parameters, cooking loss and shear force) were determined. The mRNA
abundances of calpain-1 (Capn1), calpain-2 (Capn2), calpastatin (Cast), caspase 3 (Casp3), caspase 9 (Casp9), αB-crystallin (Cryab),
heat shock protein 27 (Hsp27), heat shock protein 40 (Hsp40) and heat shock protein 70 (Hsp70) were detected by quantitative
PCR. Capn1, Cast, Cryab and Hsp27 protein expression was investigated by ELISA. The Sk group had the leanest carcasses.
The meat of the Pu group was the darkest (P < 0.05) and the toughest (P < 0.05). The redness of meat increased with the age of
the goats. The Sk group showed lower mRNA abundances for the Capn2/Cast ratio, Casp3, Cryab, Hsp27, Hsp40 and Hsp70 than
the Pu group (P < 0.05). Intermediate values were found for the Ch group. Similar results were highlighted for the protein
expression of Cryab and Hsp27. The experiment acknowledged a differentiation of the experimental groups based on performance,
carcass and meat characteristics, and the genes considered. Moreover, Sk and Pu groups, characterized by a different tenderness of
their meat, were clearly discriminated by a different expression of the Hsp.

Keywords: calpains, caspases, heat shock proteins, PCR, ELISA method

Implications population with values that exceeded 37% and 11% in

Goat husbandry is widespread worldwide and its importance
could increase in the future to satisfy the food demand of a
growing world population and the demand for quality and
healthy products by the consumer. Our study led to a deeper
knowledge of the relationships between certain genes considered
and the quality of meat, helping the farmers to obtain a product
meeting consumers’ expectation. In particular, our experiment
has highlighted the importance of some genes involved in goat
meat tenderness, opening the possibility to consider them as
markers of this fundamental aspect of meat quality.
Introduction
Goat husbandry is widespread worldwide, and in the last 10
years, there has been a 19% increase of the world’s caprine
†
E-mail: elena.sacca@uniud.it
Africa and Asia, respectively. Moreover, these continents will
have a population increase of more than 80% over the next
20 years (Food and Agriculture Organization of the United
Nations, 2018). Therefore, goat meat can play a role in
satisfying the growing demand of products of animal origin
on these continents. Goat meat can also satisfy the demand
for healthy products by consumers in developed countries. In
fact, goat meat has a low intramuscular fat content ranging
between 0.6% and 2.6%, is rich in protein of high biological
value and is high in beneficial fatty acids, such as α-linolenic
and CLA (Webb et al., 2005). The consumption of goat meat
is linked to cultural and geographical factors. For marketing
purposes, goat meat could be divided into two classes: one
that was derived from young suckling animals (i.e. capretto,
cabrito) producing carcasses of 6 to 12 kg, and chevon, one
derived from older goats producing carcasses of 16 to 22 kg.
In some countries of the Mediterranean basin, such as Italy,
capretto/cabrito production is traditional and still the main

