Evaluating genetic markers for muscling (tm-qtl)

Summary of research and results:

The objectives and results from the project are madeunder the two main themes: (i) Evaluation of direct and indirect effects of 3 muscling QTL/genes (ii) Evaluation of VIA and its use to measure the effects of the muscling genes/QTL, and the project had following objectives.

Objective 1.1: to evaluate the direct (lean meat yield) and indirect (growth, carcass composition, especially meat eating quality, animal health and animal welfare) effects of the TM-QTL, in a purebred terminal sire line of Texel sheep

Objective 1.2: to evaluate the direct and indirect effects in crossbred lambs produced out of Mule ewes mated to Texel rams heterozygous for TM-QTL

Objective 1.3: to evaluate the direct and some indirect effects in crossbred lambs produced out of Mule ewes inseminated with semen from New Zealand Poll Dorset rams (supplied by Catapult Genetics) carrying the LM-QTL

Objective 1.4: to evaluate the direct and some indirect effects in crossbred lambs produced out of Mule ewes inseminated with semen of New Zealand Texel rams (supplied by Innovis Genetics) carrying the MM-QTL

Objective 1.5: to evaluate the direct effects of TM-QTL and MM-QTL in crossbred lambs out of Welsh Mountain ewes (lambs produced by Innovis Genetics) inseminated with semen of rams heterozygous for the TM-QTL and MM-QTL

Objective 1.6: to introgress both the TM-QTL and MM-QTL into purebred Inverdale-Texel sheep, and TM-QTL into other Terminal Sire breeds (e.g. Suffolk and Charollais) using marker assisted backcrossing, with the aim of producing a small number of carrier rams for more widespread introduction of the QTL within these breeds

Objective 2.1: to investigate the associations of observations using a VIA – system with: a) carcass composition measured by CT; b) carcass dissection results

Objective 2.2: to predict the relationships between VIA observations and eating quality traits

Objective 2.3: to use the TM-QTL as an example for a QTL affecting carcass and possibly meat quality and estimate its effect on VIA characteristics

Objective 2.4: to work towards the development of optimal sustainable breeding programmes to improve meat and carcass quality

(i) Evaluation of direct and indirect effects of 3 muscling QTL/genes

a) TM-QTL: This QTL on chromosome 18, originally identified in purebred UK Texel sheep, had been reported to increase UMD muscle depth by around 4% to 7%. Muscling in other parts of the body had not been investigated.

• The QTL increased loin muscling by 4-11% in heterozygous animals (crossbreds out of Mule ewes and purebred Texels) when they inherit the mutation from the ram.
• Phenotypic effects on muscling were largely restricted to the loin area, with only minor effects on other parts of the carcass.
• TM-QTL appeared to show polar overdominance for muscling traits, implying that only heterozygous animals inheriting the mutated allele from the ram showed increased muscling.
• Homozygous lambs had increased body weights (from birth to slaughter by ~7 to 8%) and higher carcass weights (by 9% or 1.4kg). Unlike the muscling effects, body growth seemed to show an additive mode of inheritance.
• There was a small but significant increase in shear force (a measure of MQ) at meat ageing < 7 days, although meat quality was still within acceptable limits for consumer preference. Any effects of TM-QTL on meat quality disappeared after 7 days ageing time.
• TM-QTL carrier lambs had slightly higher incidence of birth difficulties in purebred Texels, with no effect in crossbred lambs. Additionally, if dams that carry TM-QTL need lambing assistance, it is more likely to need a higher grade of assistance. However, if TM-QTL was of interest to industry any selection for TM-QTL could be done in conjunction with selection for lambing ease using the lambing ease EBV now available for Texels. The project showed also that genetic variance is sufficient to select successfully for lambing ease.

b) LM-QTL (LoinMax): This QTL also found on chromosome 18 was found to segregate in Australian Poll Dorset sheep (Banks, 1997). It had been reported to increase loin muscle area and weight by 11% and 8%, respectively from studies conducted in New Zealand but had not been evaluated in crossbred lambs out of UK Mule ewes.

