antioxidants

Article
MARGRA Lamb Eating Quality and Human
Health-Promoting Omega-3 Long-Chain
Polyunsaturated Fatty Acid Profiles of Tattykeel
Australian White Sheep: Linebreeding and
Gender Effects
Shedrach Benjamin Pewan 1,2 , John Roger Otto 1 , Robert Tumwesigye Kinobe 1 ,
Oyelola Abdulwasiu Adegboye 3 and Aduli Enoch Othniel Malau-Aduli 1, *
1 Animal Genetics and Nutrition, Veterinary Sciences Discipline, College of Public Health, Medical and
Veterinary Sciences, Division of Tropical Health and Medicine, James Cook University,
Townsville, QLD 4811, Australia; [email protected] (S.B.P.); [email protected] (J.R.O.);
[email protected] (R.T.K.)
2 National Veterinary Research Institute, Private Mail Bag 01 Vom, Plateau State, Nigeria
3 Australian Institute of Tropical Health and Medicine, College of Public Health, Medical and
Veterinary Sciences, Division of Tropical Health and Medicine, James Cook University,
Townsville, QLD 4811, Australia; [email protected]
* Correspondence: [email protected]; Tel.: +61-747-815-339




Received: 16 October 2020; Accepted: 11 November 2020; Published: 12 November 2020


Abstract: Health-conscious consumers increasingly demand healthier, tastier, and more nutritious
meat, hence the continuous need to meet market specifications and demand for high-quality lamb.
We evaluated the longissimus dorsi muscle of 147 Tattykeel Australian White (TAW) sheep fed on
antioxidant-rich ryegrass pastures exclusive to MAGRA lamb brand for meat eating quality parameters
of intramuscular fat (IMF) content, fat melting point (FMP) and omega-3 long-chain polyunsaturated
fatty acids (n-3 LC-PUFA). The aim was to assess the impact of linebreeding and gender on pasture-fed
lamb eating quality and to test the hypothesis that variation in healthy lamb eating quality is a function
of lamb gender and not its antioxidant status or inbreeding coefficient (IC). After solid-phase extraction and
purification, phenolics and antioxidant enzyme activities were analysed by high-performance liquid
chromatography and mass spectrometry. IMF and fatty acid composition were determined using solvent
extraction and gas chromatography, respectively. IC was classified into low (0–5%), medium (6–10%)
and high (>10%) and ranged from 0–15.6%. FMP and IMF ranged from 28 to 39 ◦ C and 3.4% to 8.2%,
with overall means of 34.6 ± 2.3 ◦ C and 4.4 ± 0.2%, respectively, and n-3 LC-PUFA ranged from “source”
to “good source” levels of 33–69 mg/100 g. Ewes had significantly (P < 0.0001) higher IMF, C22:5n-3
(DPA), C22:6n-3 (DHA), C18:3n-6, C20:3, C22:4n-6, C22:5n-6, total monounsaturated (MUFA), PUFA and
Σn-3 fatty acids and lower total saturated fatty acids (SFA) and FMP, than rams. As IC increased, there
were no differences in FMP and IMF. Folin–Ciocalteu total phenolics, ferric reducing antioxidant power
and antioxidant activities of glutathione peroxidase, catalase and superoxide dismutase enzymes did
not differ by either gender or IC. This study provides evidence that IC is inconsequential in affecting
antioxidant status, IMF, FMP and n-3 LC-PUFA in linebred and pasture-fed TAW sheep because the
observed variation in individual fatty acids was mainly driven by gender differences between ewes
and rams, hence the need to accept the tested hypothesis. This finding reinforces the consistent healthy
eating quality of MARGRA lamb brand from TAW sheep regardless of its linebred origin.

Keywords: antioxidants; Tattykeel Australian White; MARGRA lamb; meat quality; longissimus dorsi
muscle; omega-3 LC-PUFA; fat melting point; intramuscular fat; inbreeding coefficient; gender

Antioxidants 2020, 9, 1118; doi:10.3390/antiox9111118 www.mdpi.com/journal/antioxidants

Antioxidants 2020, 9, 1118 2 of 20

1. Introduction
The Food and Agriculture Organisation (FAO) of the United Nations defines meat quality as the
constitutional standard of lean-to-fat ratio and palatability indices that include visual appearance,
aroma, drip loss, colour, texture, pH, intramuscular fat content, fatty acid and fat melting point
profiles, tenderness, flavour and juiciness [1]. Meat and Livestock Australia (MLA) describes the
entire processes of feeding culminating in the finishing of animals including their genetic constitution,
husbandry practices and handling, as all affect the overall quality of meat [2]. Fat melting point (FMP),
intramuscular fat (IMF) content (marbling) and fatty acid (FA) profile all influence eating quality and,
ultimately, consumer preferences for consistent, safe, nutritious and tasty lamb with a healthy FA
composition [3]. Globally, meat is regarded as one of the main sources of animal protein [4,5] and lamb
is known to be highly nutritious and digestible [6], fortified with essential amino acids, iron, zinc,
selenium, fatty acids and vitamins A, B6 and B12 [5]. Lamb also has relatively low lipid and saturated
fat contents compared to meat from other ruminants [7], and its marbling, tenderness, juiciness, aroma
and colour attributes have been known to influence consumer liking [8], carcass [9], meat assignment
into quality grades [10], consumer food choices [11] and nutritional value [12]. It is therefore very
important that sheepmeat producers guarantee the consistency of their lamb products in order to meet
consumer preferences and adapt to the dynamics of purchasing decisions based on meat eating quality.
FMP dictates fat firmness. Soft fat has a low melting point and vice versa [13]. From a nutritional
perspective, fats with low melting points consist of high levels of unsaturated fatty acids, and, conversely,
fats with high melting points have comparably higher saturated fatty acids [14,15]. IMF content or
marbling is a main determinant of meat eating quality in most carcass grading systems [16]. As the
IMF increases, so does the eating quality [17] because it influences meat palatability and contributes
significantly to juiciness, flavour and tenderness [18,19]. Consumers therefore prefer meat with low FMP,
moderate IMF and fatty acid composition with proportionately more of the health-promoting omega-3
long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Given that humans and other vertebrates lack
the capacity to synthesize n-3 LC-PUFA because they lack the enzyme ∆15 desaturase, they must
obtain these from dietary intake sources in order to meet their daily requirement of 500 mg of n-3
LC-PUFA [20]. Lamb producers can tap into the omega-3 functional meat market niche by matching
their sheep breeding and production system to meet this health-conscious consumer preference.
Ryegrass is a popular grass species in pasture-based grazing production systems in Australia and
New Zealand. Ryegrass contains many phenolic compounds such as gallic and salicylic acids (phenolic
acids), tannins, coumarins, flavonoids, α-tocopherol, lignans, xanthones and anthocyanidins [21,22].
These phenolic compounds in ryegrass serve as natural antioxidants, anti-inflammatory and anti-septic
agents [23] that enhance meat oxidative stability and quality attributes such as nutritive value, flavour
and colour. Luciano et al. [24,25] reported that antioxidants in dietary tannins from fresh herbage
improved colour stability in Comisana lambs by halting myoglobin oxidation in the muscle and reducing
meat colour deterioration. In lambs grazing ryegrass, phenolics and antioxidant enzyme activities have
been demonstrated to impact oxidative stability in the Longissimus thoracis et lumborum muscle [26],
liver and plasma [27]. Research investigations of perceived sheepmeat eating quality sensory scores [28]
and demographic influences [29] on Australian, American and Chinese consumers demonstrated a
consistent consumer response to production factors of muscle type, sire, age, and sex. Evidence in the
published literature [30] indicates that meat eating quality and fatty acid (FA) composition of lipids in
tandem with variable fat deposition at the attainment of maturity, vary in the muscles of sheep due to
differences in breed [31–34], physiological status, breeding systems [35], grass-fed versus concentrate
feeding [36,37], and sex [38,39].
Linebreeding is a sheep breeding practice of mating closely related animals that can be traced
back to one common ancestor with highly desirable attributes. The Tattykeel Australian White (TAW)
sheep are renowned for producing the remarkably unique high-eating-quality MARGRA lamb brand,
and were developed from more than a decade of rigorous selection, culling and linebreeding of
Texel, Van Rooy, Dorper and Poll Dorset with an extensive utilisation of natural mating, artificial
Antioxidants 2020, 9, 1118 3 of 20

insemination and embryo transfer. Linebreeding increases the frequency of desirable alleles, selection
intensity and homozygosity, hence a tight culling regime and close monitoring of the inbreeding
coefficient are key breeding management practices that ensure uniformity and consistency in TAW
lamb eating quality. A comprehensive review of omega-3 long-chain polyunsaturated fatty acids (n-3
LC-PUFA) metabolism and meat eating quality in TAW lambs [30] previously identified knowledge
gaps in using Longissimus dorsi muscle biopsy sampling of ram and ewe lambs to directly determine
the impact of linebreeding and gender on n-3 LC-PUFA, IMF and FMP contents while the animals
are young and alive for early selection and breeding purposes. It also recommended the need for
further research to better understand the genetic and nutritional interactions between dietary n-3
LC-PUFA oil supplements versus pasture grazing, finishing performance, carcass traits and the unique
eating quality of TAW lambs to afford industry players the opportunity to consistently meet consumer
preferences as well as key demand and supply determinants of profitability. This paper aims to fill
some of these knowledge gaps by assessing the impact of linebreeding and gender on pasture-fed lamb
eating quality consistency in antioxidant status, IMF, FMP, n-3 LC-PUFA and to test the hypothesis
that variation in healthy lamb eating quality will be a function of lamb gender and not its antioxidant
status or inbreeding coefficient (IC) as an index of linebreeding.