1
Saccà, Corazzin, Bovolenta and Piasentier
meat product from goats. Less frequently, chevon is con-
sidered for consumption. Conversely, meat from heavier
animals are preferred in other parts of the world, such as
North America.
Tenderness is the most important factor influencing con-
sumer satisfaction for meat (Argüello et al., 2012). The low
acceptability of some consumers toward goat meat is due to
the frequent lack of tenderness (Webb et al., 2005). Tender-
ness is affected by many factors, such as the species, breed
and age of the animal, and, at the muscle level, by the amount
of collagen, intramuscular fat content, pH postmortem decline,
and the size and type of muscle fibers. Based on what is
known in cattle, many enzymatic systems are involved in
protein degradation and are taken into consideration in the
postmortem meat tenderization process (Kemp et al., 2010;
Saccà et al., 2018). Among endogenous proteinases, calpains
play a primary role in meat tenderness development during
aging. Calpain-1 and calpain-2 are calcium-activated pro-
teases that can hydrolyze the myofibrillar and cytoskeletal
proteins (such as troponin-T, α-actinin, myosin and titin,
nebulin, filamin and desmin) and cause structural changes
during meat aging. Conversely, the high activity of their spe-
cific inhibitor, calpastatin, is related to the low degradation of
myofibrillar proteins and low meat tenderness (Koohmaraie
and Geesink, 2006).
Becila et al. (2010) provided evidence that the hypoxic/
ischemic conditions that occur during the slaughter of animals
could activate muscle cell death postmortem via an apoptotic
process. There is growing interest in investigating the invol-
vement of apoptosis in meat tenderization. Caspases are the
main enzymes responsible for apoptosis. They can be initia-
tors (such as caspases 8 and 9) or effectors (such as caspases
3 and 7). The predominant pathway of apoptosis in post-
mortem skeletal muscle occurs with the action of caspases
9 and 3 (Herrera-Mendez et al., 2006). Initiator caspases are
activated in response to environmental stresses and can
activate effector caspases that can cleave specific substrates,
including myofibrillar and cytoskeletal proteins (Brad Kim
et al., 2018). Despite this capacity to degrade a large number
of muscle proteins, caspases role in the meat tenderization
process is still debated (Saccà et al., 2015).
However, the state of stress caused by the ischemia that
characterizes postmortem conditions could also activate
some cell survival processes that involve increasing the
concentration of several heat shock proteins (Hsp). In rela-
tion to their physiological role as chaperones, anti-apoptotic
and anti-oxidative factors, heat shock proteins are believed
to be involved in the transformation of muscle to meat
(Cassar-Malek and Picard, 2016). In particular, Hsp27 can
interrupt the apoptosis cascade at many levels, and together
with other small Hsp, such as αB-crystallin and Hsp20, it can
stabilize and protect muscle proteins (such as desmin and
titin) to calpains action (Lomiwes et al., 2014). Hsp70 to 40
have been identified as regulators and suppressors of the
apoptotic process by several authors.
Despite numerous reports on beef, scarce information is
available on the expression of the genes involved in the meat
2
tenderization process in goat, and, according to the biblio-
graphy, even less information is available on the variation of
the expression of these genes during animal growth (Lin
et al., 2017). It was hypothesized that the quality and the
tenderness in particular, of goat meat at different ages could
have a genetic origin. Therefore, the aim of this trial was to
assess meat quality traits and the expression of tenderness-
related genes, such as calpains, calpastatin, caspases and
Hsp, at both the messenger RNA (mRNA) and protein levels,
in the longissimus lumborum (LL) of young goats at
different ages.
Material and methods
Animals and treatments
Thirty-two young male goats of Alpine breed born on com-
mercial farms of the Friuli Venezia Giulia region (N-E Italy) at
the end of winter were considered. Animals were randomly
allotted into three groups, which included 9 suckling kids
(Sk), 16 chevons (Ch) and 7 post-puberal goats (Pu). After
birth, the kids were suckled by their dams up to weaning,
when the Sk group was slaughtered. After weaning, the
remaining kids were transferred to the experimental farm
and kept in a pen with straw bedding until slaughter.
Animals were fed ad libitum on a mixed diet comprising
400 g/day of meadow hay (913 g/kg dry matter (DM), 85 g/kg
DM ash, 87 g/kg DM CP, 23 g/kg DM ether extract, 646 g/kg
DM NDF) and 800 g/day of commercial concentrate. The
concentrate was made up of cereals by-products (wheat
middlings, distillers corn grains, wheat bran, 377 g/kg), cer-
eals grains (corn and barley, 304 g/kg), sunflower (100 g/kg),
soybean (80 g/kg) and rapeseed meal (28 g/kg), grape by-
products (22 g/kg), carob (10 g/kg), minerals, vitamins and
molasses (79 g/kg). The proximate analysis was as follows:
867 g/kg DM, 80 g/kg DM ash, 32 g/kg DM ether extract,
238 g/kg DM NDF, 177 g/kg DM CP, 269 g/kg DM starch and
7.67 MJ/kg DM net energy for fattening. The milk intake of Sk
was not measured. The young goats were weighed bi-weekly
(from birth to slaughter for the Sk group and from weaning to
slaughter for the Ch and Pu groups) to determine live weight
gain and on the day before slaughtering to determine the live
weight at slaughter. The slaughter occurred at an average
age of 5.4 ± 0.15 weeks for the Sk group, 17.1 ± 0.55 weeks
for the Ch group and 34.3 ± 0.47 weeks for the Pu group. The
animals in the Sk, Ch, and Pu groups were in the impuberal,
pre-puberal and post-puberal phases, respectively.
Sample collection and first measurements
For all analysis, the LL of the right side of the carcass was
considered. After slaughter, samples (5 g) of LL muscle were
obtained within 20 min of exsanguination, frozen in liquid
nitrogen and stored at −80°C until mRNA and protein
analysis.
Twenty-four hours after slaughter, the carcasses were
weighed to determine the cold carcass weight and visually
evaluated for subcutaneous and kidney fat deposition. In

