Matthew J Forrest
email: mjforrest@ucsd.edu

Martin A Schlaepfer

Nothing a Hot Bath Won't Cure: Infection Rates of Amphibian Chytrid Fungus Correlate Negatively with Water Temperature under Natural Field Settings

Dramatic declines and extinctions of amphibian populations throughout the world have been associated with chytridiomycosis, an infectious disease caused by the pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd). Previous studies indicated that Bd prevalence correlates with cooler temperatures in the field, and laboratory experiments have demonstrated that Bd ceases growth at temperatures above 28uC. Here we investigate how small-scale variations in water temperature correlate with Bd prevalence in the wild. We sampled 221 amphibians, including 201 lowland leopard frogs (Rana [Lithobates] yavapaiensis), from 12 sites in Arizona, USA, and tested them for Bd. Amphibians were encountered in microhabitats that exhibited a wide range of water temperatures (10-50uC), including several geothermal water sources. There was a strong inverse correlation between the water temperature in which lowland leopard frogs were captured and Bd prevalence, even after taking into account the influence of year, season, and host size. In locations where Bd was known to be present, the prevalence of Bd infections dropped from 75-100% in water ,15uC, to less than 10% in water .30uC. A strong inverse correlation between Bd infection status and water temperature was also observed within sites. Our findings suggest that microhabitats where water temperatures exceed 30uC provide lowland leopard frogs with significant protection from Bd, which could have important implications for disease dynamics, as well as management applications. There must be quite a few things a hot bath won't cure, but I don't know many of them -Sylvia Plath, ''The Bell Jar'' (1963).

Introduction

Chytridiomycosis, an infectious disease caused by the pathogenic chytrid fungus Batrachochytrium dendrobatidis (Bd), is a primary factor in worldwide amphibian declines and species extinctions [START_REF] Stuart | Status and trends of amphibian declines and extinctions worldwide[END_REF], [START_REF] Kilpatrick | The ecology and impact of chytridiomycosis: an emerging disease of amphibians[END_REF]. Bd belongs to a group of virulent multi-host pathogens that have had profound effects on entire communities and ecosystems [START_REF] Fisher | Global emergence of Batrachochytrium dendrobatidis and amphibian chytridiomycosis in space, time, and host[END_REF]. In fact, Bd has been called ''possibly the most deadly invasive species on the planet (excluding humans)'' [START_REF] Rohr | Evaluating the links between climate, disease spread, and amphibian declines[END_REF]. Although amphibian susceptibility to Bd and chytridiomycosis is species-specific, environmental conditions also appear to modify host-disease dynamics [START_REF] Kilpatrick | The ecology and impact of chytridiomycosis: an emerging disease of amphibians[END_REF]. The prevalence of Bd (i.e. the proportion of infected animals) and the virulence of chytridiomycosis are particularly influenced by temperature [START_REF] Woodhams | Life-history trade-offs influence disease in changing climates: strategies of an amphibian pathogen[END_REF]. Field studies conducted in disparate geographic regions show Bd infections are generally more severe in winter months, and when hosts are found in cooler temperatures [START_REF] Bradley | Chytridiomycosis in native Arizona frogs[END_REF]- [START_REF] Voordouw | Prevalence of the pathogenic chytrid fungus, Batrachochytrium dendrobatidis, in an endangered population of northern leopard frogs, Rana pipiens[END_REF].