2. Materials and Methods

2.1. Animal Ethics

The use of animals and all procedures performed in this study were approved by the James Cook
University Animal Ethics Committee (Permit No. A0015657) in compliance with the Australian Code
for Care and Use of Animals for Scientific Purposes (Eighth edition, 2013).

2.2. Animals and Experimental Design

The animals used in this study comprised a cohort of 100 ewe and 47 ram lambs at the Tattykeel
Australian White stud farm in Black Springs, Oberon, New South Wales, Australia, grazing the same
ryegrass pastures in separate paddocks. They were all 10-month-old lambs, with an average liveweight
of 36.8 ± 0.3 kg (range of 36–38 kg for rams), 37.4 ± 0.4 kg (range of 37–38 kg for ewes), and an overall
mean body condition score of 2.5 ± 0.01. Carcass performance and meat quality characteristics of
TAW had been published [30]. An a priori power analysis was conducted using G-Power to justify
an appropriate sample and effect size. As depicted in Figure 1, to achieve a statistical power of 95%
with a critical F-value of 2.5, a minimum total sample size of 146 lambs was sufficient for a large effect
size, and a two-sided significance level of 0.05. Therefore, the cohort of 100 ewe and 47 ram lambs
at the Tattykeel Australian White stud farm in Black Springs, Oberon, New South Wales, Australia
grazing the same ryegrass pastures in separate paddocks used in this study, was a sufficient and
statistically robust experimental design. Total digestible nutrients [40] and metabolisable energy [41]
were computed from the nutritive composition of the ryegrass (Table 1) analysed by the Association of
Official Analytical Chemists (AOAC) wet chemistry procedure.

Table 1. Nutrient and phenolic antioxidant compositions of ryegrass pastures grazed by Tattykeel
Australian White lambs 1 .

Nutrient Composition (% DM)

DM 20.7
CP 19.0
ADF 26.5
NDF 30.9
EE 1.8
Ash 6.8
%TDN 62.5
DE (Mcal/kg) 2.8
ME (MJ/kg) 9.4
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ME (MJ/kg) Table 1. Cont. 9.4

Phenolic Antioxidants:
Nutrient Composition (% DM)
FCTP (mg GAE/g) 1.631
Phenolic Antioxidants:
FRAP (mmol Fe2+ E/g) 6.572
FCTP (mg GAE/g) 1.631
1DM: dry matter; NDF: neutral detergent 2+ fibre; ADF: acid detergent fibre; EE: ether extract; CP: crude
FRAP (mmol Fe E/g) 6.572
protein; %TDN [40]: total digestible nutrients, calculated as (% of DM) = 82.38 − (0.7515 × ADF [% of
1 DM: dry matter; NDF: neutral detergent fibre; ADF: acid detergent fibre; EE: ether extract; CP: crude protein;
DM]).[40]:
%TDN ME total
[41]:digestible
metabolizable energy,
nutrients, calculated
calculated as (% ofby converting
DM) %TDN×to
= 82.38 − (0.7515 ADF digestible energy
[% of DM]). (DE
ME [41]:
[Mcal/kg] = %TDN
metabolizable × 0.01 × 4.4)
energy, calculated which was
by converting converted
%TDN as MEenergy
to digestible = (DE(DE
(Mcal/kg)
[Mcal/kg] × =0.82)
%TDN× 4.185;
× 0.01FCTP:
× 4.4)
which was convertedtotal
Folin–Ciocalteu as ME = (DE (Mcal/kg)
phenolics; GAE:× gallic
0.82) × acid
4.185; equivalents;
FCTP: Folin–Ciocalteu
FRAP: total phenolics;
ferric reducing GAE: gallic acid
antioxidant
equivalents; FRAP: ferric reducing antioxidant power.
power.

Figure 1. G-Power analysis for statistical power, effect and sample size.

Figure 1. Procedure
2.3. Muscle Biopsy Sampling G-Power analysis for statistical power, effect and sample size.

Longissimus dorsi muscle biopsy samples were taken from the 12th–13th rib interface following the
2.3. Muscle Biopsy Sampling Procedure
procedure described by Malau-Aduli et al. [42] and are shown in Figure 2. Briefly, the animal was
Longissimus
directed dorsi muscle
into a weighing chutebiopsy samples were
with collapsible sidestaken from head
and some the 12th–13th
restraint. rib
Theinterface following
Longissimus dorsi
the procedure described by Malau-Aduli et al. [42] and are shown in Figure
muscle area on the back of the animal between the 12th and 13th ribs was shaved with a small2. Briefly, the animal was
directed into a weighing chute with collapsible sides and some head restraint. The
electric clipper and cleaned with 90% ethanol and chlorhexidine. About 15 mL of a local anaesthetic Longissimus dorsi
musclelignocaine
agent, area on the back
was of the animalintramuscularly.
administered between the 12thFive andminutes
13th ribsfollowing
was shaved thewith a small electric
administration of
clipper and cleaned with 90 % ethanol and chlorhexidine. About 15 mL of a local
the anaesthetic, a 5–7 cm incision was made with a scalpel blade and about 5 g of the underlying anaesthetic agent,
lignocaine
fat was administered
and Longissimus dorsi muscle intramuscularly.
was sampled. The Fivewound
minuteswasfollowing
closed via the
3–4administration of the
interrupted sutures
anaesthetic, a 5–7 cm incision was made with a scalpel blade and about 5 g of the
using surgilon thread. An anti-bacterial aerosol, Cetrigen, was applied to the sutured area on the underlying fat and
Longissimus dorsi muscle was sampled. The wound was closed via 3–4 interrupted
skin to promote wound healing, prevent flies and the animal was released back to the paddock. sutures using
surgilon
No thread. An
post-operative anti-bacterial
complications wereaerosol, Cetrigen,
reported waswas
as healing applied
rapid.to The
the sutures
suturedwerearea removed
on the skin to
after
promote wound healing, prevent flies and the animal was released back to the
10–14 days. The muscle biopsy sample was immediately placed in a plastic bag on dry ice, flushed paddock. No post-
operative
with complications
nitrogen were reported
gas and transferred into aas healing
mobile was rapid. Samples
refrigerator. The sutureswere were removedfrozen
transported after 10–14
and
days. The
stored at −20muscle
◦ biopsy further
C pending sample analysis
was immediately placed inThe
in the laboratory. a plastic
musclebag on drywere
biopsies ice, analysed
flushed with
for
nitrogen gas and transferred into
IMF content, FMP and FA composition. a mobile refrigerator. Samples were transported frozen and stored
at −20 °C pending further analysis in the laboratory. The muscle biopsies were analysed for IMF
content, FMP and FA composition.
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Antioxidants 2020, 9, x FOR PEER REVIEW 5 of 20

Muscle FAC – Malau-Aduli et al. (1998)

Figure 2.
Figure Muscle biopsy
2. Muscle biopsy sampling
sampling technique
technique in
in Tattykeel
Tattykeel Australian
Australian White
White sheep.
sheep.