particular, fat cover and kidney fat were assessed visually
using the available photographic standards, together with
the description of each, following the procedure of Colomer
Rocher et al. (1987). Ultimate pH was measured at five
points/loin using a pH-meter (HI 8424; Hanna Instruments,
Padova, Italy) equipped with a glass electrode (5232; Crison
Instruments, Barcelona, Spain). The samples for fat content
analysis were taken from the LL and immediately stored at
−20°C until analysis. After 48 h of aging at 4°C, the color of
the LL was measured by obtaining a 2 cm slice of the loin and
allowing for a blooming time of 45 min. Measurements were
obtained on five points/slice using a portable spectro-
photometer, Minolta CM 2600d (Konica Minolta, Tokyo,
Japan), with an 8 mm aperture, Standard Illuminant D65
light source and 10 viewing angle geometry. The values
recorded, according to the standard conditions of the Com-
mission Internationale de I’Eclairage (1986), included light-
ness (L*), redness (a*) and yellowness (b*). At the same time,
samples of LL were collected and aged at 4°C for 7 days until
cooking loss and Warner–Bratzler Shear Force (WBSF)
determination.
Cooking loss and Warner-Bratzler Shear Force
determinations
Slices of LL muscle of 2 cm thickness were cooked in plastic
bags using a water bath until they reached 75°C at the core
(Honikel, 1998). The internal temperature was monitored by
a thermocouple (Thermocouple Thermometer HD 9016; Delta
Ohm, Padova, Italy). Each slice of meat was weighed before
and after cooking (dried with paper). The cooking loss was
calculated as the difference between the weight before and
the weight after cooking and expressed as a percentage of
the initial weight.
The WBSF test was performed on cooked meat after
cooking loss determination. Seven cross-section cylinders
15 mm in diameter were cut along the fibers axis for each LL
sample. The cylinders were sheared perpendicularly of muscle
fibers using a Warner–Bratzler device equipped with a tri-
angular hole (60°) in the shear blade (1 mm thickness),
mounted on a Lloyd TA Plus texture analyzer (Lloyd, Borgnor
Regis, UK) and operating at a crosshead speed of 100 mm/
min. From the WB deformation curves, two parameters were
recorded: the peak yield force (PY, in N), which is the max-
imum peak yield of the force-deformation curve, and the final
yield (FY, in N), which is the second peak yield of the force-
deformation curve. The first peak is taken as a measurement
of the myofibrillar component of tenderness and the second
is taken as a measurement of the connective tissue compo-
nent of tenderness (Møller, 1981).
Intramuscular fat content analysis
Two grams of minced meat were homogenized in 40 ml of
chloroform–methanol mixture (2 : 1 v/v) using an Ultra-
Turrax homogenizer (T 25 basic; IKA, Staufen, Germany) and
were subsequently filtered under a vacuum through What-
man filter paper (1820-047; GE Healthcare Life Sciences,
Pittsburgh, PA, USA). The extract was washed with 10 mL of
Tenderness-related gene expression in young goats
0.88% (w/v) KCl, mixed vigorously for 1 min and then left
overnight at room temperature. The organic phase was
separated and the solvents were evaporated at 40°C with a
Univapo Vacuum Concentrator (100EHC; Elettrofor Scientific
Instruments, Rovigo, Italy). The fat amount was calculated by
the difference between the weights before and after eva-
poration and expressed as grams of lipids per 100 g of
muscle tissue.
RNA expression analysis
Ribonucleic acid analysis was made in according to previous
experiments (Saccà et al., 2015), with some modifications, in
particular regarding the sequence of some primers used.
RNA extraction. Total RNA was extracted from 40 mg of
muscle (powdered with a mortar and a pestle in liquid
nitrogen) using the RNeasy Fibrous Tissue Mini Kit (Qiagen,
Hilden, Germany) according to the manufacturer’s protocol,
DNase treatment included. The concentration and purity of
extracted RNA were assessed using a NanoDrop 2000c
spectrophotometer (Thermo Fisher Scientific, Waltham, MA,
USA). The integrity of RNA (presence of intact 18S and 28S
ribosomal RNA bands) and absence of genomic DNA were
assessed by agarose gel (1% in tris-borate ethylendiamine-
tetracetic acid (TBE) buffer) electrophoresis, comparing with
molecular weight standards.
Retro-transcription. To obtain complementary DNA (cDNA),
the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA, USA)
was used. Each 20 μl of reaction contained 4 μl of reverse-
transcription reaction mix, 1 μl of iScript reverse transcriptase
(moloney murine leukemia virus-derived RNase H + ), the
volume of RNA solution necessary to produce a final con-
centration of 50 ng/μl RNA, and nuclease-free water to final
volume. The mixture was held for 5 min at 25°C, 1 h at 42°C
and 5 min at 85°C before being cooled on ice.
Primers validation. A qualitative PCR was carried out on six
randomly chosen samples to validate the primers pairs
(Supplementary Table S1) specificity for all the genes exam-
ined: calpain-1 (Capn1), calpain-2 (Capn2), calpastatin
(Cast), caspase 3 (Casp3), caspase 9 (Casp9), αB-crystalline
(Cryab), heat shock protein 27 (Hsp27), heat shock protein
40 (Hsp40), heat shock protein 70 (Hsp70) and reference
genes. Polymerase chain reaction was performed using the
Bio-Rad CFX96 system (Bio-Rad) on a reaction volume of
20 μl containing 0.3 μl of each forward and reverse primer
(0.3 μM), 10 μl of iQ SYBR Green Supermix (Bio-Rad), 8.4 μl
of sterile water and 1 μl of cDNA. Amplification conditions
included one cycle of 3 min at 95°C, 40 PCR cycles (15 s at
95°C, 30 s at 60°C), 30 s at 72°C and 1 min at 95°C, followed
by a melt curve of 55°C to 95°C with increments of 0.5°C
every 5 s. Unspecific amplifications and primer-dimers for-
mation were excluded by checking melt curves. Amplicons
length (Supplementary Table S1) was verified by agarose gel
(1.5% in TBE buffer) electrophoresis, comparing with mole-
cular weight standards.
3
Saccà, Corazzin, Bovolenta and Piasentier
Quantitative PCR. Quantitative PCR was performed on each