In the laboratory Bd cultures grew and reproduced at temperatures between 4-25uC, with maximal growth at 17-25uC, but growth ceased at temperatures above 28uC [START_REF] Piotrowski | Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians[END_REF]. Incubation of Bd cultures at 30uC for 8 days killed 50% of colonies [START_REF] Piotrowski | Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians[END_REF], and 100% mortality occurred within 96 hours at 32uC and within 4 hours at 37uC [START_REF] Johnson | Survival of Batrachochytrium dendrobatidis in water: quarantine and disease control implications[END_REF]. Similarly, Bd does not persist in amphibian hosts above certain temperature thresholds. In laboratory experiments, short-term exposure to temperatures between 27 and 37uC successfully cleared Bd infections from five species of adult frogs with no reported side effects [START_REF] Berger | Effect of season and temperature on mortality in amphibians due to chytridiomycosis[END_REF], [START_REF] Woodhams | Emerging disease of amphibians cured by elevated body temperature[END_REF]- [START_REF] Chatfield | Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs[END_REF]. Although Bd is susceptible to certain antifungal agents when tested in vitro, there are few proven methods for clearing infections in adult amphibians, and acute drug toxicity can be a problem for tadpoles and juveniles [START_REF] Pessier | Management of disease as a threat to amphibian conservation[END_REF]- [START_REF] Berger | Treatment of chytridiomycosis requires urgent clinical trials[END_REF]. Therefore, heat treatments may be a superior alternative to currently available antifungal drugs for captive animals infected with Bd [START_REF] Chatfield | Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs[END_REF], [START_REF] Berger | Treatment of chytridiomycosis requires urgent clinical trials[END_REF]. However, whether amphibians in the wild can also be cleared of Bd by short-term exposure to elevated temperatures remains unknown.

Several species of leopard frogs naturally inhabit geothermal ecosystems in the southwestern United States, including endangered and threatened species. For example, all naturally occurring populations of the relict leopard frog (Rana [Lithobates] onca) are now associated with perennial geothermal springs in Nevada with source temperatures exceeding 30uC [START_REF] Bradford | Population status and distribution of a decimated amphibian, the relict leopard frog (Rana onca)[END_REF], and geothermal spring sites in New Mexico are particularly important breeding habitats for the threatened Chiricahua leopard frog (Rana [Lithobates] chiricahuen-sis) [START_REF] Scott | The tadpoles of five species of New Mexican leopard frogs[END_REF]. Observations from a previous study indicated that Bd was less prevalent in frogs inhabiting geothermal waters in Arizona, USA [START_REF] Schlaepfer | High prevalence of Batrachochytrium dendrobatidis in wild populations of lowland leopard frogs Rana yavapaiensis in Arizona[END_REF]. Consequently, we tested whether Bd is negatively associated with water temperature by sampling amphibian populations from several geothermal ecosystems, as well as non-geothermal sites.

All seven native Arizona ranid species have experienced significant population declines and local extinctions [START_REF] Clarkson | Status of Leopard Frogs (Rana pipiens Complex: Ranidae) in Arizona and Southeastern California[END_REF], and chytridiomycosis appears to be an important contributory factorparticularly during the winter months [START_REF] Bradley | Chytridiomycosis in native Arizona frogs[END_REF], [START_REF] Sredl | Understanding and mitigating effects of chytrid fungus to amphibian populations in Arizona[END_REF]. Lowland leopard frogs (Rana [Lithobates] yavapaiensis) have been extirpated from nearly half of their historic geographic range [START_REF] Clarkson | Status of Leopard Frogs (Rana pipiens Complex: Ranidae) in Arizona and Southeastern California[END_REF], and populations continue to decline or disappear from additional sites [START_REF] Sredl | Rana yavapaiensis (Platz and Frost[END_REF].

Identifying environments and climactic conditions that provide natural refuges from Bd will benefit imperiled amphibian populations [START_REF] Puschendorf | Distribution models for the amphibian chytrid Batrachochytrium dendrobatidis in Costa Rica: proposing climatic refuges as a conservation tool[END_REF], [START_REF] Walker | Factors driving pathogenicity vs. prevalence of amphibian panzootic chytridiomycosis in Iberia[END_REF], and may provide some susceptible species with opportunities to evolve evolutionary responses to the pathogen [START_REF] Tobler | Within-and among-population variation in chytridiomycosis-induced mortality in the toad Alytes obstetricans[END_REF]. Information on the environmental limitations of Bd in the wild is critical to the conservation of amphibians affected by this disease [START_REF] Muths | Distribution and environmental limitations of an amphibian pathogen in the Rocky Mountains, USA[END_REF], yet a clear understanding of how temperature modulates host-disease dynamics in the field has remained elusive to date [START_REF] Savage | Disease dynamics vary spatially and temporally in a North American amphibian[END_REF], [START_REF] Knapp | Nowhere to hide: impact of a temperature-sensitive amphibian pathogen along an elevation gradient in the temperate zone[END_REF]. Geothermal settings provide unique opportunities to examine the effects of a wide range of environmental temperature on chytridiomycosis host-disease dynamics in wild amphibian populations. To our knowledge, this study is the first to utilize steep natural temperature gradients in field settings, and measure water temperature at the precise time and place where each amphibian was captured, thereby providing valuable information collected at a fine spatial scale to help elucidate the thermal restrictions of Bd under natural field conditions.