2.4. Determination of Intramuscular Fat

2.4. Determination of Intramuscular Fat
The procedures of Holman et al. [43] and Flakemore et al. [44] were utilised for IMF determination.
The procedures of Holman et al. [43] and Flakemore et al. [44] were utilised for IMF
Briefly, the muscle sample was homogenised and 1g transferred to a labelled 50 mL plastic tube
determination. Briefly, the muscle sample was homogenised and 1g transferred to a labelled 50 mL
containing 20 mL of chloroform: methanol (2:1) solvent and shaken vigorously for 5 min. A filter paper
plastic tube containing 20 mL of chloroform: methanol (2:1) solvent and shaken vigorously for 5 min.
was used to collect the filtrate in another labelled 50 mL tube. Approximately 5 mL of 10% KCl was
A filter paper was used to collect the filtrate in another labelled 50 mL tube. Approximately 5 mL of
added to the filtrate to precipitate and separate the inorganic and lipid fractions into two distinct layers.
10 % KCl was added to the filtrate to precipitate and separate the inorganic and lipid fractions into
The upper inorganic layer was removed and discarded, while the lower lipid layer was transferred
two distinct layers. The upper inorganic layer was removed and discarded, while the lower lipid
into a clean, dry, pre-weighed and labelled ceramic crucible and evaporated in a laminar fume hood
layer was transferred into a clean, dry, pre-weighed and labelled ceramic crucible and evaporated in
over a heating block. The crucible was cooled and further dried in a desiccator for 10–20 min before
a laminar fume hood over a heating block. The crucible was cooled and further dried in a desiccator
it was re-weighed. Samples were analysed in duplicates to allow for replication and reproducibility.
for 10–20 min before it was re-weighed. Samples were analysed in duplicates to allow for replication
Intramuscular fat percentage was calculated as:
and reproducibility. Intramuscular fat percentage was calculated as:
[(Final
[(Finalcrucible weight)−− (Initial
crucibleweight) (Initial crucible
crucible weight)/(Initial
weight)/(Initial sample weight)]×× 100.
sampleweight)]

2.5. Determination of Fat Melting Point

2.5. Determination of Fat Melting Point
The procedures of Holman et al. [43] and Flakemore et al. [44] were utilised for FMP determination.
Thetheprocedures
Briefly, of Holman
crucible containing et al. [43]IMF
the extracted andwasFlakemore
placed in an et oven
al. [44] were
at 100 utilised
◦ C for aboutfor
1–2FMPmin
to melt the fat. Using air suction, the melted fat was sucked into a thin capillary tube and placedfor
determination. Briefly, the crucible containing the extracted IMF was placed in an oven at 100 °C in
about
a 1–2 minfor
refrigerator to about
melt the 10 fat.
minUsing airfat
for the suction, the melted
to solidify. The fatfat wasinsucked
level into a thin
the capillary tubecapillary
was markedtube
and placed
with in a refrigerator
an indelible for about
pen. The capillary 10 was
tube min attached
for the fat
to to solidify. The and
a thermometer fat level in thesuspended
vertically capillary tube
in a
was marked with an indelible pen. The capillary tube was attached to a thermometer
beaker containing 80 mL of cold water, gradually heated over a heating block and closely observed and vertically
suspended
until the fat in a beaker
melted and containing 80 mL
“slipped” (rose of cold
above water,within
the mark) gradually heated over
the capillary tube.a The
heating block and
temperature at
closely observed until the fat melted and “slipped” (rose above the mark) within
which this slip occurred was recorded as the fat melting point. Samples were analysed in duplicates to the capillary tube.
The temperature
allow at and
for replication which this slip occurred
reproducibility. was recorded
TAW lamb has a veryaslow thefat
fatmelting
meltingpoint
point.andSamples were
can be liquid
analysed in duplicates to allow◦ for replication
at room temperature of 25–28 C as shown in Figure 3. and reproducibility. TAW lamb has a very low fat
melting point and can be liquid at room temperature of 25–28 °C as shown in Figure 3.
Antioxidants 2020, 9, 1118 6 of 20
Antioxidants 2020, 9, x FOR PEER REVIEW 6 of 20

Figure 3. Tattykeel Australian White intramuscular fat (liquid at room temperature) indicating a low
fat melting point (FMP).

2.6. Determination of
2.6. Determination of Fatty
Fatty Acid
Acid Composition
Composition
Fatty
Fatty acid
acidcomposition
composition including
including n-3n-3
LC-PUFA
LC-PUFA analysis of Longissimus
analysis dorsi muscle
of Longissimus dorsi biopsy
musclesamples
biopsy
was analysed by means of gas chromatography–mass
samples was analysed by means of gas chromatography–mass spectrophotometry spectrophotometry procedure described
procedure by
Malau-Aduli et al. [45]. Briefly, total lipids in 1 g of un-homogenised
described by Malau-Aduli et al. [45]. Briefly, total lipids in 1 g of un-homogenised muscle tissue muscle tissue samples were
extracted
samples were overnight
extractedusing a modified
overnight Bligh
using and Dyer
a modified [46]and
Bligh method.
Dyer The[46] first step The
method. wasfirst
a single-phase
step was a
overnight extraction using
single-phase overnight extraction CHCl :MeOH:H
3 using CHCl 2 O (1:2:0.8 v/v). The second step involved
3:MeOH:H2O (1:2:0.8 v/v). The second step involved
phase separation
with the addition
phase separation with the of CHCl :saline Milli-Q
3 addition of CHCl H 2 O (1:1 v/v) followed by rotary evaporation
3:saline Milli-Q H2O (1:1 v/v) followed by rotary
of the lower
chloroform ◦
evaporationphase of theatlower
40 Cchloroform
to obtain totalphaselipids.
at 40The°C to extracted
obtain totaltotal lipids.
lipids were separatedtotal
The extracted intolipids
lipid
classes by thin layer chromatography (TLC) using 100 mL of the lipid
were separated into lipid classes by thin layer chromatography (TLC) using 100 mL of the lipid extract reconstituted in hexane [42].
3
The extract
extract was spotted
reconstituted onto silica
in hexane [42].gel
TheG plates
extract(200 was×spotted
200 × 0.25ontomm ) with
silica gel G a micropipette.
plates (200 × 200 The× TLC
0.25
plate
mm ) with a micropipette. The TLC plate was developed in an acetone/petroleum ether (1:3 vol/vol)a
3 was developed in an acetone/petroleum ether (1:3 vol/vol) solvent system in a tank containing
few crystals
solvent system of butylated
in a tank hydroxytoluene
containing a few(BHT) crystals to prevent
of butylatedoxidation. Triacylglycerols,
hydroxytoluene (BHT) cholesterol
to prevent
and
oxidation. Triacylglycerols, cholesterol and free fatty acids migrated, while phospholipids The
free fatty acids migrated, while phospholipids remained at the origin of the plate. areas
remained
corresponding
at the origin of the plate. The areas corresponding to the phospholipids were scraped off the clean
to the phospholipids were scraped off the plate and each lipid class transferred to plate
screw-capped
and each lipidtest classtubes for transmethylation
transferred and eventual
to clean screw-capped testcomputation of the lipid conversion
tubes for transmethylation factor
and eventual
(LCF) of 0.912of
computation onthethelipid
basisconversion
of g fatty acids/g
factortotal
(LCF) lipids (0.083onphospholipids,
of 0.912 the basis of g0.829 fatty triacylglycerols
acids/g total lipids and
0% cholesterol because cholesterol does not contain any fatty acids). An
(0.083 phospholipids, 0.829 triacylglycerols and 0 % cholesterol because cholesterol does not contain aliquot from each total lipid
extract
any fatty wasacids).
used for Antransmethylation
aliquot from each withtotal
MeOH:CHCl
lipid extract3 :HClwas (10:1:1
usedv/v)for 2 h at 80 ◦ C. Fatty with
fortransmethylation acid
methyl esters (FAME) were extracted three times using hexane:CHCl
MeOH:CHCl3:HCl (10:1:1 v/v) for 2 h at 80 °C. Fatty acid methyl esters (FAME) were extracted three
3 (4:1 v/v). A known concentration
of an internal
times standard (19:0)
using hexane:CHCl was added in a 1500 µL vial containing the extracted FAME. The FAME
3 (4:1 v/v). A known concentration of an internal standard (19:0) was added
were
in a analysed
1500 μL on viala 7890B gas chromatograph
containing the extracted (Agilent
FAME. The Technologies,
FAME were Paloanalysed
Alto, CA,on USA) equipped
a 7890B gas
with an Equity TM -1 fused 15 m silica capillary column with 0.1 mm internal diameter and 0.1 µm film
chromatograph (Agilent Technologies, Palo Alto, CA, USA) equipped with an Equity -1 fused 15 m TM

thickness (Supelco,
silica capillary column Bellefonte,
with 0.1 PA,
mmUSA),
internala flame
diameterionisation
and 0.1detector,
μm film athickness
split/splitless injector
(Supelco, and an
Bellefonte,
Agilent
PA, USA), Technologies 7683 B Series
a flame ionisation autosampler.
detector, The gas
a split/splitless chromatograph
injector and an Agilentconditions were: splitless
Technologies 7683 B
mode injection; carrier gas He; initial oven temperature 120 ◦ C and then increased to 270 ◦ C at flow
Series autosampler. The gas chromatograph conditions were: splitless mode injection; carrier gas He;
rates 10 ◦ C/min
initialofoven and to120
temperature 310°C ◦ C at 5 ◦ C/min. The Agilent Technologies ChemStation software (Palo
and then increased to 270 °C at flow rates of 10 °C/min and to 310 °C
at 5 °C/min. The Agilent Technologies ChemStation software (Palo Alto, CA, USA) was used to
quantify fatty acid peaks. The fatty acid identities were confirmed by gas chromatograph–mass
Antioxidants 2020, 9, 1118 7 of 20