• LM-QTL has similar effects on loin muscling to TM-QTL. It increased loin muscling by 8-14% in carrier lambs inheriting QTL from the sire in UK crossbred lambs sired by NZ Poll Dorset LoinMAX carrier rams.
• Phenotypic effects were restricted to the loin area, with little evidence for effects on live or carcass weights
• There were no negative effects of LM-QTL on lamb or ewe health or welfare for Poll Dorset sired crossbred lambs out of Mule ewes. It remains unknown whether TM-QTL and LM-QTL are allelic or not and whether they might have additive effects on loin muscling.
• Heterozygous carriers have higher lean tissue (by ~3%) and muscle areas (2 to 5%) and total fat weight is decreased (by 6-16%). Increased CT-measured muscle densities in heterozygous lambs have been observed which is indicative of lower intramuscular fat content.
• Carrying two copies of MM was associated with a significant positive effect on 8 week weight, a negative effect on UFD and a substantial decrease in MLC fat class. Two copies of MM-QTL also had a strong positive impact on VIAestimated weight of the hind leg, chump and loin primal cuts, as well as the muscularity of the hind leg and loin regions, and loin muscle width, depth and area.

c) MM-QTL (MyoMax): During the timeframe of this project Clop et al. (2006) identified the causal mutation of this QTL as*1232G > A. The mutation is located on chromosome 2 in the region of the myostatin gene. Polymorphisms in this gene in several other mammalian species result in “double-muscling. However, from the results of studies carried out in New Zealand, the effects of this ovine mutation seem small to moderate. Phenotypic effects of this QTL were investigated in crossbred lambs out of Mule and Welsh Mountain ewes.

• In contrast to TM-QTL and LM-QTL, the phenotypic muscling effects of MMQTL are not confined to the loin area. In general, lean tissue growth is increased and fat growth is reduced. This mutation seems to act mostly in a partial recessive way, indicating that heterozygous animals are closer to the homozygous wildtype animals and larger effects occur only in homozygous carrier animals.
• Heterozygous carriers have higher lean tissue (by ~3%) and muscle areas (2 to 5%) and total fat weight is decreased (by 6-16%). Increased CT-measured muscle densities in heterozygous lambs have been observed which is indicative of lower intramuscular fat content.
• Carrying two copies of MM was associated with a significant positive effect on 8 week weight, a negative effect on UFD and a substantial decrease in MLC fat class. Two copies of MM-QTL also had a strong positive impact on VIAestimated weight of the hind leg, chump and loin primal cuts, as well as the muscularity of the hind leg and loin regions, and loin muscle width, depth and area.

(ii) Evaluation of VIA and its use to measure the effects of the muscling genes/QTL

a) Evaluation of VIA

• Previous work closely related to this project had shown that VIA, under abattoir conditions and at line speed (800 lambs/h), improved the prediction of primal meat yields compared to the current MLC EUROP carcass classification.
• VIA has been calibrated in this study against dissection. It was shown that VIA accurately and precisely reflected the value of sheep carcasses: the R² values for predicting leg, chump, loin, breast and shoulder primal joints, and total of the primals, were 0.97, 0.94, 0.91, 0.88, 0.96 and 0.99, respectively.
• Dataset of >7000 animals with pedigree information and 630 with repeated VIA measures from this project were used to estimate genetic parameters (heritabilities values and genetic correlations) for VIA-based predictions of primal weights. The repeatability for VIA traits was high (> 0.9) and h² values were low to moderate (0.08 to 0.26). High repeatability estimates of VIA traits and moderate heritabilities of the most valuable carcass joints suggest that including VIA information in breeding programs would be useful in order to improve carcass quality.
• Using the same dataset it was shown that the h² values for subjective characters (fat class, conformation) were low (h² = 0.1). This is likely to be a reflection of the subjective nature of this assessment. VIA based dimensional carcass measurements showed moderate to high h² values (0.20-0.53), suggesting that using this VIA dimensional information in the evaluation of purebred terminal sire breeds is likely to improve conformation on crossbred lambs.