Results

We sampled 221 post-metamorphic anurans belonging to five species, of which the lowland leopard frog (n = 201) was the most common (Table 1). The primary result was a significant negative association between Bd infection status of lowland leopard frogs and water temperature. Overall, the temperature of the water in which we captured Bd-positive (Bd+) individuals (mean = 19.8 60.67uC, n = 50) was significantly cooler than for Bd-negative (Bd2) individuals (mean = 25.860.50uC, n = 151; 2-sample t-test: df = 108, t = 7.1, p,0.001; Fig. 1A). The negative association between water temperature and Bd infection status was apparent in the univariate test, but also after accounting for the influence of other significant factors such as Distance to Solstice (i.e., seasonality) and Year (Table 2).

To control for a possible absence of Bd from certain sites (Table 1) we repeated the binomial logistic regression analysis excluding samples from Markham Creek, Upper Hassyampa, El Dorado Hotspring and BHP Hotwell, where more than one individual was tested, and Bd had not been detected. The analysis on the restricted dataset (n = 148) yielded a qualitatively identical result for the binomial regression, with Bd prevalence declining sharply from 100% at 10-15uC until 30uC, where it approaches zero (Fig. 1B). Our subsequent analyses include data from all sites because our samples sizes at any particular site were too small to be confident that Bd was truly absent (Table 1).

Distance to Solstice and Year were also significantly associated with Bd prevalence (Table 2). The positive association between Bd prevalence and Distance to Solstice illustrates that individuals were more likely to be Bd+ during early spring and late fall sampling than during the summer, even after taking into account water temperatures (Table 2). Bd prevalence also varied between years, with the prevalence in 2009 being significantly lower than in 2004 and 2010 once the time of year was taken into account (Table 2). Because slightly different methods for assaying Bd were used in the 2004 study, we repeated all analyses using only the 2009 and 2010 data and found qualitatively similar results.

Size of host (SVL) was not significantly associated with Bd prevalence in the binomial regression (Table 2). Nevertheless, other results suggest that the relationship between Bd and host may change with size. We observed that seven of the eight Bd+ individuals that were captured in waters warmer than 25uC were juveniles (#50 mm SVL). Furthermore, the mean water temperature in which Bd+ juveniles were captured (21.661.31uC, n = 20) was marginally higher than that of Bd+ adults (18.760.64uC, n = 30; t-test: df = 28, t = 2.00, p = 0.056). Our results suggest that juveniles and adults may differ in their susceptibility to Bd or in their behavior once infected.

Bd prevalence clearly varies between sites (Table 1), and we wanted to ensure that the correlation between water temperature and Bd prevalence was not confounded by site characteristics. When Site was included in the binomial logistic model, Water Temperature remained a significant factor, but certain site-specific coefficients could not be estimated (likely because of small sample sizes). We therefore undertook an additional analysis in which we compared AICc values (Akaike information criterion corrected for finite sample size) of two logistic binomial models with Sex, SVL, Year and Site as factors, but where Water Temperature was present only in the first model. The much lower AICc value (139.1 vs. 158.3) associated with the first model, which included Water Temperature, strongly suggests a significant correlation between Bd prevalence and water temperature, even when accounting for variation in Bd between sites.

The negative relationship between Bd prevalence and water temperature was also apparent within the Secret Spring and Mammoth Hot Well sites, where sampling covered a wide range of water temperatures from the relatively constant, elevated temperatures at their sources to more variable, ambient temperatures in the distal portions of the ecosystems. Bd was detected at these sites, but more commonly in individuals located in cooler waters. At both sites, the prevalence of Bd was significantly lower in water temperatures of 30uC and above (Secret Spring: 19/30 Bd+ in water ,30uC, and 0/4 Bd+ in water $30uC; two-tailed Fisher's exact test; p = 0.0294; Mammoth Hot Well: 13/45 Bd+ in water ,30uC, and 1/26 Bd+ in water $30uC; two-tailed Fisher's exact test p = 0.0124). In 2009 we mapped the location of each individual captured at these two locations. Eight of the nine Bd+ individuals (89%) at Mammoth Hot Well were found in waters cooler than 27uC (Fig. 2), and all 15 Bd+ individuals (100%) at Secret Spring were found in waters cooler than 20uC (Fig. 3).