sample for every gene examined using the same instrument,
reaction reagents and volumes, and amplification conditions
that were used in the primers validation phase. Each sample
was analyzed in triplicate and relative gene expression was
calculated according to the efficiency-corrected (Pfaffl, 2001)
2−ΔΔCq method (Livak and Schmittgen, 2001). Cyclophilin, β-
actin, Glyceraldehyde-3-P dehydrogenase (GAPDH) and
Ribosomial Protein Large P0 (RPLP0) were treated as refer-
ence genes. Since all the reference genes showed an average
expression stability (M value) lower than 1 after GeNorm
analysis, their geometric mean was used for the normal-
ization of data (Vandesompele et al., 2002). Data are pre-
sented as the fold-change ratio with the Sk group as
reference. Primers efficiencies (Supplementary Table S1)
were calculated and verified using the standard curve
obtained by amplification of serial dilutions of the pooled
cDNA (Pfaffl, 2001).
ELISA analysis of calpain-1 and calpastatin
Protein extraction. Two hundred milligrams of the frozen
meat sample were homogenized on ice for 1 min using an
Ultra-Turrax T25 basic (IKA, Staufen, Germany) in 1 ml of ice-
cold extraction buffer (50 mM Tris/HCl, pH 8.3, 10 mM
ethylendiaminetetracetic acid (EDTA) and 0.05% v/v 2-mer-
captoethanol) and 10 µl of Protease Inhibitor Cocktail
(P8340; Sigma-Aldrich, Saint Louis, MO, USA). The homo-
genate was centrifuged at 30 000 g for 1 h at 4°C and the
supernatant was collected. Protein concentrations of the
muscle extracts were determined using Bradford reagent
(Sigma-Aldrich).
Quantification of calpain-1 and calpastatin. Capn1 and Cast
were quantified by ELISA. Muscle extracts were adjusted to
concentrations of 10 µg/ml protein with Tris-buffered saline
(TBS). One hundred microliters of samples was dispensed
into 96-well Costar® high binding polystyrene plates (3590;
Corning Inc., NY, USA) in duplicate and incubated for 2 h at
37°C. Following coating, the contents of the wells were
discarded and the wells were blocked with 200 μl/well of 1%
bovine serum albumin (BSA)-TBS for 1 h at room tempera-
ture. After that, wells were washed three times with TBS
containing 0.01% Tween-20 (TTBS) and then incubated for 1 h
at 37°C with 100 ml/well of the mouse monoclonal anti-
calpain-1 antibody (C0355; Sigma-Aldrich), diluted to 10 µg/ml
in 1% BSA-TTBS, or with 100 ml/well of the mouse monoclonal
anti-calpastatin antibody (SAB1402139; Sigma-Aldrich),
diluted to 15 µg/ml in 1% BSA-TTBS. Wells were then washed
three times with TTBS and the goat anti-mouse IgG HRP
conjugate (A4416; Sigma-Aldrich), diluted to 1 : 500 in 1%
BSA-TTBS, was applied for 1 h at 37°C. Following five washes
with TTBS, 100 ml/well of 3,3’,5,5’-tetramethylbenzidine
(TMB) substrate (T0440; Sigma-Aldrich) was applied. After
approximately 10 min at room temperature, the reaction was
stopped by adding 100 ml/well of 2 N H2SO4. The absorbance
was measured at 450 nm using a Tecan Sunrise plate reader
(Tecan Group Ltd., Männedorf, Switzerland). The relative
4
quantity of Capn1 and Cast was measured by optical
density (OD). Data are presented as the fold-change ratio
with the Sk group as a reference. The mean intra-assay CV
for Capn1 and Cast were 4.8% and 6.1%, respectively,
whereas the mean inter-assay CV for Capn1 and Cast were
10.5% and 12.7%, respectively. The limit of detection was
0.2020 OD for Capn1 and 0.2210 OD for Cast. The linearity
(R 2) of the calibration line was 0.981 for Capn1 and 0.985
for Cast.
ELISA analysis of heat shock protein 27 and αB-crystallin
Protein extraction. Two hundred milligrams of the frozen
meat sample were homogenized on ice for 1 min using an
Ultra-Turrax T25 basic (IKA, Staufen, Germany) in 1 ml of ice-
cold extraction buffer (25 mM HEPES pH 7.5, 0.1% Triton
X-100, 5 mM MgCl2, 1 mM EDTA and 1 mM 2-mercap-
toethanol) and 10 µl of Protease Inhibitor Cocktail (P8340;
Sigma-Aldrich) to produce the whole muscle homogenate.
The homogenate was centrifuged at 20 000 g for 10 min at
4°C, and the supernatant was collected (sarcoplasmic frac-
tion). Protein concentrations of the muscle extracts were
determined using Bradford reagent (Sigma-Aldrich).
Quantification of heat shock protein 27 and αB-crystallin.
Hsp27 and Cryab were quantified by ELISA. The sarcoplasmic
fraction was adjusted to concentrations of 10 µg/ml protein
with coating buffer (10 mM Na2HPO4, 15 mM NaCl; pH 7.4).
One hundred microliters of samples was dispensed into 96-
well Costar® high binding polystyrene plates (3590; Corning
Inc.) in duplicate. Plates were placed on an oscillating shaker
overnight at 4°C. Following coating, the contents of the wells
were discarded, and the wells were blocked with 200 μl/well
of 1% BSA wash buffer (10 mM Na2HPO4, 15 mM NaCl,
0.1% Tween 20; pH 7.4) for 1 h at room temperature, and
then washed three times with 200 µl/well wash buffer. Fol-
lowing washing, 100 μl/well of the mouse monoclonal anti-
body anti-Hsp27 (WH0003315M4; Sigma-Aldrich), diluted to
5 µg/ml in 1% BSA in assay buffer (0.01 M PBS, 0.01%
Tween 20) or anti-Cryab (WH0001410M1; Sigma-Aldrich),
diluted to 10 µg/ml in 1% BSA in assay buffer, were added
and incubated for 2 h at room temperature and then washed
as previously described. The goat anti-mouse IgG HRP con-
jugate (A4416) was diluted to 1 : 1000 in assay buffer (1%
BSA), and 100 μl/well aliquots were dispensed and incubated
for 1 h at room temperature. Following washing, 100 μl/well
of 3,3’,5,5’-TMB substrate (T0440; Sigma-Aldrich) was dis-
pensed and incubated for 30 min. The reaction was stopped
by dispensing 100 μl/well of 2 N H2SO4. The absorbance was
measured at 450 nm using a Tecan Sunrise plate reader
(Tecan Group Ltd., Männedorf, Switzerland). The relative
quantity of Hsp27 and Cryab was measured by OD. Data are
presented as the fold-change ratio with the Sk group as a
reference. The mean intra-assay CV for Cryab and Hsp27
were 4.4% and 4.8%, respectively, whereas the mean inter-
assay CV for Cryab and Hsp27 were 8.9% and 9.7%,
respectively. The limits of detection was 0.1505 OD for Cryab
 Tenderness-related gene expression in young goats

