a zigzag from my tide I'll be discussing a transition a muscle to meet and briefly reviewing the energy metabolism and oxidative processes underlying this transition you can see I have three co-authors on this paper and there's a couple points I want to make with this none of us really that or at all success will operate in a vacuum when we talk about muscle myopathies about beginning to later my talk you really need to acknowledge some of the groups and really make contributions to this the Arkansas group with KC and and include and CUDA pond and their contributions operating to let together and collectively my co-authors here just mcquewick is just a fantastic muscle biologist he and I have Co co-chaired co-advised about ten graduates together Lewis pariahs is that ever important statistician and so I would recommend that if you have questions over our model development later on don't ask me I'll give the ESA's email and then Jackie Griffin is that ever-important worker B as a graduate student so you know you can have ideas and things but you really need that person that really puts the attraction to things I want to start my talk with an overview of the wing and flight muscles as can be seen you not burn you know the underlying super core Aquarius or the pectoral miner is responsible for raising the wing during the process of flight the overlying a much larger pectoralis major is the muscle that is needed for pulling the wing down during flight and so I like this picture because it shows the ever increasing thickness in the pectoralis major and again a wild bird during the course of flight and one of the things that's important is you can actually see in this picture the transition and the increase in thickness as the pectoralis major approaches the sternum I worked for a major Braille or breeding company for five years before going to Ohio State and I spent quite a bit of time the selection team and back in the early days where our yield was just becoming important we used to actually be able to select for confirmation in the breast muscle in those early days back in the mid 80s when we were trying to develop yield strain birds and improve the conformation so really we have were very effective improving yield but largely while that was done in the early days by palpation because you only had a thin layer of skin over the development pectoralis muscle in my next slide here I just want to go through a little bit of the fiber typing that's inherent to our breast muscles basically the pectoralis major is what we call white muscle it's predominantly type 2b fibers very very glycolytic very anaerobic which is very important and post-harvest metabolism as opposed to the red muscles and the drum and the thigh very high in myoglobin very high in my Oconto mitochondrial content and and are very aerobic okay these are the muscles that really have to work all the time versus the flight muscles and birds which are only needed periodically those of you that know me realize that the literature is there for a reason and so we really want to go back through and understand the research and what's kind of contributed to where we are in the state of muscle biology today we need to go back to some of the early literature George and nake 1958 these are the first folks that really quantify the different contribution of fibers in the the muscle of pigeons south or in hope in 1969 these are really the first folks that truly identified the mitochondria as having the complete enzyme picture complete glycolytic potential in breast muscle up until this time there was contention that when you isolated mitochondria you could actually look at glycolysis occurring but there was some contention because people were assuming that during the process of isolating the mitochondria some of the glycolytic enzymes were coming other tissues that were contaminating the mitochondrial preps so really 69 if when we first confirmed for the first time that we're actually getting a lot of our glycolysis occurring in mitochondria and breast muscle Rosser and George this is probably the most heavily cited paper where basically they characterize fiber typing over multiple species is an excellent review if you're really entering the history of muscle fiber typing and converts and finally Riveter dr. Bhatti referred to him earlier 2017 paper dr. Botkin was one of the at all's on this paper and reverter was really the one that went in and confirmed the fact that you had mitochondria more less mitochondria pectoralis major than you had in other tissues but they also showed the fact that breast muscle mitochondria content was negatively correlated with breast muscle yield so what they saw or excuse me yeah negative correlated with breast muscle yield solo breast muscle mitochondrial content was associated with more muscular burbs so again we're increasing the pectoralis major size during the course of cycling for yield or actually decreasing mitochondrial content which will leave you know I hypothesis later in terms of some of these relationships contributing to the development of some of our anomalies I don't often actually have a written script for talks but I want to make sure I stayed on time and I covered the the pertinent points in this so please excuse me as I go through this the next slide is that is a very common slide showing the the process of pH drop post-harvest we go from the top of about 63 to 64 we see a very very rapid drop in pH until we eventually get out most of the rapid drop is occurring here most of the rapid drop is occurring here in the first hours until finally about 24 hours later we have our ultimate pH and you can see this like gray line here represents a slight delay in the pH decline in carcasses that are actually refrigerated immediately post harvest so this basically shows that if you do have you somewhat higher temperature that will accelerate the glycolytic process what I really want to point out here however everywhere but the slide what I want to appear however is look at the variability along each of your points in pH decline and this variability was actually used to develop two lines a high pH line and a low pH line so you can see that in the high pH line and I believe this was after six generations