We also tested 20 individuals of four other species: 17 American bullfrogs (Rana [Lithobates] catesbeiana), 1 canyon tree frog (Hyla arenicolor), 1 Woodhouse's toad (Bufo [Anaxyrus] woodhousii woodhousii), and 1 Sonoran toad (Bufo [Incilius] alvarius) (Table 1). All were Bd2, with the exception of two American bullfrogs captured at 16uC.

Discussion

This study provides evidence that the probability that a lowland leopard frog is infected with Bd is significantly negatively correlated with water temperature at the point of capture (Fig. 1A and1B). Previous field studies documented a correlation between Bd prevalence and seasonality, with higher infection rates during cool seasons [6]- [START_REF] Voordouw | Prevalence of the pathogenic chytrid fungus, Batrachochytrium dendrobatidis, in an endangered population of northern leopard frogs, Rana pipiens[END_REF], [START_REF] Savage | Disease dynamics vary spatially and temporally in a North American amphibian[END_REF]. In this study, we documented that the inverse relationship between water temperature and Bd prevalence also holds true within seasons. Furthermore, the association between temperature and Bd prevalence was apparent even within bodies of water, with Bd+ frogs largely absent from water warmer than 25uC (Figs. 2 and3). This result appears surprising given the short distances involved, but the observed pattern suggests that these frogs may have relatively small home ranges, or that the detectability and status of the disease changes rapidly with environmental conditions. Although other factors such as water chemistry may potentially affect Bd infection rates, our results strongly suggest a functional link between water temperature and Bd infection status in frogs.