Alto, CA, USA) was used to quantify fatty acid peaks. The fatty acid identities were confirmed by gas
chromatograph–mass spectrometric (GC/MS) analysis using a Finnigan Thermoquest GCQTM GC/MS
fitted with an on-column injector and Thermoquest Xcalibur software (Austin, TX, USA). The gas
chromatograph (GC) was equipped with an HP-5 cross-linked methyl silicone-fused silica capillary
column (50 m × 0.32 mm internal diameter) which is of similar polarity to the column described above.
The carrier gas was helium (head pressure 30 kPa) and GC conditions had been previously described
by Miller et al. [47]. Fatty acid percentages were computed as follows: FA% = [(individual fatty acid
area) ∗ (100)]/(sum total area of fatty acids). Fatty acid contents were calculated as follows: FA mg/100 g
= (Total lipid) ∗ (LCF [0.912]) ∗ ([%FA]/100) ∗ 1000, where 0.912 was the derived lipid conversion factor
similar to the one cited by Clayton [48].

2.7. Extraction and Purification of Phenolic Compounds

Solid-phase extraction, purification and analysis of phenolics in the ryegrass utilised the procedure
described in detail by López-Andrés et al. [27]. Briefly, 2.5 g of the ryegrass was chopped to pass
through a 1 mm sieve and homogenised at 4000 r.p.m. in 15 mL of acetone/water 70/30 v/v for 1 min and
sonicated for 6 min in a water bath. Homogenates were centrifuged at 4 ◦ C for 15 min at 3000× g and the
supernatants filtered with Whatman filter papers. About 10 mL of the filtered supernatant was acidified
with 0.5 M H2 SO4 and loaded onto reversed phase cartridges (C18 Sep-Pak Vac WAT043395, WATERS,
Milan, Italy) preconditioned with methanol and distilled water to disrupt polyphenol-binding protein.
The extracted phenolics were eluted with 2 mL methanol and stored in a −30 ◦ C freezer until ready for
Folin–Ciocalteu (FCTP) and ferric reducing antioxidant power (FRAP) assays using a double-beam
spectrophotometer (model UV-1601, Shimadzu Corporation, Milan, Italy) to measure the absorbance
of the samples at 725 nm and 593 nm, respectively. Details of both assay procedures have been
described [27] and will not be repeated herein.

2.8. Antioxidant Enzyme Activities

Antioxidant activities of glutathione peroxidase, catalase and superoxide dismutase enzymes in the
muscle were assayed as described by Petron et al. [26]. Briefly, about 5 g of the Longissimus dorsi muscle
was homogenised in 25 mL of 0.005 M phosphate buffer (pH 7.0) and centrifuged at 4 ◦ C for 20 min at
7000 g. The supernatant fraction was filtered through glass wool and used to determine glutathione
peroxidase, catalase and superoxide dismutase enzyme activities. By measuring the inhibition of
pyrogallol autoxidation, total superoxide dismutase (SOD) activity (Cu–Zn SOD + Mn SOD) was
determined where one unit was taken as the activity that inhibits the reaction by 50% [1]. To determine
glutathione peroxidase enzyme activity, the oxidation of NADPH at 22 ◦ C was used. The assay medium
(3 mL) consisted of 1 mM reduced glutathione, 0.15 mM NADPH, 0.15 mM H2 O2 , 40 mM potassium
phosphate buffer (pH 7.0), 0.5 mM EDTA, 1 mM NaN3 , 1.5 units of glutathione reductase, and 300 µL
of the muscle extract. Absorbance at 340 nm was recorded over 3 min. An extinction coefficient of
6300 M−1 cm−1 was used for calculation of NADPH concentration. One unit of glutathione peroxidase
enzyme activity was defined as the amount of extract required to oxidize 1 µmol of NADPH per min
at 22 ◦ C [1]. Catalase enzyme activity was performed as described by Petron et al. [1]. About 2 mL
of the Longissimus dorsi muscle supernatant (2 mL) was reacted at room temperature (∼22 ◦ C) with
1 mL of 30 mM H2 O2 in 0.05 M phosphate buffer (pH 7.0), and the reaction (H2 O2 decomposition) was
monitored by measuring the absorbance at 240 nm during the initial 30 s. An extinction coefficient of
0.040 cm2 µmol−1 was used for calculation of H2 O2 splitting. One unit (U) of catalase activity was
defined as the amount of extract needed to decompose 1 µmol of H2 O2 per min [26].

2.9. Statistical Analysis

IC as an index of linebreeding, estimates the probability that two alleles in an individual lamb
will be homozygous (HH or hh) rather than heterozygous (Hh) because the parents are related and
have one common ancestor. In other words, IC measures the extent to which two genes at any locus in
Antioxidants 2020, 9, 1118 8 of 20

an individual lamb are identical by descent from the common ancestor(s) of the two parents. IC was
computed as:
FX = Σ[(1/2)n+1 (1 + FA )] (1)

where Fx = IC of lamb X, Σ = summation, n = number of common ancestors connecting the parents of

lamb X and FA is the IC of the common ancestor A.
Fatty acids, IMF, FMP, antioxidants and enzyme activities were analysed as dependent variables
using multivariate analysis of variance (MANOVA) after fitting the fixed effects of gender and IC
in General Linear Model procedures (PROC GLM) using Statistical Analysis System software (SAS)
version 9.4 (SAS Institute, Cary, NC, USA) [49]. First-order interactions between gender and IC were
initially tested but later dropped from the final model due to non-significance. The initial full statistical
model used for the analysis was:

Y = µ + Gi + Bj + (GB)ij + eijk (2)

where Y = dependent variable (FMP, IMF, FA, antioxidants and enzyme activities), µ = overall mean,
Gi = Gender, Bj = Inbreeding Coefficient, (GB)ij = first-order interaction between gender and inbreeding
coefficient, and eijk = residual error. Level of significance threshold was set at p < 0.05 and differences
between least square means were established using Tukey’s pairwise comparison test.

3. Results

3.1. Nutrient Composition of the Grazed Ryegrass Pasture, Muscle Phenolics and Antioxidant Enzyme Activities
The ewe and ram lambs utilised in this study grazed high-quality ryegrass whose nutrient
composition and antioxidant status is presented in Table 1 and fatty acid profile in Table 2. The low dry
matter is indicative of fresh pasture with high moisture content, while the high phenolic antioxidants,
crude protein, low neutral detergent and high metabolisable energy are all indicative of high palatability,
digestibility and total digestible nutrients from the ryegrass pastures that are typical during spring.
There were no significant differences due to gender (Table 3) and inbreeding coefficient (Table 4) in
total phenolics and antioxidant enzyme activities of glutathione peroxidase, catalase and superoxide
dismutase in the Longissimus dorsi muscle of these ryegrass pasture-fed lambs.

Table 2. Fatty acid composition of grazed ryegrass pasture.

Fatty Acid % Total Fatty Acids

14:0 0.6
15:0 0.2
16:1n-9c 0.0
16:1n-7c 0.2
16:0 15.7
17:0 0.5
18:2n-6 LA 14.8
18:3n-3 ALA 57.6
18:1n-9c 1.0
18:1n-7c 0.2
18:1n-7t 0.0
18:0 0.1
20:4n-6 ARA 0.0
20:5n-3 EPA 0.0
20:3n-6 0.1
20:4n-3 0.1
20:2n-6 0.1
20:0 1.6
22:5n-6 DPA-6 0.0
Antioxidants 2020, 9, 1118 9 of 20

Table 2. Cont.