of selection you had no differences in body weight you had no differences in breast yield no differences in abdominal fat but what you do see are major differences in glycolytic potential ultimate ph and all of these indices of breast muscle quality so I think it just emphasizes there's so much variability in this case in pH decline you can actually have a strong genetic component and what you're essentially doing here is you're creating one set of birds which have a PSC type condition or another set of birds which have a DF D but it just emphasizes that the role of mitochondria the role of post-harvest pH decline is very very important in product quality but I would also take from this that this also has a reference to pre-harvest treated treatment of birds for instance if you can accelerate glycolysis of glycogen prior to prior to harvest through heat treatment or things of this sort it's really important that it is just a process of processing in which we got our pH declines the glycogen will also be metabolism will also be metabolized before harvest so what a transition now to a picture's worth a thousand words okay and I'm sure that you've seen many many pictures over time of the genetic increases Jerry Havenstein has a sure that people often cite this came from Chang and I want to make this the transition here now you can easily see the genetic increases we've had in carcass yield with time and you can see here again the somewhat disproportional increase in the pectoralis major what I have at the bottom of this slide is one of the current hypotheses this came from a paper by Petrucci and by Rousseau 2015 that the increase in incidence of breast muscle biopsies is a result of selection for body weight and extremes in phenotype in terms of breast muscle phenotype this was a paper by Bayley in 2015 Bailey who is an a vegan geneticist it's a very busy slide began what they did was they had two lines a line a and a line be big differences in body weight in parentheses here are some differences in age what I want to point out here is for each of these traits body weight carcass weight breast yield deep pectoral myopathy percentage green muscle disease which I'll be describing a little bit later woody breast and white striping that the fast growing line with the great increase in breast yield had an increased incidence in these my aunt myopathies and again the slow growing line the levels are much lower what these values represent in parentheses are a number of observations so certainly the authors certainly had a lot of observations from which they came up with their conclusions one of the conclusions is paper was at the genetic correlations between breast muscle myopathies body weight and breast yield were very very low so they concluded that future selection for body weight breast yield would not necessarily increase the risk for the expression of breast muscle myopathies one things I want to point out however is that breast yield is a ratio it's a weight of the weight of the muscle over your body weight or carcass Li and one of the things that's been missing in some of our papers is the fact that we don't feel necessarily it's the weight of the muscle what I want to spend the rest of my presentation on but it's the components and it's the confirmation in that muscle that contributes to weight that is really the critical potential predictor of some of these myopathies so again we developed a hypothesis that a lot of the onset of the pectoralis major breast muscle myopathies is a result of what we call compartment syndrome now compartment syndrome was first identified by Siller this is a Siller paper 1985 which was actually a paper that came from a poultry science symposium in 85 Silla referred back to work with Martindale and others back in the late 70s and you can see here in the quote supercorps Aquarius muscle lies and rigid compartment so right here is the pectoralis minor it lies in a rigid compartment on the one side you have the sternum on the other side you have the pectoralis overriding major and what they basically said that at this time in the late 70s deep pectoral myopathy was most commonly observed in turkeys Martindale actually went in into some very very neat work in terms of actually surgically ablating some of the blood supply to the muscle and was able to very clearly show the ontogeny of a deep pectoral myopathy like condition so what Siller it all came up with was the reason for this was this muscle couldn't go anywhere after exertion it increased 20% in time 20% in size but there was nowhere for it to go so you had a pulling of blood it could not be drained out eventually had a nice jami'a followed by a necrosis so this really has been the model that Siller proposed has been the base of what we have been working on work on breast muscle myopathies now to get into compartment syndrome a little bit we need to understand that within a developing muscle we have several layers of what I call inelastic fashion at the level of the independent fibre okay we have a button demise iam epimysium surrounds the entire muscle the Paramecium basically is surrounding muscle fiber bundles all of these are contiguous collagen layers and the reason I have this slide here is it basically emphasizes that each of these different levels of fashio connective tissue surrounding the fiber the muscle fiber bundles and the entire muscle a really very inelastic can be very stiff so we know we've had tremendous increases in selection for muscle size there's been a tremendous amount of work looking at satellite cells their contribution to muscle size muscle regeneration but what all I remind you is that a lot of the satellite cell work has been done in vitro in Satellite cells in the muscle itself do not operate in a vacuum and I was really excited yesterday when I was listening to Jessica Starkey from Auburn talked about some of her work and her attempts really to kind of look at proliferation of satellite cells in vivo because I really think that's where we need to look at this process of muscle growth and muscle regeneration because our hypothesis is that it's the regeneration process after an original necrosis that's leading to some of our myopathies so Mary Decker unless you're an