Previous laboratory work conducted on other species indicates that amphibian hosts are able to clear Bd if infected individuals experience temperatures between 27-37uC. Caging great barred frogs (Mixophyes fasciolatus) at 27uC (n = 8) cured 50% of the frogs, which remained healthy, and tested negative for Bd when the experiment was terminated [START_REF] Berger | Effect of season and temperature on mortality in amphibians due to chytridiomycosis[END_REF]. Orange-eyed treefrogs (Litoria chloris) cleared Bd within 16 hours after being caged at an environmental temperature of 37uC (n = 10) [START_REF] Woodhams | Emerging disease of amphibians cured by elevated body temperature[END_REF]. Western chorus frogs (Pseudacris triserata) collected in Arizona, cleared Bd following incubation at 32uC for 5 days (n = 6) [START_REF] Retallick | Strain differences in the amphibian chytrid Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection[END_REF]. American bullfrogs and northern cricket frogs (Acris crepitans) cleared Bd after being subjected to 30uC for 10 consecutive days, after which only one frog remained infected (n = 28) [START_REF] Chatfield | Elevated temperature as a treatment for Batrachochytrium dendrobatidis infection in captive frogs[END_REF]. Heat treatments can also clear larval amphibians of Bd; 7 out of 8 tadpoles of the midwife toad (Alytes obstetricans) cleared Bd infection when exposed to temperatures higher than 26uC for 5 days [START_REF] Geiger | Elevated temperature clears chytrid fungus infections from tadpoles of the midwife toad, Alytes obstetricans[END_REF]. Our results are consistent with these findings, despite having been conducted in uncontrolled environments. Indeed, the vast majority of R. yavapaiensis (51/52; 98%) captured in water warmer than 30uC were Bd2 (Figs. 1A,2, 3). Overall, our results are consistent with the hypothesis that warm waters exclude Bd from infecting R. yavapaiensis hosts, although we cannot rule out all hypothetical alternatives. In addition to the well-documented negative effects of warm waters on Bd, temperature is also strongly linked to amphibian immune system responses. Declines in amphibian immune defenses as temperatures decrease are well documented [START_REF] Green | Effect of temperature on serum complement levels in the leopard frog, Rana pipiens[END_REF]- [START_REF] Rollins-Smith | Activity of antimicrobial skin peptides from ranid frogs against Batrachochytrium dendrobatidis, the chytrid fungus associated with global amphibian declines[END_REF], which may explain why amphibians are particular susceptible to pathogens such as Bd that survive and grows at low temperatures [START_REF] Berger | Effect of season and temperature on mortality in amphibians due to chytridiomycosis[END_REF], [START_REF] Piotrowski | Physiology of Batrachochytrium dendrobatidis, a chytrid pathogen of amphibians[END_REF], [START_REF] Raffel | Negative effects of changing temperature on amphibian immunity under field conditions[END_REF]. Conversely, at warmer temperatures amphibians may be less susceptible to Bd and chytridiomycosis due to greater effectiveness of the immune response [START_REF] Andre | Effect of temperature on host response to Batrachochytrium dendrobatidis infection in the Mountain Yellow-legged Frog (Rana muscosa)[END_REF], [START_REF] Murphy | Temperature, hydric environment, and prior pathogen exposure alter the experimental severity of chytridiomycosis in boreal toads[END_REF]. Thermal impacts on Bd, and on the amphibian immune system have important ramifications for the ecology of chytridiomycosis and its impacts on wild amphibian populations [START_REF] Kriger | Large-scale seasonal variation in the prevalence and severity of chytridiomycosis[END_REF]. We currently do not know if habitat choice is altered by a host's disease state, nor whether infected lowland leopard frogs deliberately seek out warmer sites to clear Bd. However, such speculation appears well supported, as diseased or parasitized amphibians and other ectotherms have been shown to actively seek temperatures above their thermal optima in order to generate 'behavioral fevers' that enhance host immune response or reduce pathogenic activity [START_REF] Woodhams | Emerging disease of amphibians cured by elevated body temperature[END_REF], [START_REF] Vaughn | Fever in the lizard Dipsosaurus dorsalis[END_REF], [START_REF] Rowley | Behaviour of Australian rainforest stream frogs may affect the transmission of chytridiomycosis[END_REF]; although see [START_REF] Han | Ancient behaviors of larval amphibians in response to an emerging fungal pathogen, Batrachochytrium dendrobatidis[END_REF]. For example, green tree frogs (Hyla cinerea) behaviorally elevated their body temperature 2uC following inoculation with a pathogenic bacterium [START_REF] Kluger | Fever in the frog Hyla Cinerea[END_REF]. More directly, captive boreal toads (Bufo [Anaxyrus] boreas) with severe Bd infections shifted resting positions towards heat strips, suggesting a strategy to combat Bd infection [START_REF] Murphy | Temperature, hydric environment, and prior pathogen exposure alter the experimental severity of chytridiomycosis in boreal toads[END_REF]. Behavioral fever response to Bd has also been observed in wild populations. The average body temperature of a population of Panamanian golden frogs (Atelopus zeteki) increased 2.4uC following exposure to Bd, suggesting that the frogs exhibited a population-wide behavioral fever response during the epidemic [START_REF] Richards-Zawacki | Thermoregulatory behaviour affects prevalence of chytrid fungal infection in a wild population of Panamanian golden frogs[END_REF]. The odds of Bd infection decreased with increasing body temperature, demonstrating that even slight environmental or behavioral changes have the potential to affect an individual's vulnerability to infection [START_REF] Richards-Zawacki | Thermoregulatory behaviour affects prevalence of chytrid fungal infection in a wild population of Panamanian golden frogs[END_REF].

Since A.D. 79, when Pliny the Elder documented frogs inhabiting the hot springs of Pisa in his seminal work ''Naturalis Historia'', amphibian populations have been observed in geothermal ecosystems around the world, including in Algeria [START_REF] Mason | Studies on the fauna of an Algerian hot spring[END_REF], Taiwan [START_REF] Wu | Thermal tolerance and thermoregulation by Taiwanese Rhacophorid tadpoles (Buergeria japonica) living in geothermal hot springs and streams[END_REF],