Table 1 Animal and carcass characteristics and lipid content of the Table 2 Ultimate pH, color characteristics, cooking loss (CL) and
longissimus lumborum of the young goats Warner–Bratzler Shear Force (WBSF) parameters of the longissimus
lumborum of the young goats
Sk Ch Pu SEM P-value
Sk Ch Pu SEM P-value
Animal (no.) 9 16 7 – –
Age at slaughter (weeks) 5.4 17.1 34.3 – – pHu 5.87 5.86 5.79 0.034 0.63
LWG (g/day) 196a 134b 124b 5.28 <0.01 L* 40.7a 39.4a 30.5b 0.477 <0.01
LW (kg) 11.4c 22.7b 32.4a 0.485 <0.01 a* 4.2c 6.1b 6.9a 0.165 <0.01
CCW (kg) 5.4c 10.0b 14.8a 0.277 <0.01 b* 11.5a 11.3a 9.1b 0.160 <0.01
Subcutaneous score (points) 1.6b 2.2a 2.0a
1
0.060 <0.01 CL (%) 18.1 16.6 15.2 0.902 0.51
Kidney score2 (points) 1.8 2.2 2.1 0.084 0.12 WBSF parameters (N)
Intramuscular fat (%) 1.4b 2.8a 2.2ab 0.160 <0.01 PY 28.0b 30.9b 38.2a 1.444 0.04
FY 25.2 24.9 30.8 1.327 0.18
Sk = suckling kids; Ch = chevons; Pu = post-puberal goats; LWG = live weight
gain (from birth to slaughter for Sk; from weaning to slaughter for Ch and Pu); Sk = suckling kids; Ch = chevons; Pu = post-puberal goats;
LW = live weight at slaughter; CCW = cold carcass weight. pHu = ultimate pH; L* = lightness; a* = redness; b* = yellowness; CL = cooking
a,b,c
Values within a row with different superscripts differ significantly at loss; PY = peak yield force; FY = final yield force.
P ⩽ 0.05. a,b
Values within a row with different superscripts differ significantly at P ⩽ 0.05.
1
From 1 (low fat cover) to 5 (very high fat cover) (Colomer Rocher et al., 1987).
2
From 1 (little) to 3 (excessive) (Colomer Rocher et al., 1987).
(b*) were higher in the Sk and Ch groups compared to the Pu
and 0.1210 OD for Hsp27. The linearity (R 2) of the calibration group (P < 0.05). Moreover, the Sk group showed the lowest
line was 0.979 for Cryab and 0.988 for Hsp27. redness (a*) value (P < 0.05). The cooking loss percentage
was not different among age classes (P > 0.05), and even for
Statistical analysis WBSF_FY, the difference between experimental groups did
Data analysis was performed using R software (3.4.0 ver- not reach the threshold of significance (P > 0.05). Con-
sion, R core team, 2017) with the packages emmeans versely, WBSF_PY was significantly lower in the Sk and Ch
(Lenth, 2018), multcomp (Hothorn et al., 2008) and mixOmics groups compared with the older group (P < 0.05).
(Le Cao et al., 2017). The normality of the data distribution
was tested using the Shapiro–Wilk test. The effect of Messenger RNA and protein expression
goat meat type on fatness scores was studied using The gene expression results in terms of mRNA and protein
the Kruskal–Wallis test and the non-parametric Mann– are shown in Tables 3 and 4, respectively. Since Cast is the
Whitney U-test. For the other variables, when appropriate, competitive inhibitor of Capn1 and Capn2, the calpain
non-parametrically distributed data were transformed for proteolytic system (Capn1, Capn2, Cast) is reported as
parametric testing. These variables were subjected to a Capn1/Cast and Capn2/Cast in tables. The Capn1/Cast ratio
one-way ANOVA with ‘goat type’ as a fixed effect. Tukey’s was similar between experimental groups, both in terms of
correction, adapted for an unequal sample size, was used mRNA abundance (P > 0.05) and protein expression
for multiple comparisons. Partial Least Squares Dis- (P > 0.05). The Capn2/Cast mRNA ratio was lower in younger
criminant Analysis (PLS-DA; Chevallier et al., 2006) was animals compared to the Pu group (P < 0.05). The Sk group
applied to check the efficacy of the data on in vivo perfor- also showed a lower level of Casp3 mRNA than the oldest
mances, carcass, meat and gene expression (X matrix) to goats (P < 0.05), and Casp9 mRNA abundance was sig-
discriminate the goat types, the matrix (Y matrix) of which nificantly different between the Sk and Ch groups (P < 0.05).
was made by defining a dummy variable for each of them. For Cryab and Hsp27, a significant increasing trend
The association networks between X and Y data sets were (P < 0.05) was observed with animal age. The expression of
generated using the ‘network’ function of the mixOmics Hsp40 was lower in the Sk group than in the Pu group
package (Le Cao et al., 2017). (P = 0.05); moreover, the expression of Hsp70 was lower in
the Sk group than in the older groups (P < 0.05). These
results are similar to those obtained with ELISA analysis; in
Results fact, the Cryab protein level in the Sk group was the lowest
Animal and meat characteristics among the three groups, and the Hsp27 level in the Sk group
The animal and carcass characteristics, together with the lipid was significantly lower than that in the Pu group (P = 0.05).
content of LL, are summarized in Table 1. The Sk group showed
a higher live weight gain compared to the other age classes Multivariate analysis
(P < 0.05). Regarding fat deposits in carcasses and meat, Sk A multivariate analysis was performed to verify the possibi-
was the leanest group (subcutaneous fat, P < 0.05), while for lity that animals’ features (in vivo performance, carcass and
the intramuscular fat, it was leaner than the Ch group. meat characteristics, and genes expression) considered could
The ultimate pH and physical parameters of LL meat are differentiate the experimental groups. A PLS-DA was thus
shown in Table 2. The ultimate pH values were similar carried out, and the loadings of X (standardized values of the
between groups (P > 0.05). Lightness (L*) and yellowness animals’ features) and Y (goat types) variables along