ardent track-and-field fan or you're old enough to be on the AARP listserv you may not have heard where Mary Decker she was the queen of track and field back in the 70s Mary Decker had compartment syndrome and when Jackie Griffin was talking about compartment syndrome and some of our lab meetings it you know it struck a bell somewhere in the back of mine and I realized that back from my day as a runner the Mary Decker had compartment syndrome it almost derailed her career her calf muscles were so big that they were pressing up against the sheath to the point where she could not run so he did in 1977 she had a somewhat rare surgery where they actually went in and they slit the sheath on her cats it allowed the muscle to expand the sheath repaired itself and she went on to have a fabulous career in the early 80s in track and field so again it's just another example of where this compartment syndrome this inelastic fashio that doesn't allow a muscle to expand in this case causes an awful lot of pain but chickens with breast muscle myopathies don't have the option of getting their sheaths slit so we need to think in terms of how to develop the birds so they can actually have muscle expansion basically coordinated with the muscle sheath now this paper right here is from a review by man 2011 and it really fits very very well into the hypothesis we've generated now this is in mice and we need to realize that a lot of really good muscle growth and muscle repair work is not done in poultry species but but basically what man summarized was that is a dystrophic muscle and what he show is here is what he calls a chronic injury so you have a chronic and dystrophic muscle a chronic sublevel of inflammation as this inflammation persists you start then getting some cells coming in some leukocytes and ultimately you get accumulation in collagen deposition secondary to this chronic inflammation so it's not the same thing as in the above panel where it's an acute injury and again this somewhat supports if you look at my summary down here the satellite cell population is either exhausted over time or loses the capacity for repair and the muscle tissue progressively is replaced by atom some fibrotic tissue and isn't this very similar to what we're seeing at least visually and what's happening in our breast muscle myopathies we do have this inflammation over time we do have some extremists and the Krause is occurring followed by an appearance of lipid and also fibrosis or collagen during the repair process so again I think that some of the models that we want to look at we would be well-served to go into the human or the the other species where they have using other models to try to explain where it's seeing in chickens so again this is a slide from Jackie Griffin's original proposal for her PhD based on compartment syndrome and basically what it shows is the fact you have an increase in inter muscular pressure this increase in her muscular pressure basically from the ne from the the inflexible epimysium a Paramecium goes in one of two directions you can either have it going down here to restricted mudflow blood flow and is jami'a or decrease in oxygen transport to the tissue and a localized hypoxia from this original insult again you get increase in hypoxia you get down you actually have an increase in calcium infiltration altered calcium homeostasis in the muscle oxidative stress ultimately an immune response followed by altered altered setting or repair and the fibrosis condition so again putting together here our hypothesis so we have a compartment like syndrome occurring in this muscle leading to our myopathies so what Jackie did she had a large experiment we put Birds in 27 in nine pens and from two days through 46 days we sampled Birds every other day she noticed her first incidence of white striping at about 16 so from 16 days through 46 days she took pictures of each muscle and these are actually just images that she developed presentation purposes but we had a rank one which was a mild case of white striping superficial hemorrhages much deeper hemorrhages and then roses white striping and and necrotic tissue so from 16 days to 46 days each muscle tissue sample 27 each day she gave one of these ranks and you can see they had quite a large n number from 77 and our rank 4 down to 86 in our rank 1 we had to terminate the experiment at 46 days because our birds had such a high incidence of white striping it really became a welfare concern for us within each of these with each of these these muscles she measured these 10 parameters parameters of pectoralis major depth yield weight body weight pectoralis minor weight with length and within each of these then you can see that as you went from a rank one to a rank four there were significant differences that you went up in rank in each of these parameters we ran all possible two-way correlations and any correlations that were greater than 65 we did not use those values and our ultimate model generation just so we wanted to have independent we wanted to establish the independence of the individual traits without compounding effects of two-way correlations what I want to point out here is you ran all possible independent variables created all possible models and these are the top ten models that we were generated the thing I want to point out here is a number it's a not important but the weight of the pectoralis major was only important in one of the ten predicted models body weight was not significant factor in any of the 10 best models so certainly this partially supports the Bayley hypothesis sets sucking for bodyweight continued suck before bodyweight probably is not going to have a strong effect predictive effect or correlated effect on breast muscle myopathies and the weight of the major is probably not a major factor as well but what we did find in these in these models were that pectoralis major depth pectoralis minor with and pest pectoralis major yield where our three largest predictors of increasing in ranked score so what I want to point out here is this is our ranked one as we increase muscle depth okay it gave us a giver a given predictor for the predisposition for breast muscle myopathies so again it gets back to the point that it is the