Honor Calnan

RNA‐binding proteins (RBPs) play a pivotal role in post‐transcriptional regulation of gene expression, critically influencing skeletal myogenesis, muscle growth, and regeneration. Despite the recent identification of RBP Rps27l (ribosomal protein S27‐like) as a regulator affecting myogenic proliferation and differentiation, its functions and regulatory mechanisms in skeletal muscle development remain largely unknown. In this study, it is observed that muscle‐specific Rps27l knock‐in (M─KI) mice exhibit significantly increased muscle mass, enlarged myofiber size, a higher proportion of fast‐twitch myofibers, and enhanced muscle regeneration capabilities compared to wild‐type controls. Overexpression of Rps27l promotes myoblast proliferation while inhibiting differentiation in skeletal muscle cells. Mechanistically, it is revealed that the expression of Rps27l is negatively regulated by SIX4, a myogenic transcription factor. The N‐terminal intrinsically disordered region of RPS27L facilitates liquid‐liquid phase separation (LLPS) and interacts with IGF1 to collaboratively regulate myogenesis. The findings uncover the novel regulatory roles of RPS27L in skeletal muscle and highlight the significance of RPS27L‐driven LLPS in myogenesis.

In 2016 an Australian project, the Advanced Livestock Measurement Technologies project (ALMTech), was initiated to accelerate the development and implementation of technologies that measure lean meat yield and eating quality. This led to the commercial testing, and implementation of a range of new technologies in the lamb, beef, and pork industries. For measuring lean meat yield %, these technologies included dual energy X-ray absorptiometry, hand-held microwave systems, and 3-D imaging systems. For measuring beef rib-eye traits and intramuscular fat %, both pre- and post-chilling technologies were developed. Post-chilling, a range of camera systems and near infrared spectrophotometers were developed. While pre-chilling, technologies included insertable needle probes, nuclear magnetic resonance, and X-ray systems. Initially these technologies were trained to predict the pre-existing traits already traded upon within industry. However, this approach was limiting because the technologies could measure attributes that were either non-existent in the trading language, were superior as calibrating standards, or more accurately reflected value than the pre-existing trait. Therefore, we introduced IMF% into the trading language for both beef and sheep meat, and carcase lean%, fat%, and bone% for sheep meat. These new technologies and the traits that they predict have delivered multiple benefits. Technology provider-companies are instilled with the confidence to commercialise due to the provision of achievable accreditation standards. Processors have the confidence to invest in these technologies and establish payment grids based upon their measurements. And lastly, it has enhanced data flow into genetic databases, industry data systems (MSA), and as feedback to producers.

The value of precise dual energy X-ray absorptiometry (DEXA) cut weight predictions to lamb allocation to cut plans is unknown. Lambs (n = 191) varying in carcase weight (HSCW) and GR (tissue depth over the 12th rib) were DEXA scanned and boned out to weigh retail cuts. Cut weights were predicted using HSCW; HSCW + GR; HSCW + DEXA and HSCW + DEXA image components in GLM models. DEXA improved cut weight predictions in most cuts (P < 0.05). A dataset of 10,000 carcases was then simulated using the associations between HSCW, GR and cut weights, before being truncated to 4500 lambs representing onel day's HSCW distribution. A lamb Carcase Optimisation Tool scenario was developed with 2–3 cut options per carcase section and cut weight thresholds applied to several cuts. Processing costs, market values and actual cut weights were input into the Optimiser to determine carcase allocation to cut options for optimised profits. This scenario was repeated using the predicted cut weights to determine the cut misallocations caused. DEXA-predicted cut weights produced 16.7% and 8.0% less misallocations than HSCW and GR. DEXA produced 20.8% and 14.3% less misallocations than HSCW and GR in shortloins, and 25.5% and 12.9% less in hindquarters. While cut misallocations have little direct impact on total profits, as product is over and under-valued when misallocated, reducing cut misallocations will improve processor compliance when sorting carcases into cut plans- reducing their need to retrim, downgrade and repackage product or the erosion of customer confidence caused by supplying product not meeting market specifications.