Fatty Acid % Total Fatty Acids

22:6n-3 DHA 0.0
22:5n-3 DPA-3 0.0
22:0 1.0
23:0 0.3
24:0 0.9
P
SFA 20.9
P
MUFA 4.9
P
PUFA 73.1
P
n-3 LC-PUFA 0.1
P
n-3 PUFA 58.0
P
n-6 PUFA 15.2
P
other FA 1.0
n-6/n-3 0.3
LA, linoleic acid; ALA, α-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; DPA,
docosapentaenoic acid; ARA, arachidonic acid; ΣSFA, total P saturated fatty acids; ΣMUFA, total monounsaturated
fatty acids; and P
total polyunsaturated fatty acids (ΣPUFA). SFA is the sum of 14:0, 15:0, 16:0, 17:0, 18:0, 20:0, 21:0,
22:0, 23:0, 24:0; MUFA is the sum of 14:1, 16:1n-13t, 16:1n-9, 16:1n-7, 16:1n-7t, 16:1n-5c, 17:1n-8+a17:0, 18:1n-9,
18:1n-7t,
P 18:1n-5, 18:1n-7, 18:1a, 18:1b, 18:1c, 19:1a, 19:1b, 20:1n-11, 20:1n-9, 20:1n-7, 20:1n-5, 22:1n-9, 22:1n-11, 24:1n-9;
PUFA is the sum of 18:4n-3,
P 18:3n-6, 18:2n-6, 18:3n-3, 20:3, 20:4n-3, 20:4n-6, 20:5n-3, P 20:3n-6, 20:2n-6, 22:6n-3,
22:5n-3, 22:5n-6, 22:4n-6; n-3 LC-PUFA is the sum P of 20:5n-3, 20:4n-3, 22:6n-3, 22:5n-3; n-3 PUFA is the sum of
18:3n-3, 18:4n-3, 20:4n-3, 20:5n-3, 22:6n-3, 22:5n-3; n-6 PUFA is the sum of 18:2n-6, 18:3n-6, 20:4n-6, 20:3n-6, 20:2n-6,
22:5n-6, 22:4n-6; other FA is the sum of other individual FA present at <0.1% except ARA, DHA, EPA, and DPA.
P

Table 3. Effect of gender (Means ± s.d.) on fat melting point, intramuscular fat, fatty acids, antioxidant
phenolics and enzyme activities in the Longissimus dorsi muscle of ryegrass-fed Tattykeel Australian
White (TAW) lambs 1 .

Variable Ram (n = 47) Ewe (n = 100) Overall (n = 147) P-Value

Fat Melting Point (◦ C) 35.5 ± 1.5 34.2 ± 2.4 34.6 ± 2.3 0.0001
Intramuscular fat (%) 3.4 ± 0.3 4.4 ± 1.4 4.1 ± 1.3 0.0001
FCTP (mg GAE/g) 1.142 ± 0.0036 1.171 ± 0.0042 1.156 ± 0.0039 0.4723
FRAP (mmol Fe 2+ E/g) 5.481 ± 0.0172 5.605 ± 0.0198 5.543 ± 0.0185 0.2982
GSH-Px (U/g) 0.085 ± 0.0012 0.091 ± 0.0024 0.088 ± 0.0018 0.0921
Cat (U/g) 39.8 ± 1.3 40.1 ± 1.5 40.0 ± 1.4 0.0882
SOD (U/g) 63.8 ± 5.7 64.9 ± 6.1 64.4 ± 5.9 0.1566
Fatty Acids (mg /100 g)
C12:0 0.1 ± 0.5 0.0 ± 0.0 0.0 ± 0.3 0.1453
C13:0 3.2 ± 5.4 0.8 ± 3.2 1.5 ± 4.2 0.0009
C14:0 438.7 ± 492.2 153.8 ± 291.9 244.9 ± 389.7 0.0001
C14:1 11.9 ± 15.7 3.0 ± 5.8 5.8 ± 10.9 0.0001
C15:0 168.6 ± 172.2 46.1 ± 98.0 85.3 ± 138.3 0.0001
C16:0 3321.2 ± 2631.5 1093.0 ± 1457.6 1805.4 ± 2170.2 0.0001
C16:1 260.7 ± 237.0 94.6 ± 134.1 147.7 ± 189.5 0.0001
C17:0 321.0 ± 307.3 109.1 ± 237.7 176.8 ± 279.1 0.0001
C17:1 233.0 ± 247.8 75.3 ± 137.5 125.7 ± 194.0 0.0001
C18.0 2692.0 ± 2283.4 1016.9 ± 1649.3 1552.5 ± 2025.2 0.0001
C18:1 4942.6 ± 4041.0 1920.3 ± 2489.0 2886.6 ± 3368.4 0.0001
C18:2 n-6 LA 423.5 ± 266.6 125.4 ± 80.0 220.7 ± 214.9 0.0001
C18:3 n-3 ALA 262.6 ± 209.0 72.5 ± 81.6 133.3 ± 161.9 0.0001
C18:3 n-6 2.1 ± 4.3 2.4 ± 6.5 2.3 ± 5.9 0.8014
C18:4 n-3 3.7 ± 8.6 1.6 ± 10.1 2.3 ± 9.7 0.2262
CLA 117.9 ± 129.7 63.7 ± 190.0 81.1 ± 174.4 0.0788
C19:1 39.1 ± 41.4 14.3 ± 26.3 22.2 ± 33.8 0.0001
C20:0 20.3 ± 18.4 7.3 ± 10.8 11.4 ± 15.0 0.0001
C20:1 26.3 ± 28.4 8.1 ± 12.2 13.9 ± 20.7 0.0001
C20:2 n-6 7.9 ± 8.9 2.1 ± 3.1 4.0 ± 6.2 0.0001
Antioxidants 2020, 9, 1118 10 of 20

Table 3. Cont.

Variable Ram (n = 47) Ewe (n = 100) Overall (n = 147) P-Value

C20:3 8.4 ± 4.1 11.7 ± 12.7 10.6 ± 10.8 0.0839
C20:3 n-6 8.8 ± 4.9 6.1 ± 2.1 7.0 ± 3.5 0.0001
C20:4 n-3 4.8 ± 7.3 2.1 ± 1.5 3.0 ± 4.5 0.0005
C20:4 n-6 36.4 ± 14.2 33.7 ± 8.0 34.6 ± 10.4 0.1473
C20:5 n-3 (EPA) 26.0 ± 8.5 24.3 ± 5.2 24.9 ± 6.5 0.1402
C21:0 2.0 ± 2.5 0.6 ± 1.4 1.0 ± 1.9 0.0001
C22:0 2.8 ± 3.3 2.3 ± 1.3 2.5 ± 2.2 0.1342
C22:1 0.4 ± 1.2 0.8 ± 1.6 0.7 ± 1.5 0.1613
C22:4 n-6 0.6 ± 1.2 1.4 ± 0.5 1.2 ± 0.9 0.0001
C22:5 n-3 (DPA) 22.5 ± 11.6 25.2 ± 8.0 24.4 ± 9.4 0.097
C22:5 n-6 0.0 ± 0.1 0.2 ± 0.2 0.1 ± 0.2 0.0001
C22:6 n-3(DHA) 5.8 ± 3.7 8.3 ± 2.7 7.5 ± 3.2 0.0001
C23:0 2.5 ± 2.3 2.5 ± 0.7 2.5 ± 1.4 0.7207
C24:0 2.2 ± 2.0 2.8 ± 0.9 2.6 ± 1.4 0.008
C24:1 n-9c 1.7 ± 2.1 3.9 ± 1.8 3.2 ± 2.1 0.0001
EPA+DHA 31.9 ± 11.3 32.6 ± 7.0 32.4 ± 8.5 0.6265
EPA+DHA+DPA 54.4 ± 21.8 57.9 ± 13.6 56.7 ± 16.7 0.2388
SFA 6971.2 ± 5684.7 2434.4 ± 3700.9 3884.9 ± 4896.6 0.0001
MUFA 2120.3 ± 2772.9 5515.7 ± 4577.1 3205.9 ± 3786.7 0.0001
PUFA 380.7 ± 331.9 931.1 ± 614.7 556.7 ± 510.0 0.0001
PUFA/SFA 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.1 0.0036
P
n-3 PUFA 134.1 ± 96.5 325.5 ± 231.7 195.3 ± 176.7 0.0001
P
n-6 PUFA 479.3 ± 279.5 171.3 ± 85.2 269.8 ± 224.3 0.0001
n-6/ n-3 PUFA 1.6 ± 0.5 1.4 ± 0.3 1.5 ± 0.4 0.0001
1 FCTP: Folin–Ciocalteu Total Phenolics; GAE: Gallic acid equivalents; FRAP: Ferric reducing antioxidant