5
Saccà, Corazzin, Bovolenta and Piasentier

Table 3 Fold change ratio in relative RNA expression of the Table 5 Estimated loadings on the two first components of a Partial
tenderness-related genes in longissimus lumborum of the young goats Least Squares Discriminant Analysis model for the classification of the
goat types according to the age and the physiological condition
Sk Ch Pu SEM P-value (suckling kids (Sk); chevons (Ch); and post-puberal goats (Pu); Y
matrix), based on their performance, carcass and meat characteristics,
Capn1/Cast 1.00 0.99 1.06 0.066 0.08
and gene expression profile (X matrix)
Capn2/Cast 1.00a 1.02a 1.08b 0.067 0.03
Casp3 1.00a 1.51ab 1.78b 0.134 0.02 Component 1 Component 2
Casp9 1.00a 1.38b 1.19ab 0.133 <0.01
Cryab 1.00a 2.21b 5.83c 0.284 <0.01 X matrix1
Hsp27 1.00a 1.86b 3.07c 0.196 <0.01 LWG 0.304 −0.187
Hsp40 1.00a 1.14ab 1.45b 0.089 0.05 Subcutaneous fat score −0.189 0.385
Hsp70 1.00a 1.73b 2.33b 0.180 <0.01 Kidney fat score −0.135 0.166
Intramuscular fat −0.180 0.352
Sk = suckling kids; Ch = chevons; Pu = post-puberal goats; Capn1 = calpain-1;
Capn2 = calpain-2; Cast = calpastatin; Casp3 = caspase 3; Casp9 = caspase 9;
pHu 0.054 0.093
Cryab = αB-crystallin; Hsp27 = heat shock protein 27; Hsp40 = heat shock pro- L* 0.272 0.402
tein 40; Hsp70 = heat shock protein 70. a* −0.329 0.078
a,b,c
Values within a row with different superscripts differ significantly at b* 0.239 0.369
P ⩽ 0.05. CL 0.089 0.015
WBSF PY −0.164 −0.152
Table 4 Fold change ratio in relative protein quantity of the longissimus WBSF FY −0.087 −0.197
lumborum of the young goats Capn1/Cast2 −0.099 −0.243
Capn2/Cast2 −0.126 −0.211
Protein Sk Ch Pu SEM P-value
Casp32 −0.207 0.060
Capn1/Cast 1.00 0.83 0.78 0.061 0.32 Casp92 −0.165 0.356
Cryab 1.00a 1.58b 1.68b 0.145 0.03 Cryab2 −0.339 −0.134
Hsp27 1.00a 1.14ab 1.20b 0.031 0.05 Hsp272 −0.337 −0.034
Hsp402 −0.150 −0.105
Sk = suckling kids; Ch = chevons; Pu = puberal goats; Capn1 = calpain-1; Hsp702 −0.305 0.043
Cast = calpastatin; Cryab = αB-crystallin; Hsp27 = heat shock protein 27. Capn1/Cast3 0.174 −0.081
a,b
Values within a row with different superscripts differ significantly at P ⩽ 0.05.
Cryab3 −0.181 0.120
Hsp273 −0.184 0.050
component 1 and component 2 are reported in Table 5. The Y matrix4
two components explained about the 42% of the original Sk 0.770 −0.303
variance of the animal features. The first component is Ch −0.187 0.754
Pu −0.610 −0.583
positively correlated with live weight gain, and negatively
correlated with a* and with the mRNA abundance of Cryab, LWG = live weight gain; pHu = ultimate pH; L* = lightness; a* = redness;
Hsp27 and Hsp70; conversely, the second component is b* = yellowness; CL = cooking loss; WBSF PY = Warner–Bratzler Shear Force,
peak yield force; WBSF FY = Warner–Bratzler Shear Force, final yield force;
positively correlated with subcutaneous fat score, intramus- Capn1 = calpain-1; Capn2 = calpain-2; Cast = calpastatin; Casp3 = caspase 3;
cular fat level, L*, b* and the mRNA abundance of Casp9. At Casp9 = caspase 9; Cryab = αB-crystallin; Hsp27 = heat shock protein 27;
mRNA level, the Capn1/Cast and Capn2/Cast ratios showed Hsp40 = heat shock protein 40; Hsp70 = heat shock protein 70.
1
Explained variance, 31% Component 1, 11% Component 2.
very similar loadings on both components 1 and 2. Moreover, 2
Messenger RNA.
for the Y matrix values, along the component 1 the Sk group 3
Protein.
4
is well separated from the Pu group, while the Ch group is Explained variance, 46% Component 1, 54% Component 2.
well separated from the other two groups on component 2.
The association networks between the X (in vivo perfor- Discussion
mances, meat and carcass characteristics and gene expres- Animal and meat characteristics
sion profile) and Y (goat type) data sets are reported in In our experiment, weaning seems to be the phase during
Figure 1. In this figure the negative association of the which goats tend to gain the most weight relative to time
youngest group, Sk, with a* (−0.68), Cryab mRNA (−0.69), when compared with any other phase in the growth pro-
Hsp70 mRNA (−0.71) and Hsp27 mRNA (−0.67), the positive cess. Rojo Rubio (2016) showed a live weight gain of
association of the same group with the live weight gain 163.2 g/day at weaning for Alpine breed, a value similar to
(0.67), the negative association of the oldest group, Pu, with that is reported in the present study, considering that the
L* (−0.82) and b* (−0.72), and the positive association of Pu authors previously cited weaned the kids at a higher BW of
with Cryab mRNA (0.69) are evident. The scores of the indi- 20 kg. The cold carcass weight in relation to the live weight
vidual animals are plotted in Figure 2; the mRNA abundance of the three classes is in line with the literature data
can discriminate the youngest animals from the oldest ones regarding Alpine breed (Borgogno et al., 2015). The fat
(Sk v. Pu); intermediate characteristics were found for the Ch level results confirm the lean nature of goat carcasses
group. (Webb et al., 2005). In our trial, Sk was the leanest group