China [START_REF] Feng | Ultrasonic communication in frogs[END_REF], and Chile [START_REF] Me ´ndez | Divergence in morphometric and life history traits in two thermally contrasting Andean populations of Rhinella spinulosa (Anura: Bufonidae)[END_REF]. There are hundreds of geothermal watersheds throughout western North America, Central America, and Eastern Africa [START_REF] Waring | Thermal springs of the United States and other countries of the world -a summary[END_REF], many of which are located within the historic ranges of vulnerable species of amphibians [START_REF]IUCN Red List of Threatened Species[END_REF]. Although geothermal ecosystems make up only a small fraction of most landscapes, they may be demographically important if they provide amphibians with even partial protection from temperature-sensitive diseases such as chytridiomycosis during pandemic events. Models suggest that the key to long-term persistence with Bd is survival of at least some fraction of infected adults-if some individuals survive the initial epidemic, it is possible that the infected amphibian population will persist in a new endemic state [START_REF] Briggs | Investigating the population-level effects of chytridiomycosis: an emerging infectious disease of amphibians[END_REF], [START_REF] Briggs | Enzootic and epizootic dynamics of the chytrid fungal pathogen of amphibians[END_REF].

Geothermal ecosystems may confer disease-protection to other amphibian species besides lowland leopard frogs. For example, in our study we sampled 20 individuals of other species in water temperatures ranging from 16-36uC (Table 1). Only two (10%) of these individuals were Bd+, and both individuals were captured at 16uC. Furthermore, geothermal ecosystems in Yellowstone National Park appear to be protecting boreal toads from redleg, a potentially fatal bacterial disease [START_REF] Hawk | Amphibian declines in the Greater Yellowstone Ecosystem: do thermally influenced waters protect boreal toads from bacterial disease?[END_REF]. Finally, the relict leopard frog Rana (Lithobates) onca, a close relative to the lowland leopard frog [START_REF] Jaeger | Rediscovering Rana onca: evidence for phylogenetically distinct leopard frogs from the border region of Nevada, Utah, and Arizona[END_REF], [START_REF] Ola ´h-Hemmings | Phylogeography of declining relict and lowland leopard frogs in the desert Southwest of North America[END_REF], is a rare species whose survival may be contingent on geothermal watersheds. The relict leopard frog was once thought to be extinct, but several populations were rediscovered in the 1990's, and all naturally occurring R. onca populations are now associated with perennial geothermal springs with source temperatures exceeding 30uC [START_REF] Bradford | Population status and distribution of a decimated amphibian, the relict leopard frog (Rana onca)[END_REF].

Elucidating the relationship between temperature and Bd prevalence has important implications for effective conservation, and reintroductions of threatened and endangered native amphibians [START_REF] Muths | Distribution and environmental limitations of an amphibian pathogen in the Rocky Mountains, USA[END_REF]. No methods are currently available to treat amphibian populations against Bd in the wild; therefore susceptible species may persist only where conditions are not favorable for Bd or for chytridiomycosis outbreaks [START_REF] Puschendorf | Distribution models for the amphibian chytrid Batrachochytrium dendrobatidis in Costa Rica: proposing climatic refuges as a conservation tool[END_REF]- [START_REF] Tobler | Within-and among-population variation in chytridiomycosis-induced mortality in the toad Alytes obstetricans[END_REF]. Our findings indicate that geothermal waters 25uC-37uC appear to provide amphibians with significant protection from Bd and, by extension, chytridiomycosis. While some species may not tolerate high temperatures, there is often a wide range of water temperatures present in geothermal ecosystems (Figs. 2 and3). It may also be possible to experimentally augment temperatures in non-geothermal environments, thereby creating Bd-free microhabitats that can provide infected individuals with opportunities to clear themselves of the pathogen. Geothermal watersheds appear to represent habitats of exceptional conservation value for some amphibians, and emphasizing protection and restoration efforts, as well as native species translocations into suitable geothermal ecosystems could help recover threatened and endangered species.

Figure 1 .