structure the topography so to speak of this muscle that's having the greatest effect on the predisposition for myopathy and there really this is support in the whitter provides some of the folks that have actually been looking at woody breast in the sense the fact that the thickest part of the muscle as I pointed out up near the sternum okay in that plight muscle the thickest part of the muscle is usually where we see the the initial ontogeny and progression of the defect but the other thing she found was that and again I want to point out initially here that these bars the color in these bars are backwards okay so this should be our rank one here and that should be our rank four because what this is saying is that the increase in values of each of these factors increases the odds of following any lower rank so what I'm saying it here is we're going from a pectoralis major length of almost fourteen up to six up to seventeen the longer the muscle the greater the odds that you will have a rank of one or a less severe rank whereas up here pectoralis major depth the deeper or thicker the muscle and remember all of these observations are between 16 and 46 days okay so this really covers a wide range of muscles in this it's not simply a fixed time when you have ten Donal Birds and ten myopic Birds my Pathak Birds so again pectoralis major with pectoralis major length the wider and the longer the muscle the greater the chance you will not have one of these myopathies you'll be in more of the lower ranks so we think again with technology out there we need to develop a way of actually assessing muscle depth cranial muscle depth and we think this would be one of the fact that we could use as a predictor so one of my last couple asides what I want to point out is that we went to some we use an ANA string technology which is basically allows us to jockey isolate the RNA from all of these different muscles and all the ages and I just want to point out that here we have hypoxia-inducible factor one alpha here we have carbonic anhydrase and this is decorated now if you read the papers by muta ring and the group from Delaware and a couple of other papers in the literature they've shown pretty conclusively that genes associated with oxidative stress are much higher in Maya Pathak Birds versus normal Birds what we're finding here is that again this covers all the ages we had so this is actually 2 through 46 days for each of these 3 each of these three proteins and I remember that our ranks were only generated from 16 to 46 days so what I want to point out here is that I don't know why we're seeing the significant decline hypoxia this will factor 1-alpha until about 11 to 14 days but beginning at this point you start seeing a linear increase in hif-1 alpha again an indices of oxidative stress beginning carbonic anhydrase has been pointed out by a number of authors mew turin being one as actually h IH if' one alpha actually induces carbonic anhydrase this is muscle specific carbonic anhydrase so again everything I'm showing here is consistent with the literature in a sense of up regulated cytokine tore up regulated proteins associated with oxidative stress the thing that we're showing however is that some of this up regulation of these genes associated with oxidative stress are occurring well before we're starting to see some of our first myopathies occurring so we think this is getting back to that Mouse that mouse data in that mouse picture I showed you we think this supports the concept of a low-grade inflammation and a low greatest Rima and optative stress in these muscles as the bird gets older and gets heavier get progressively you get into a more mild Pathak condition but it's beginning at a young age as suggested here by protein up regulation and again here Dekker in and against Andy bellman has shown that in woody breast tissue versus normal tissue you have an increase in Decorah --tx pression decorat is a heparin proteoglycan very very important in collagen cross-linking okay it works with fgf2 at increase in collagen cross-linking and so what we think is happening here and you can see this is our number four ranked we think what's happening here is again supporting the concept of low-grade inflammation ongoing at younger ages that increasing that dec Renee is responding to the hypoxia we're having a necrosis we're having a repair process here in which case a lot of our woody breasts were our collagen is actually the result of muscle repair muscle fibrosis increasing so really our transcription data here falls in line with what's in the literature what we're showing here I think the significance of our data is that we're starting to see an insult occurring within the tissue itself before we're actually seeing clinical or phenotypic science of muscle myopathy so basically this is just a more complicated picture of Jackie's hypothesis pointing out the processes that are occurring in normal muscle we have a stream you know we have oxidative stress and then we have essentially disaggregation of muscle that's appearing in many of the histologic types of things associated with these muscle myopathies and that fibrosis really is part of the repair process that is then leading to the woody part of the woody breast and we really need to look at these earlier stages before the phenotypic signs are occurring to really understand and to possibly address what is driving our muscle down this road so with that we've made a lot of progress I think the real question now is how do we solve this we need to be able to predict it first we I do think significantly that there is a genetic relationship here we need to incorporate into selection program it's easy for me to say because I'm a nutritionist but we need to have some handle on the the phenotypic depth width of the muscle and not just the weight of the muscle we need to take into consideration how it's developing where the stresses are occurring as we move forward because we certainly have a market out there that's not going to allow us to take a lot of muscle off breast yield breast muscle are still very very important but I think we could have rearranged the form of this muscle and possibly help ameliorate some of these myopathies thank you very much [Music]