power. Antioxidant enzyme P activities of GSH-Px: glutathione peroxidase, Cat: catalase (Cat) and SOD:
superoxide dismutase. SFA sum of saturated P FAs: C12:0+C13:0+C14:0CC14:0+C15:0+C15:0+C15:0+C16:0+
C17:0+C18:0+C20:0+C21:0+C22:0+C23:0+C24:0;P MUFA sum of monounsaturated FAs: C14:1+C16:1+C17:1+
C18:1+C19:1+C20:1+C21:1+C22:1+C24:1. PUFA is the sum of polyunsaturated FA: C18:2 n-6+ C18:3 n-3+ C18:3Pn-6+
C18:4 n-3+CLA+C20:2 n-6+C20:3+C20:3 n-6+C20:4 n-3+C22:4 n-6+C20:5 n-3+C22:5 n-3+C22:5 n-6+C22:6 P n-3. n-6
PUFA is the sum of n-6 PUFA: C18:2 n-6+C18:3 n-6 +C20:2 n-6+C20:4 n-6+C20:3 n-6+C20:4 n-6+C22:5 n-6. n-3 PUFA is
the sum of n-3 PUFA: C18:3 n-3+C18:4 n-3+C20:4 n-3+C20:5 n-3+C22:5 n-3+C22:6 n-3.

Table 4. Effect of inbreeding coefficients (Means ± s.d.) on fat melting point, intramuscular fat, fatty
acids, antioxidant phenolics and enzyme activities in the Longissimus dorsi muscle of ryegrass-fed
TAW lambs 1 .

Inbreeding Coefficient (%)

Low (0–5) Medium(6–10) High (Above 10)
Variable P-Value
(n = 49) (n = 49) (n = 49)
Fat Melting Points (◦ C) 34.9 ± 2.1 34.2 ± 2.4 34.8 ± 2.8 0.225
Intramuscular fat 4.1 ± 1.4 4.0 ± 1.2 4.1 ± 1.0 0.9148
FCTP (mg GAE/g) 1.271 ± 0.0014 1.269 ± 0.0019 1.290 ± 0.0014 0.8524
FRAP (mmol Fe 2+ E/g) 6.018 ± 0.0027 6.083 ± 0.0045 6.102 ± 0.0086 0.2352
GSH-Px (U/g) 0.091 ± 0.0041 0.086 ± 0.0036 0.089 ± 0.0062 0.0896
Cat (U/g) 40.5 ± 1.8 40.1 ± 1.6 40.7 ± 1.9 0.1843
SOD (U/g) 64.8 ± 5.7 65.0 ± 5.9 64.5 ± 5.3 0.0972
Fatty Acids (mg/100 g)
C12:0 0.0 ± 0.4 0.0 ± 0.0 0.0 ± 0.0 0.6757
C13:0 2.3 ± 4.6 0.7 ± 3.5 0.0 ± 0.0 0.0574
C14:0 340.3 ± 479.9 127.5 ± 176.7 94.6 ± 50.8 0.0033
C14:1 7.7 ± 11.9 3.6 ± 9.4 2.4 ± 1.7 0.0609
C15:0 116.6 ± 152.0 47.7 ± 112.7 27.1 ± 22.9 0.0074
C16:0 2377.1 ± 2509.5 1100.6 ± 1410.2 923.7 ± 527.9 0.0013
C16:1 194.1 ± 215.9 90.9 ± 135.4 71.8 ± 38.3 0.0033
C17:0 241.4 ± 322.4 98.9 ± 193.4 61.0 ± 32.9 0.006
Antioxidants 2020, 9, 1118 11 of 20

Table 4. Cont.

Inbreeding Coefficient (%)

Low (0–5) Medium(6–10) High (Above 10)
Variable P-Value
(n = 49) (n = 49) (n = 49)
C17:1 165.6 ± 202.9 78.3 ± 178.8 46.6 ± 26.2 0.0174
C18.0 2130.6 ± 2447.3 840.2 ± 938.8 655.2 ± 288.8 0.0004
C18:1 3704.7 ± 3831.0 1885.3 ± 2423.3 1552.9 ± 690.9 0.0036
C18:2 n-6 (LA) 271.1 ± 244.2 158.9 ± 156.5 139.0 ± 65.3 0.0053
C18:3 n-3 (ALA) 170.3 ± 179.0 89.0 ± 129.2 63.7 ± 33.0 0.0067
C18:3 n-6 3.1 ± 7.8 1.3 ± 0.9 1.5 ± 0.8 0.032
C18:4 n-3 3.1 ± 11.5 1.4 ± 7.0 0.4 ± 0.3 0.5311
CLA 114.4 ± 219.4 40.4 ± 75.8 24.9 ± 13.7 0.032
C19:1 29.4 ± 37.4 13.6 ± 27.5 9.1 ± 5.2 0.0139
C20:0 15.5 ± 17.7 6.5 ± 8.4 5.2 ± 2.7 0.001
C20:1 18.4 ± 22.3 8.7 ± 17.9 5.2 ± 2.2 0.0124
C20:2 n-6 5.3 ± 7.2 2.2 ± 4.3 2.0 ± 2.1 0.0092
C20:3 11.0 ± 14.3 10.2 ± 2.7 9.4 ± 2.7 0.8782
C20:3 n-6 7.6 ± 3.9 6.2 ± 2.7 5.6 ± 1.1 0.8782
C20:4 n-3 3.6 ± 5.3 2.3 ± 3.1 1.6 ± 0.6 0.171
C20:4 n-6 32.8 ± 11.6 36.6 ± 8.4 38.0 ± 8.3 0.0737
C20:5 n-3 (EPA) 24.3 ± 7.0 25.8 ± 5.9 23.0 ± 4.8 0.3091
C21:0 1.6 ± 2.3 0.4 ± 0.9 0.1 ± 0.2 0.0009
C22:0 3.0 ± 2.7 1.8 ± 1.1 1.6 ± 0.8 0.0055
C22:1 0.8 ± 1.9 0.6 ± 0.9 0.3 ± 0.3 0.5598
C22:4 n-6 1.2 ± 1.0 1.2 ± 0.7 1.3 ± 0.7 0.9005
C22:5 n-3 (DPA) 24.7 ± 11.5 23.9 ± 5.7 23.3 ± 5.0 0.8515
C22:5 n-6 0.1 ± 0.2 0.2 ± 0.2 0.2 ± 0.3 0.3705
C22:6 n-3(DHA) 7.4 ± 3.8 7.6 ± 2.4 7.5 ± 3.1 0.9816
C23:0 2.7 ± 1.7 2.7 ± 1.0 2.0 ± 1.1 0.2737
C24:0 2.7 ± 1.5 2.5 ± 1.2 2.3 ± 1.3 0.674
C24:1 n-9c 2.9 ± 2.1 3.5 ± 2.1 3.7 ± 2.1 0.2498
EPA+DHA 31.8 ± 9.5 33.4 ± 7.1 30.6 ± 7.6 0.4708
EPA+DHA+DPA 56.5 ± 19.8 57.3 ± 11.9 53.8 ± 12.4 0.8738
SFA 5231.3 ± 5786.8 2228.5 ± 2775.3 1772.7 ± 913.9 0.0007
MUFA 4123.6 ± 4281.0 2084.4 ± 2782.6 1691.9 ± 761.3 0.0036
PUFA 680.1 ± 577.2 407.1 ± 373.2 341.5 ± 105.3 0.0037
PUFA/SFA 0.2 ± 0.1 0.2 ± 0.1 0.2 ± 0.0 0.0781
P
n-3 PUFA 233.4 ± 196.1 149.9 ± 141.9 119.6 ± 27.5 0.0114
P
n-6 PUFA 321.2 ± 255.7 206.6 ± 162.3 187.6 ± 71.6 0.0067
n-6/ n-3 PUFA 1.5 ± 0.4 1.5 ± 0.3 1.5 ± 0.3 0.8487
1 Abbreviations are the same as in Table 3.