b) Capability of VIA to measure phenotypic effects of the muscling QTLs/genes

• TM-QTL: Neither the current industry carcass evaluation system for conformation and fatness nor the standard VIA system was able to identify the effect of TM-QTL in the loin region.
• Calibrating the VIA system against CT measurements resulted in improved VIA prediction equations for primal meat yields.
• The improved‘ VIA system showed moderate potential to estimate loin muscle traits measured by CT and to detect, partially, the effect of the TMQTL on these traits. VIA measured carcass compactness‘ was found to be significantly increased in carrier lambs.
• LM-QTL: As the phenotypic effects of this QTL were very similar in magnitude to those of the TM-QTL, it was not surprising to find that VIA did not detect significant differences in loin muscling between wildtype and heterozygous carrier lambs. The improved VIA was able to detect a significant increase in loin weight of heterozygous carrier animals compared with non-carriers.
• MM-QTL: As MM shows predominately a partial recessive action, the small phenotypic changes in muscling between heterozygous and wild lambs were too small to be reliably detected by VIA. However, the larger effects (primal weights and muscularity measures) seen in homozygous carrier lambs were detected by VIA.

All milestones were met in full, albeit with a short, no-cost extension because of maternity leave of key scientists. An additional tasks: bolt on project‘ on the effect of different ageing times on MQ was also undertaken.

Additional output arising from project

• Establishment of a database for the SAC/IBERS purebred Texel flock: There is pedigree information from well phenotyped purebred Texels in the TM-QTL database (incl. 2010 born SAC lambs):

Number of animals in pedigree: 3294 of which 2692 (2325) animals were born between 2005 (2006) and 2010. The number of sires in the pedigree is 156, with 49 having more than 9 offspring.

Additionally there is ultrasound scan information (e.g. loin muscle depth) for 2002 (1767) animals born between 2005 (2006) and 2010. Many of the lambs will have also CT and meat quality MQ traits measured.

• Establishment of a tissue bank: Altogether there are 4470 blood or tissue samples from the TM-QTL project in the SAC freezer; of those there are 2824 purebred Texel samples representing ~1760 animals. This could be used for SNP or other genotyping for association and/or validation studies and for further fine mapping.
•  Use of data in PhD theses: Data from the project has also been used for the following PhD theses, one of which(i) has been defended already) and is included in the list of publications (page 7):

(i) Rius-Vilarrasa, E. (2009). Evaluation of a Video Image Analysis system for the prediction of carcass and meat quality in genetic improvement programmes. PhD Thesis, IEB, University of Edinburgh and Scottish Agricultural College

(ii) Masri, A.Y. (2011). Use of muscling QTL in sheep breeding. PhD Thesis, IEB, University of Edinburgh and Scottish Agricultural College

(iii) Matheson, S.M. (2011). Genetic selection for health and welfare traits in lambs. PhD Thesis, IEB, University of Edinburgh and Scottish Agricultural College

(iv) Craigie, C.R. (2012). Investigation into adding value to the meat production system, through prediction of carcass and meat eating quality using no destructive technologies such as Near-Infrared (NIR) spectroscopy and Video

Image Analysis (VIA). PhD thesis, Massey University, Palmerston North and Scottish Agricultural College

(v) Donaldson, C.L. (2013). The genetic basis of spine characteristics and relationships to meat yield in different sheep breeds and crosses. PhD Thesis, IEB, University of Edinburgh

Relevance to industry for future commercial exploitation

In brief, the direct effects of TM-QTL on loin muscling (4-11% increase in highly priced part of the carcass) and growth make it an interesting candidate for exploitation within the UK sheep industry, especially since it does not have any major negative impacts on eating quality. The muscling benefits were limited to the loin part of the carcass.

However, the paternal polar overdominance pattern of inheritance will require a suitable and affordable DNA test to be developed to identify carrier animals to achieve this. This will necessitate further research to fine map and identify the specific mutation involved so that a commercial SNP test can be developed. In contrast the muscling effects, the growth effects of TM-QTL show a partial recessive mode of inheritance, with two copies of the QTL required for maximum benefit. Such differences have implications for exploitation within the stratified industry structure. Benefitting fully from the growth/carcass weight benefits, but losing the muscling benefits, will require the QTL to be introgressed into the dam line (Mule ewe) as well fixed within terminal sires; however, exploiting the loin muscling effects will require it to be fixed in an homozygous state just within terminal sire breeds to derive maximum commercial benefit. This again requires developing a suitable and affordable DNA test to identify carrier animals.

The magnitude of any negative side effects of TM-QTL on welfare traits and MQ does not preclude its use, but suggests the need for vigilance and continued attention.