The development of a novel rapid dual energy X-ray absorptiometry (DEXA) system provides the opportunity to improve measurement of beef carcase composition. A prototype rapid DEXA system was built in a shipping container to scan 51 beef carcases selected for a wide range in weight and fatness. One side of each carcase was spray chilled and the other conventionally chilled overnight before being quartered for DEXA scanning and then being cut into 16 pieces for CT scanning to determine carcase composition. Spray chilling did not impact DEXA prediction of CT composition, with the DEXA system describing 89%, 95%, and 87% of the variation in beef carcase CT lean %, fat % and bone %, with a root mean square error of prediction of 2.31 lean %, 2.15 fat %, and 1.12 bone % units. These results demonstrate that the novel rapid DEXA system has excellent capacity to predict CT composition in beef carcases.

The rapid browning of lamb meat on retail display reduces its appeal to consumers and thus the marketability of lamb meat. Predicting the rate of meat browning on retail display would allow retailers to effectively manage this issue. The ability of bloomed meat color at the start of retail display to predict meat browning over subsequent retail display was investigated in lamb loin meat. Mixed breed lambs (n = 4404) produced at 5 sites over 5 yr were slaughtered at ∼23kg carcass weight and measured for loin pH at 24 h, myoglobin, iron and zinc concentrations, isocitrate dehydrogenase activity and intramuscular fat. Loin meat was aged for 5 d before being re-sliced and overwrapped for color measurement over a 72 h simulated retail display. Meat redness (R630/R580) was measured after blooming and every 24 h across display using a Hunterlab spectrophotometer. Simple and partial correlation coefficients between initial and subsequent R630/R580 measures over the display were low (≤ 0.4). Accounting for key muscle traits influencing meat color such as pH24, myoglobin, iron or intramuscular fat concentration did not improve these correlations between bloomed meat color and subsequent meat color over retail display. Therefore bloomed meat color at the start of display is not a useful predictor of meat browning after 24 h of retail display. Alternatively, correlations between 24, 48, and 72 h R630/R580 were > 0.8, suggesting that meat color measured from 24 h of display can accurately predict subsequent retail meat browning.

Rapid browning limits the retail display of lamb meat, particularly following extended chilled storage that is typical of international shipment. Lamb stored chilled for short periods browns more rapidly on retail display when the meat has high levels of marbling (intramuscular fat), a high pH or greater oxidative capacity. In contrast, high muscle vitamin E concentration reduces browning of short stored lamb meat on retail display. However, the capacity of dietary vitamin E supplementation to reduce retail browning in lamb meat following extended chilled storage is unknown. Additionally, the ability of vitamin E to mitigate the negative impacts of intramuscular fat, pH and oxidative capacity on meat browning following chilled storage is unknown. Sixty six industry sires of Terminal, Merino and Maternal breed types were bred with Merino and Merino-cross dams to produce 132 lambs. The two lambs from each sire were divided into two vitamin E treatment groups that were housed in 12 pens (6 pens per treatment) for 8 weeks leading up to slaughter. Control lambs (6 pens of 11 lambs) were fed a pelleted ration containing basal levels of vitamin E (30 mg/kg feed) while supplemented lambs (6 pens of 11 lambs) were fed the same ration with increased vitamin E content (275 mg/kg feed). After slaughter, the m. longissimus lumborum was sampled for measurement of vitamin E concentration, intramuscular fat, pH, oxidative enzyme isocitrate dehydrogenase activity, and retail meat colour. Colour samples were vacuum packaged for storage at −1°C for 5, 35 and 70d, before being re-sliced, wrapped and placed under stimulated retail display where meat redness (R630/R580) was measured 24 hourly for 72 h. Lamb pens supplemented with vitamin E produced redder loin meat at 24, 48 and 72 h of display compared to control pens of lambs (P < 0.05), regardless of storage time. Increasing muscle vitamin E concentration from 0.8 to 4 mg/g of loin muscle improved meat redness to a slightly greater extent in meat stored for 35 d than 5 or 70 d (P < 0.05). High intramuscular fat reduced redness in 5 day stored lamb (P < 0.05) and increasing muscle vitamin E had a greater effect improving the display colour in this highly marbled meat. These results demonstrate that dietary vitamin E supplementation will improve the display colour of highly marbled or long stored lamb meat.