3.2. Intramuscular Fat Content (IMF)

IMF ranged from 3.4% to 8.2%, but ewe lambs had significantly higher IMF (4.4 ± 1.4%) than
ram lambs (3.4 ± 0.3%) as shown in Figure 4A. Irrespective of gender, the overall IMF was 4.1 ± 1.3%
(Table 3). As shown in Table 4, IC as an index of linebreeding was classified into low (0–5%), medium
(6–10%) and high (>10%) and ranged from 0% to 15.6%. As IC increased, there were no differences in
IMF (Table 4 and Figure 4B).
3.2. Intramuscular Fat Content (IMF)
IMF ranged from 3.4 to 8.2 %, but ewe lambs had significantly higher IMF (4.4 ± 1.4 %) than ram
lambs (3.4 ± 0.3 %) as shown in Figure 4A. Irrespective of gender, the overall IMF was 4.1 ± 1.3 %
(Table 3). As shown in Table 4, IC as an index of linebreeding was classified into low (0–5 %), medium
(6–10 %) and
Antioxidants high
2020, (>10 %) and ranged from 0 to 15.6 %. As IC increased, there were no differences
9, 1118 12 ofin
20
IMF (Table 4 and Figure 4B).

Antioxidants 2020, 9, x FOR PEER REVIEW 12 of 20

Figure 4. Variation in intramuscular fat (IMF) percentage of Tattykeel Australian White (TAW):
(A) gender;
Figure (B) Inbreeding
4. Variation Coefficient
in intramuscular fat(IC).
(IMF) percentage of Tattykeel Australian White (TAW): (A)
gender; (B) Inbreeding Coefficient (IC).
3.3. Fat Melting Point (FMP)
3.3. Fat
FMPMelting Point
ranged 28 to 39 ◦ C, but ewe lambs had significantly lower FMP (34.26 ± 2.43 ◦ C)
(FMP)
from
◦ C) as shown in Figure 5. Irrespective of gender, the overall FMP was
thanFMP
ram ranged from ±
lambs (35.5 281.5
to 39 °C, but ewe lambs had significantly lower FMP (34.26 ± 2.43 °C) than
34.6lambs ◦
± 2.3 (35.5
C (Table
ram ± 1.53).
°C)Similar
as shown to in
IMF, IC was
Figure not significantly
5. Irrespective associated
of gender, withFMP
the overall FMPwas
(Table
34.64±and
2.3
Figure 4B).
°C (Table 3). Similar to IMF, IC was not significantly associated with FMP (Table 4 and Figure 4B).

Figure 5. Variation in fat melting point (FMP) percentage of Tattykeel Australian White (TAW):
Figure 5. Variation in fat melting point (FMP) percentage of Tattykeel Australian White (TAW): (A)
(A) gender; (B) Inbreeding Coefficient (IC).
gender; (B) Inbreeding Coefficient (IC).
3.4. Faty Acid Composition
3.4. Faty Acid Composition
The fatty acid composition in ewe and ram lambs in mg /100 g tissue is shown in Table 3. It shows
that The
ewe fatty
lambsacid
hadcomposition in <
significantly (P ewe and higher
0.0001) ram lambs in mg
C22:5n-3 /100 C22:6n-3
(DPA), g tissue is shownC18:3n-6,
(DHA), in TableC20:3,
3. It
shows thatC22:5n-6,
C22:4n-6, ewe lambs had significantly
MUFA, (P ˂ 0.0001)
PUFA and Σn-3 higher
and lower SFAC22:5n-3 (DPA),
fatty acids than C22:6n-3
ram lambs.(DHA), C18:3n-
Although n-3
6,LC-PUFA
C20:3, C22:4n-6, C22:5n-6,
ranged from MUFA,
“source” PUFA
to “good and levels
source” Σn-3 of
and lower
33–69 /100 g
mgSFA fatty acids thanlambs,
in individual ram lambs.
overall,
Although n-3 LC-PUFA ranged from “source” to “good source” levels of 33–69 mg /100 g in
individual lambs, overall, there were no gender differences in the health-promoting EPA, DPA,
EPA+DHA and EPA+DHA+DPA (Table 3). As IC increased, there were no differences in C20:5n-3
(EPA), DHA, DPA, EPA+DHA, EPA+DHA+DPA and ∑n-6/ ∑n-3 ratio, while increases in C18:3n-3
(ALA), MUFA, PUFA, C18:1, C18:2n-6, C18:3N-6, ∑n-3 PUFA and ∑n-6 PUFA were observed as IC
Antioxidants 2020, 9, 1118 13 of 20

there were no gender differences in the health-promoting EPA, DPA, EPA+DHA and EPA+DHA+DPA
(Table 3). As IC increased, there were no differences in C20:5n-3 (EPA), DHA, DPA, EPA+DHA,
P P
EPA+DHA+DPA and n-6/ n-3 ratio, while increases in C18:3n-3 (ALA), MUFA, PUFA, C18:1,
P P
C18:2n-6, C18:3N-6, n-3 PUFA and n-6 PUFA were observed as IC decreased from high to low
(Table 4).

4. Discussion
Consumer preferences, behaviours, perceptions and satisfaction with the eating quality of meat
products are intricately linked to flavour, odour, colour, aroma, taste and juiciness [50,51]. Previous
studies [52–57] identified diet-related “pastoral flavour” in lamb, also described as “milky”, “barnyard”,
“sheepy” or “faecal” flavour, to negatively impact consumer liking. It is thought that this unpleasant
“pastoral flavour” originates from skatole (3-methylindole) and indole derivatives from the degradation
of tryptophan, 4-methylphenol and other branched chain fatty acids in the rumen [58]. Lamb has also
been reported to have a distinct age-related “mutton flavour” and aroma associated with the three
branched chain fatty acids 4-methylnonanoic, 4-methyloctanoic and 4-ethyloctanoic acids [59]. Other
previous studies had demonstrated that the nutritional background of pasture-fed ruminants confers a
higher muscle α-tocopherol antioxidant status compared to those on concentrate based diets [60–64].
Several internal and external factors influence the quantity and quality of lipids in animal products
due to genetics-nutrition interactions in the expression of genes controlling fat metabolism [65] and
these include the key attributes of fat melting point, intramuscular fat and fatty acid composition.
The Folin–Ciocalteu total phenolics, ferric reducing antioxidant power and antioxidant enzyme
activities of glutathione peroxidase, catalase and superoxide dismutase values in the present study
were consistent with those reported in the Longissimus thoracis et lumborum muscle [26], liver and
plasma [27] of lambs grazing ryegrass. However, in our present study, the observation that none of
the dietary phenolic compounds and antioxidant enzyme activities detected in the Longissimus dorsi
muscle were affected by the lamb gender (Table 3) or inbreeding coefficient (Table 4) suggests that
gender and linebreeding had no direct impact on the antioxidant status and deposition mechanism in
the muscle tissue of MARGRA lambs. This implies that TAW sheep grazing ryegrass rich in phenolics
contributed to an improved overall meat oxidative stability with similar deposition and bioavailability
in ewe and ram lambs irrespective of inbreeding coefficient.

4.1. FMP
The FMP range of 28–39 ◦ C and an overall mean of 34.6 ± 2.3 ◦ C obtained for TAW in the present
study is well below the range of 40.6–48.0 ◦ C and 41.5–44.0 ◦ C reported by Flakemore et al. [13]
and Holman et al. [43] in purebred and crossbred Merino, Dorset, Black and White Suffolk sheep.
The presence of double or triple bonds in the FA structure leads to lower melting points because the
higher the proportions of MUFA and PUFA, the more easily such bonds can be broken and the lower
the fat melting points compared to the more stable SFA with high FMP. Smith et al. [66] reported that
these SFA contributed to an elevation in the hardness of fat in beef, while Flakemore et al. [13] reported
that the softness or hardness of fat has safety implications for meat processors and boning room
personnel in abattoirs. From our results in the present study (Table 3), the lesser the SFA concentration
and more MUFA and PUFA implied a low FMP, indicating that TAW is not only a healthier meat
product for consumers, but also a safe product to meat processors in the abattoir due to ease of
processing. It was also apparent that elevated proportions of SFA, especially palmitic (C16:0) and
stearic (C18:0) acids in ram lambs, could have been the reason for the higher FMP than in ewe lambs.
While the underpinning reasons behind the observed sex differences in FMP are not definitive from
our present study, we can speculate that hormonal differences between ram and ewe lambs could
have possibly had an indirect influence on FMP through IMF. This is because it has been reported
that in intact bovine males, testosterone binds to receptors within the muscle and increases amino
acid incorporation into protein, thus increasing muscular development, growth rate and muscle mass
Antioxidants 2020, 9, 1118 14 of 20

without simultaneous increases in IMF [67,68]. The lesser the IMF, the higher the FMP, and the higher
the IMF as seen in TAW ewe lambs, the lower the FMP. This would seem to explain why, in the current
study, the ewe lambs had higher IMF and lower FMP than ram lambs. Further research on the likely
underlying molecular mechanisms behind FMP and IMF variation through lipogenic genes controlling
fat metabolism like fatty acid binding protein-4, fatty acid synthase and stearoyl-CoA desaturase
currently being investigated with TAW lambs in our laboratory, would assist in shedding more light.