Negative effects on birth difficulties in pure bred Texels can be counterbalanced by selection for lambing ease for which sufficient additive genetic variance has been found in this project. Ageing carcasses for >7 days overcomes any possible deleterious effects on meat tenderness.

LM-QTL showed very similar phenotypic effects to TM-QTL, with significant increases in loin muscle weight only. However before, its use within the UK can be recommended, its mode of inheritance and potential impacts on meat eating quality require further evaluation. While it has been mapped to a similar region of chromosome 18 as TMQTL, it is not yet known whether it is allelic to TM-QTL or is a mutation in a different gene. If the latter, it is possible that the effects of these two QTL might be additive. It would be highly desirable to have this information before recommending exploitation.

This project has shown the clear advantages of MM-QTL on carcass muscling is sheep. Unlike TM-QTL, its pattern of inheritance appears to be partially recessive, meaning that the effect of two copies of the mutation give greater increases in muscling compared with one copy. Furthermore, the muscling benefits are not limited to the loin but are distributed across different parts of the carcass. Since the initiation of this project it has become clear from commercial genotype testing that the mutation is already present at very high frequency in UK-Texels (>95% animals are homozygous already). However, the frequency in other Terminal sire breeds appears to be much lower: it has yet to be identified in the Suffolk breed and only a small proportion of Charollais sheep are carriers. Introgression of MM-QTL should result in increased muscling in those breeds. Since a high proportion of lambs produced by the lowland sector in the UK are sired by Texel rams with no known problems of meat eating quality, it seems highly unlikely that this specific mutation within the myostatin gene will have negative impacts on eating quality.

Deriving maximum benefit from the exploitation of MM-QTL within the UK stratified sheep industry structure will require that slaughter lambs are homozygous for the QTL. This could only be achieved by introgression of this QTL into the hill and longwool crossing breeds that are the parental breeds of the lowland crossbred (Mule) ewe.

The refined VIA system developed within this project by calibrating VIA with CT and carcass dissection data now provides objective, accurate and precise predictions of carcass primal weights. It works under industry conditions and at slaughter line speeds and could be the backbone for a VBMS that rewards farmers providing good quality carcasses. VIA has inherent electronic data capture capability which, if combined with electronic identification of individual animals, could allow numerous carcass characteristics to be derived, stored, and then matched with other performance and pedigree data to facilitate more robust genetic parameter estimates for exploitation within sheep breed improvement programs. In contrast to subjective carcass scoring of fat class and conformation, the more objective VIA-measured traits are moderate to highly heritable, promising substantial selection response when included in a breeding programme targeting improved carcass conformation.

Due to the low magnitude of some of the phenotypic effects of the evaluated QTL/genes in this project, VIA has only a limited capability to detect these effects, restricting its potential benefit for rewarding farmers for using certain muscling QTLs. However in a synergistic approach together with Ultra Sound and CT-scanning VIA could play an important role in the genetic improvement of carcass quality.

The Problem:

Various QTL for muscling have been found in a few sheep breeds. The big question is if these QTL are effective in commercial crossbred (e.g. Texel x mule crosses) and if they have any impact on meat eating quality. If Industry adopts Video image analysis for carcase assessments this may provide valuable information for breeding, but there first needs to be a good assessment of the value of these traits (e.g. heritabilities).

Project Aims:

Comprehensive evaluation of selected QTLs with potential to increase lean meat yield, including effects on meat quality (Texel Muscling, TM – QTL, Loin Max, LM -QTL and Myo Max ,MM – QTL) Test and calibrate a VIA-system to provide more data for the objective evaluation of lamb carcases using VIA machines.

Approach:

The project will start introgressing the TM QTL to other breeds and optimise use of VIA information in breeding programmes. The project will deliver estimates of muscling QTL effects in purebred  Texel, Texel x Suffolk and Texel x Charollais crosses and Texel x mule crosses. Genetic parameters for VIA traits. It will propose use of these QTLs and VIA in sheep breeding, with economic benefits at the commercial level.

Deliverables:

This project aims to evaluate the potential to exploit QTLs in sheep breeding programmes. It will provide essential information to breeders and producers on the use of QTL information in sheep breeding. In particular, it will evaluate 2 New Zealand QTLs (LM and MM) within an UK stratified sheep industry context. It will also permit CT and dissection based calibration of a video image analysis system.