6
 Tenderness-related gene expression in young goats

Color key

-0.82 0.82
Hsp701

L* Cryab2

Casp31
Kidney s.
WBSF FY

Capn2/ Intr. Fat

Cast1
CL

b*

Sk
Capn1/ Sub. s.
Cast1 Pu
Ch

Capn1/
Cast2

Cryab1 LWG
WBSF PY
pHu

Casp91
Hsp272
Hsp271 Hsp401

a*

Figure 1 Relevance networks plot. Correlation structure after Partial Least Squares Discriminant Analysis analysis between the goat types (suckling kids
(Sk); chevons (Ch); post-puberal goats (Pu); Y matrix), and their performance, carcass and meat characteristics, and gene expression profile (X matrix).
LWG = live weight gain; pHu = ultimate pH; L* = lightness; a* = redness; b* = yellowness; CL = cooking loss; WBSF PY = Warner–Bratzler Shear Force,
peak yield force; WBSF FY = Warner–Bratzler Shear Force, final yield force; Kidney s. = fat kidney score; Intr. Fat = intramuscular fat; Sub.
s. = subcutaneous fat score; Capn1 = calpain-1; Capn2 = calpain-2; Cast = calpastatin; Casp3 = caspase 3; Casp9 = caspase 9; Cryab = αB-crystallin;
Hsp27 = heat shock protein 27; Hsp40 = heat shock protein 40; Hsp70 = heat shock protein 70; 1messenger RNA; 2protein.

regarding the subcutaneous fat level. In addition, Kaić et al.
(2012) observed a significant increase in subcutaneous
carcass fat, increasing the slaughter weight of kids from 9
to 14 to 16 to 24 weeks.
The ultimate pH values were similar between experimental
groups and in line with those reported in previous works
(Piasentier et al., 2005). Meat color is an important trait in
goat carcasses evaluation, with pink or pale red expected for
kids. The Sk group showed the lowest redness value of meat,
probably because these animals were fed exclusively on milk.
Redness reflects the presence of myoglobin and the avail-
ability of iron (Sañudo et al., 2012) in meat. An increase in
redness was observed with goat age, in accordance with the
parallel rise of muscle myoglobin content.
As previously stated, WBSF_FY and WBSF_PY are mea-
surements of the connective tissue and myofibril contribution
to meat tenderness, respectively (Møller, 1981). In our trial,
WBSF_FY was similar between experimental groups. It is
well known that collagen is the most important protein of
connective tissue, and its insolubility plays an important role
in reducing meat tenderness (Starkey, 2017). However, the
lack of difference in WBSF_FY could be due to a limited
difference in age between experimental groups, taking into
account that the Sk and Ch groups were not puberal.
Moreover, the experimental animals were too young for a
difference in tenderness due to cross-links in the collagen
molecules (Therkildsen et al., 2002) to be seen. Conversely,
since the myofibrillar component of shear force (WBSF_PY)
was significantly lower in the Sk and Ch groups compared to
the Pu group, we suppose that this was due to a different
postmortem proteolysis activity and myofibrillar degradation
in pre-puberal goats and Pu. In other words, we speculate
that sexual maturity could affects the expression of factors
related with meat tenderness. Indeed, as suggested by
Mberema et al. (2016) in cattle, androgens could influence the
transcription of calpains, which are involved in myofibrils
degradation and then in the development of the meat ten-
derness. Other authors highlighted the potential role of tes-
tosterone on sensory attributes of small ruminants’ meat,
whose levels of 4-methyloctanoic and 4-methylnonanoic acids