The colour of lamb meat on retail display is critical to consumer appeal. Consumers demand a bright red colour in lamb meat and associate dark, pale or browned meat with a lack of freshness and quality. Retailers are forces to downgrade or discount discoloured meat to prevent consumer rejection. Fresh lamb meat may be discoloured due to poor colour development with blooming, or due to browning that develops with time on retail display. The quantity of lamb meat with poor bloomed colour is unknown because lamb carcasses are not routinely graded for meat colour in Australia. The rapid browning of overwrapped lamb meat limits its retail display to only around 2 days. Retailers frequently discount lamb meat to ensure its rapid sale prior to the onset of browning and to thereby limit the economic losses associated with discounting, downgrading or wasting browned meat. Better understanding is required of the factors influencing bloomed colour of lamb meat and its stability on retail display in order to develop strategies to reduce meat discolouration. This thesis evaluates different phenotypic and genotypic factors influencing lamb meat colour in Australia. Bloomed colour (L*, a*, b*, hue angle and chroma) was measured in the loin muscle of over 8000 mixed breed lambs of known genetics produced at 8 sites across Australia over 5 years as part of the Sheep CRC’s INF experiment. The colour stability of the loin muscle was evaluated in over 4000 of these lambs using spectrophotometric measures of meat redness (R630/R580) taken over a 3 day simulated retail display. The first experiment of this thesis examines the influence of production factors and muscle traits on the bloomed colour of lamb loin. Over 40% of the 8165 loin samples were too dark for consumer acceptance, suggesting that dark meat is a substantial problem in the Australian lamb meat industry. Production factors such as lamb slaughter group, production site and year of production had substantial effects on bloomed colour, though these effects could not be attributed to changes in muscle traits such as myoglobin or pH24. Further investigation is required to better understand how production factors influence bloomed meat colour. Of the muscle traits analysed, changes in pH at 24 hours (pH24) had the greatest effect on meat a*, while myoglobin had the greatest effect on meat L*. Increasing lamb age from 140 to 400 days reduced meat L* due to increased myoglobin concentration. These results suggest that industry focus needs to shift to consideration of myoglobin concentration as well as meat pH in order to improve the bloomed colour of lamb meat, particularly meat lightness. The second experiment examines the influence of selection for IMF and lean meat yield on bloomed colour. Increasing IMF from 2 to 8% and shortloin fat weight from 100-500g were positively associated with meat L*, a*, b*, hue angle and chroma. Shortloin muscle weight was negatively associated with these colour parameters, though could largely be accounted for by correlated changes in IMF. The effect of sire breeding values for lamb weight, shortloin muscle depth and fat depth on loin L*, a*, b*, hue angle and chroma were small and varied between lamb sire type, dam breed and sex. Thus selection for increased lean meat yield in lambs will have neutral or positive effects on meat colour, while selection for increased IMF will increase the L*, a*, chroma and thereby the consumer appeal of bloomed lamb meat. The third experiment examines the influence of muscle weight and oxidative capacity on the colour of lamb loin following 72 hours of simulated retail display. Production factors such as slaughter group and site of production had the greatest magnitude effects on meat R630/R580 (redness) after 3 days of display. Increasing loin ICDH activity, reflecting muscle oxidative capacity, reduced R630/R580 at the end of display. Selection for high sire breeding values for shortloin muscle depth increased R630/R580, likely due to an associated reduction in muscle oxidative capacity. Lamb carcass weight also increased R630/R580. Genotypic factors influencing lamb size and growth rate such as sire type and dam breed further support that increased growth rate improves meat colour. These findings suggest that breeding for increased growth rate and muscle weight could improve the colour stability of overwrapped lamb on retail display. The fourth experiment examines the ability to predict the rate of lamb meat browning on display using measures of bloomed colour and information of animal factors that influence colour stability. Simple and partial correlation coefficients between initial boomed R630/R580 and subsequent R630/R580 measures over display were ≤ 0.4, despite incorporation of carcass traits including pH24, lamb age and IMF. Therefore, bloomed colour at the start of display cannot provide a useful prediction of subsequent meat browning. Correlations between 24, 48 and 72 hr measures of R630/R580 were high (> 0.8), suggesting that colour measured from 24 hours of display can provide an accurate prediction of subsequent meat colour. While predicting the rate of meat browning would allow retailers to maximise the display time of lamb, meat colour measured at 24 hrs of display is unlikely to be practical in a retail setting. The predictive ability of 24 hr colour may therefore be limited to use in the development of a breeding value for retail colour. The final experiment examines the use of dietary vitamin E supplementation to improve the colour stability of long-stored lamb meat, particularly meat with high IMF, pH24 and ICDH activity. The capacity of this antioxidant to improve the colour stability of lamb meat was expected to increase in long-stored meat with high IMF, pH24 and ICDH activity due to the anticipated increased oxidative load in this meat. Vitamin E supplementation at 275 mg/kg of feed for 8 weeks prior to slaughter increased R630/R580 throughout the display of lamb loin following 5, 35 and 70 days of storage. However contrary to expectations, vitamin E supplementation had a similar magnitude impact on meat colour stability regardless of storage time prior to the display. Vitamin E did mitigate the negative effect of high IMF on short-stored meat colour, however high IMF did not impact the colour stability of medium or long-stored meat. These results demonstrate that while vitamin E supplementation will improve the colour stability of lamb meat following short, medium or long storage, its greatest impact is on the display colour of short-stored lamb with high IMF content.