4.2. IMF
IMF influences meat palatability and contributes to its juiciness, flavour and tenderness with
direct linkage between intramuscular fat deposition and gender, age, genetics and nutrition [67–69].
The overall mean IMF of 4.4 ± 0.2% in the current study surpasses the suggested minimum Australian
threshold of 4% for lamb palatability by Pannier et al. [70] who reported an overall average IMF of
4.23 ± 0.01% in lambs from sires selected for leanness. The IMF values in TAW pasture-fed lambs
in the current study are much higher than the 1.25 ± 0.22% in lot-fed Manchega lambs reported
by Gomez-Cortes et al. [71] in contrast to the expectation that lambs sacrificed after 42 days in
the feedlot should have higher IMF. This would most likely be a combination of both genetic
and nutritional effects with TAW having a genetic predisposition for a comparatively higher and
faster rate of IMF deposition in response to ryegrass pasture feeding than other breeds such as the
Manchega lambs fed concentrate rations with high fibrous components. Published reports of gender
differences in IMF are not unanimous in their findings. For instance, while Pannier et al. [70] and
McPhee et al. [16,72] reported significant sex differences in IMF just as we also observed in TAW lambs,
Okeudo and Moss [73] did not find any differences in intramuscular lipid and fatty acid profiles of
sheep comprising four sex-types. In beef cattle, Cafferky et al. [67] stated that the higher IMF values
in steers than intact bulls are attributed to the diminished physiological effects of androgen, which
reduces plasma lipids, increases lipolysis by adipocytes and stimulates androgen receptors to directly
upregulate the lipogenic gene expression of fatty acid synthase and acetyl-CoA carboxylase [74,75]
while simultaneously downregulating the lipolytic gene expression of monoglyceride lipase and
adipose triglyceride lipase [55]. Hence, castration contributes to improved IMF deposition through
increased lipogenesis and lipid uptake while decreasing lipolysis [76]. Given the hormonal differences
between ewe and ram lambs, similar genetic, physiological and biochemical pathways may be involved,
and our lab is currently exploring the sequencing and expression of fatty acid binding protein-4,
fatty acid synthase and stearoyl-CoA desaturase genes in TAW to unravel and better understand the
underpinning mechanisms of fat metabolism.
It was quite interesting that IC had no impact on FMP and IMF (Table 3). This is very significant
from an eating quality perspective because it indicates that TAW lambs can produce consistently
high-quality MARGRA meat product with low FMP and high IMF regardless of linebreeding with IC
in the 0–15.6% range. To our current knowledge, the present study is the first of its kind to provide
a significant insight into the impact of IC on meat eating quality in lamb as the only other reported
research on inbreeding was in milking cows where Carrara et al. [77] reported significant (P < 0.04)
impact of inbreeding on milk PUFA.

4.3. Omega-3 Long-Chain Polyunsaturated Fatty Acids

The ingestion of n-3 LC-PUFA confers a number of health benefits, including inhibiting
cardiovascular diseases, cancer, and diabetes, obesity and neurodegenerative diseases such as
amyotrophic lateral sclerosis, Parkinson’s, and Alzheimer’s [78] as well as improve visual and
brain development [79]. Le et al. [80] reported that Food Standards of Australia and New Zealand
(FSANZ) guidelines stipulate that for any food or meat to be termed a ‘source’ of n-3 LC PUFA, its EPA
and DHA levels must be greater than 30 mg per 100 g per serve. TAW lambs had 32.4 ± 8.5 mg per 100 g
of muscle, thus surpassing the 30 mg limit set by FSANZ for ‘source’ claim. The main FA in pastures
is ALA, a precursor of the more potent n-3 LC-PUFA [81], especially EPA, DHA and DPA, which
Antioxidants 2020, 9, 1118 15 of 20

have important roles to play in human health. The observations that, as IC increased, there were no
differences in FMP, IMF, C20:5n-3 (EPA), DHA, DPA, EPA+DHA, EPA+DHA+DPA and Σn-6/Σn-3 ratio
and that increases in C18:3n-3 (ALA), MUFA, PUFA, C18:1, C18:2n-6 and C18:3n-6 were observed as IC
decreased, indicate that linebreeding in the 0–15.6% range is not in any way detrimental to consistency
in health-promoting n-3 LC-PUFA in TAW lambs. Such observations represent the first piece of
experimental evidence regarding the impact of IC on omega-3 FA. Our data herein provide scientific
evidence that TAW MARGRA lamb contains higher levels of health beneficial n-3 LC-PUFA than in
other Australian lamb breeds previously reported by Ponnampalam et al. [82–86], Flakemore et al. [87],
Knight et al. [88,89], De Brito et al. [90] and Fowler et al. [91]. Sex has been shown to influence heart
and muscle FA composition, although the differences were restricted to only a few FA [45]. Previous
studies have attributed FA variations due to sex as arising from sex-linked hormonal differences, which
affect development and rumen biohydrogenation [45].

5. Conclusions
The results obtained from this study provide the first detailed scientific evidence of TAW MARGRA
lamb with low SFA, high IMF, MUFA, n-3 LC-PUFA and lower FMP. Therefore, the meat from TAW
lambs provides anecdotal and scientific evidence for adequate meat oxidative stability and human
health benefits associated with n-3 LC-PUFA to consumers. This study clearly provides a scientific
confirmation of the unique meat eating quality traits of TAW lambs. Based on nutritional value to
consumers, this study reinforces the health benefits derived from consuming TAW MAGRA lamb
in view of its high EPA, DHA and DPA contents. This high n-3 LC-PUFA profile of TAW MARGRA
lamb has put this breed well ahead of others in terms of healthy meat products. Our findings clearly
show significant gender variation between ram and ewe lambs. The lower SFA and higher MUFA
and PUFA contents make MARGRA lamb fats very soft and smooth melting in the mouth without
sticking to the palate due to its low FMP and healthier composition. This study provides evidence
that IC is inconsequential in affecting antioxidant status, IMF, FMP and n-3 LC-PUFA in linebred
and pasture-fed TAW sheep. This is because the observed variation in individual fatty acids was
mainly driven by gender differences between ewes and rams, hence the need to accept the tested
hypothesis. The practical implication is that health-conscious meat consumers are reassured by the
scientific evidence herein of the consistency in the eating quality of MARGRA lamb brand from TAW
sheep regardless of its linebred origin.

Author Contributions: Conceptualization, A.E.O.M.-A.; methodology, A.E.O.M.-A., J.R.O., S.B.P., software,

A.E.O.M.-A.; validation, A.E.O.M.-A., S.B.P., J.R.O., formal analysis, S.B.P; O.A.A.; investigation, S.B.P.; resources,
A.E.O.M.-A., R.T.K. and O.A.A.; data curation, writing—original draft preparation, S.B.P.; writing—reviewing and
editing, A.E.O.M.-A., R.T.K. and O.A.A.; supervision, A.E.O.M.-A., R.T.K. and O.A.A.; project administration,
A.E.O.M.-A.; funding acquisition, A.E.O.M.-A. All authors have read and agreed to the published version of
the manuscript.
Funding: This research was funded by the Innovation Connections Research Grant from the Australian
Commonwealth Government’s Dept of Industry, Innovation, Climate Change, Science, Research and Tertiary
Education, Tattykeel Australian White Pty Ltd. and a PhD scholarship funded by the James Cook University
Postgraduate Research Scholarship (JCUPRS), Queensland, Australia, awarded to the first named author.
Acknowledgments: The authors gratefully acknowledge JCUPRS, James Cook University College of Public
Health, Medical and Veterinary Sciences (PhD scholarship), the Australian Commonwealth Dept of Industry,
Innovation, Climate Change, Science, Research and Tertiary Education (research funding), Tattykeel Australian
White Pty Ltd. (access to flock, farm resources, research funding), Commonwealth Scientific and Industrial
Research Organisation Marine and Atmosphere Hobart (fatty acid analysis) and the National Veterinary Research
Institute Vom, Nigeria (study leave approval for the first-named author).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish
the results.
Antioxidants 2020, 9, 1118 16 of 20

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