7
Saccà, Corazzin, Bovolenta and Piasentier
Ch
X-variate 2: 11% explained variance
2
Ch
Ch
Ch
Ch
0
Ch Ch
Pu
Pu
-2 Pu Pu
Pu Pu
-4
-6 -3
X-variate 1: 31% explained variance
Figure 2 Partial Least Squares Discriminant Analysis plot of the
individuals. The individual goats are divided by belonging group (suckling
kids (Sk); chevons (Ch); post-puberal goats (Pu)). In the figure, also the
group’s centroid and ellipse (95% confidence limit) were represented.
increase after sexual maturity, giving the goaty-muttony odor
and flavor (Ames, 1996; Borgogno et al., 2015).
Messenger RNA, protein expression and multivariate analysis
Calpains are proteases considered to have a key role in
postmortem tenderization because they are involved in
muscle myofibrillar proteins degradation. Calpastatin is their
specific inhibitor (Koohmaraie and Geesink, 2006), therefore,
the calpains/Cast ratio can be considered to be related to
beef tenderness. Guillemin et al. (2011), in a proteomic study
in cattle, found that in the longissimus muscle, both Capn1
and Capn2 were positively correlated with tenderness.
Instead, in our trial, the direct role of the calpains/Cast ratio
in determining muscle tenderization was not as evident
because, while the Capn2/Cast mRNA ratio was higher in the
Pu group compared to younger animals, Capn1/Cast
expression was similar between experimental groups.
Nevertheless, considering the important role of calpains in
the balance between proteosynthesis and proteolysis of
muscle fibers (Koohmaraie et al., 2002), our Capn2/Cast ratio
results suggest a lower rate of proteolysis in kids, in accor-
dance with their higher growth rate. Discussing about muscle
homeostasis, Goll et al. (2008) attribute muscle growth to a
decrease in protein degradation, principally due to an
increase in calpastatin activity.
Muscle cells die by an apoptotic process after the
slaughtering of animals (Becila et al., 2010), and the intrinsic
pathway of this process, which involves Casp9 (initiator) and
Casp3 (effector), is the major pathway in muscle cells. For
their role in apoptosis, caspases are suspected to have a role
in muscle tenderization. In fact, effector caspases are
responsible for the degradation of myofibrillar and cytoske-
letal proteins and they can be related to muscle tenderization
process and, probably because of their cooperative role with
8
calpains, also to muscle turn-over (Guillemin et al. (2011). In
our trial, caspases 3 and 9 expression increase with the age
of the animal similarly to the calpains/calpastatin ratio. Even
Ch Ch Kemp et al. (2013) highlighted the high level of cooperation
Ch between calpains and caspases in muscle homeostasis.
Ch Ch
The Hsp expression showed an increasing trend with ani-
Ch mal age both at the mRNA and protein levels. The similar,
ChCh Sk
Sk but non-exact, correspondence between mRNA and protein
Sk Sk Sk expression could be due to the post-transcriptional regula-
Ch tions or to the interactions between Hsp, as hypothesized
Sk
Sk Sk by Yang et al. (2017). It was speculated that Hsp expression
Pu Sk could be stimulated in response to muscle cell environ-
mental stress, which can occur during physiological condi-
tions or additionally during animal slaughtering. Hsp27,
together with other small Hsp, such as Hsp20 and Cryab,
can bind and protect myofibrillar proteins, such as desmin,
0 3
actin and titin, from calpains degradation, both stabilizing
denaturing proteins and functioning as competitive inhibi-
tors of the proteolytic enzymes (Lomiwes et al., 2014).
Hsp27 is also an anti-apoptotic factor that promotes cell
survival by cytochrome c sequestration and prevents acti-
vation of caspase 3 (Brad Kim et al., 2018). Hsp70 and
Hsp40 have been identified as being involved in suppres-
sion of apoptotic process, in protein folding and in the
oxidative stress response by several authors, and Hsp70 is
referred to be a good biomarker of low tenderness (Picard
et al. 2014). Based on these considerations, the higher
expression of Hsp in older animals, could have led to a
higher protection of muscular proteins, a lower degradation
of myofibrils and then to a lower tenderness. The highest
levels of Hsp expression in the Pu group are associated with
the darkest grade of meat color. The association network
(Figure 2) also confirms the negative correlation between the
Pu group and the L* and b* parameters of the meat color.
Working on cull cows, Gagaoua et al. (2017) found that L*
and b* parameters were negatively and, respectively, corre-
lated with the expression of Hsp27 and Hsp70, likely because
of their protein protection activity that affects muscle struc-
ture and light reflectance.
The multivariate analysis discriminated the experimental
groups along the first component (31% of variance
explained), mainly loaded on live weight gain, a* and mRNA
abundance of Hsp. The Ch group was placed between the
other two groups, providing a clear age trend especially for
Hsp (Table 5, Figure 1, Figure 2). Moreover, the mRNA
abundance of Hsp and the WBSF were positioned in the same
quadrant indicating an association between them. Over-
coming the individual variability, such association reached a
statistical evidence in the difference between the average
performance of pre-puberal and post-puberal groups.
Indeed, the last one combined a higher mRNA and protein
expression of Hsp with a greater hardness of the meat. The
overexpression of small Hsp, with their anti-apoptotic
properties and protective activity on myofibrillar proteins
(Cassar-Malek and Picard, 2016), could have led to a tougher
meat in older goats. The increase of Hsp with age could
represent the physiological evolution of the resistance of

animals to environmental stresses and could also be related
to the hormonal changes that occurred during puberty and
sexual maturation. Zhang et al. (2012) found that the mRNA
expression of a member of the Hsp family was higher in bulls
compared to steers and increase under testosterone addition,
suggesting its involvement in muscular maintenance after
sexual maturation. Indeed, the authors highlighted the
capacity of Hsp, in response to androgen induction, to
activate the androgen receptor in cells, leading to the
transcription of genes associated with muscle development
and, consequently, with meat characteristics.
The role of the calpain system, caspases and Hsp in muscle
cell homeostasis and in muscle to meat conversion is com-
plex and not yet fully understood. These processes need to be
considerably clarified, especially in goats where no tran-
scriptomic information about muscle development is avail-
able (Lin et al., 2017).
In conclusion, Sk and Pu groups, characterized by a dif-
ferent tenderness of their meat, were clearly discriminated by
a different expression of the Hsp. On the other hand, the
lower expression of the Capn2/Cast ratio and caspases in
younger goats could be due to the involvement of these
genes in muscle development and myofibrillar turnover.
From this point of view, further studies are needed.
Acknowledgements
The research was funded by the ERSA Agency (L.R. n. 8/2004) of
the Friuli Venezia Giulia Autonomous Region and sponsored by
the FSE ‘European Social Found’ of Friuli Venezia Giulia. The
authors thank Dr Angela Sepulcri for fat analysis.
Declaration of interest
No potential conflicts of interest were reported by the authors.
Ethics statement
The trial was carried out in accordance with EU Directive 2010/
63/EU for animal experiments. All experimental procedures and
care of the animals complied with the Italian legislation on
animal care (DL n. 26, 04/03/2014), adhered to the internal rules
of University of Udine and were authorized by the Italian Min-
istry of Health (Ministerial Decree n. 284/2012-B, 7 December
2012). Management systems and data collected on goats were
part of the routine care and periodical control. Animals were
slaughtered and dressed using standard commercial techniques
in an EU-licensed abattoir.
Software and data repository resources
None of the data presented in this manuscript were deposited in
an official repository.
Supplementary material
To view supplementary material for this article, please visit
https://doi.org/10.1017/S1751731119000405
Tenderness-related gene expression in young goats
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