The colour of bloomed m. longissimus was measured 24 h post slaughter for 8165 lamb carcasses produced over 5 years across 8 sites in Australia. Intramuscular fat across a 2 to 8% range and shortloin fat weight were positively associated with meat lightness (L*), redness (a*), yellowness (b*), hue and chroma (P < 0.01). Shortloin muscle weight was negatively associated with these meat colour parameters (P < 0.01), although this was largely accounted for by correlated changes in intramuscular fat (P < 0.01). The effect of sire breeding values for lamb weight, shortloin muscle depth and fat depth on loin L*, a*, b*, hue and chroma were small and varied between lambs of different sire type, dam breed and sex. Thus selection for lean meat yield will have neutral or positive effects on meat colour, while selection for increased intramuscular fat will make the bloomed colour of lamb meat lighter and redder.

M. longissimus colour was measured from 8165 lambs at 24 h post-mortem using a chromameter. The impact of production factors (site and year of production, slaughter group, sex, age and breed type) and muscle traits (hot carcass weight, pH24, isocitrate dehydrogenase (ICDH) activity, myoglobin, iron and zinc concentrations) on meat lightness (L*), redness (a*), yellowness (b*), hue and chroma were analysed. Greater differences in meat colour were seen between different slaughter groups and sites of production than across the range of any muscle traits. Of the muscle traits analysed, changes in pH24 had the greatest effect on meat a* (2.5 units), while myoglobin had the greatest effect on meat L* (2.9 units). The 3.1 L* unit darkening of meat with increasing lamb age (from 140 to 400 days) was accounted for by increased myoglobin concentration. These results suggest that production factors are having substantial effects on lamb colour independent of known influencing muscle traits such as myoglobin concentration and pH.