 1 Figure 1. Occurrence of Bd in lowland leopard frogs as a function of water temperature. A) Presence (1) and absence (0) of Batrachochytrium dendrobatidis (Bd) in lowland leopard frogs Rana (Lithobates) yavapaiensis as a function of water temperature across all years and sites. N = 50 Bd+ individuals and N = 151 Bd2. B) Occurrence of Bd in lowland leopard frogs as a function of water temperature, excluding four sites where Bd was not detected (see Table 1 for details). Each point represents the fraction of frogs infected and mean water temperature of groups of 10-12 individuals (ranked by water temperature, then binned). Logistic equation: y = 1/(1+e 2z ) where z = 4.56 -temp*0.226 (coefficients extracted from univariate analysis). Total sample size from sites with Bd, N = 148. doi:10.1371/journal.pone.0028444.g001

Figure 2 .

 2 Figure 2. Schematic of Mammoth Hot Well showing approximate locations of amphibians and water temperatures. Symbols show approximate locations of lowland leopard frogs (redrawn from field notes) sampled in March 2009, their Bd infection status, and the range of measured water temperatures. doi:10.1371/journal.pone.0028444.g002

Figure 3 .

 3 Figure 3. Schematic of Secret Spring showing approximate locations of amphibians and water temperatures. Symbols show approximate locations of lowland leopard frogs (redrawn from field notes) sampled in March 2009, their Bd infection status, and the range of measured water temperatures. doi:10.1371/journal.pone.0028444.g003

Table 1 .

 1 Sites sampled, Latitude and Longitude (Datum = WGS 84), dates sampled, range of water temperatures measured during sampling, species sampled, number of individuals, and Bd prevalence (percent infected) per species sampled.

	Location	Dates Sampled	H 2 O Temp (6C)	Species Sampled (n)	Species Bd Prevalence
	Aravaipa Creek	Oct 2004	17.7	Rana yavapaiensis (11)	0.64
	N32 52.725	May 2009	23.0
	W110 23.767	Feb 2010	10.5
	BHP Hotwell *	Feb 2010	24.3-28.1	R. yavapaiensis (7)	0.00
	N32 37.961
	W110 33.537
	Dankworth Pond *	Mar 2009	34.1	Rana catesbeiana (1)	0.00
	N32 43.279
	W109 42.089
	El Dorado Hot Spring *	May 2009	36.5-34.1	R. yavapaiensis (18)	0.00
	N33 29.588			Bufo alvarius (1)	0.00
	W112 56.442
	Essence of Tranquility *	Mar 2009	30.7	R. catesbeiana (1)	0.00
	N32 45.480
	W109 43.510
	Hassayampa -TNC	Oct 2004	19.7	R. yavapaiensis (8)	0.63
	N33 55.838			R. catesbeiana (2)	0.00
	W112 41.520
	Hassayampa Preserve	Oct 2004	19.7	R. yavapaiensis (6)	0.00
	N34 02.472
	W112 42.235
	San Pedro River	Mar 2009	16.0	R. yavapaiensis (1)	1.00
	N32 55.511			R. catesbeiana (3)	0.66
	W110 44.489
	Mammoth Hot Well *	Oct 2004	22.4-37.2	R. yavapaiensis (60)	0.21
	N32 41.660	Mar 2009	18.4-36.2	R. catesbeiana (10)	0.00
	W110 37.341	Feb 2010	15.1-33.4	Bufo woodhousii (1)	0.00
	Markham Creek 1	May 2009	22.4-24.0	R. yavapaiensis (29)	0.00
		Feb 2010	11.2-14.5	Hyla arenicolor (1)	0.00
	Muleshoe Hot Spring *	Aug 2004	22.8-51.2	R. yavapaiensis (27)	0.19
	N32 20.229	Mar 2009	22.0-27.0
	W110 14.331	Feb 2010	13.1-30.0
	Secret Spring

* Aug 2004 21.6-23.6 R. yavapaiensis (34) 0.56 N32 20.395 Mar 2009 13.9-38.7 W110 14.613 Feb 2010 16.6-18.3 *Indicates presence of a geothermal source at location. 1 Coordinates excluded at the request of U.S. Bureau of Land Management (Safford, AZ). doi:10.1371/journal.pone.0028444.t001

Table 2 .

 2 Logistic binary regression of infection status (0 = not infected; 1 = infected with Batrachochytrium dendrobatidis) of lowland leopard frogs Rana (Lithobates) yavapaiensis (N = 198; 3 missing SVL).Years 2009 and 2010 contrasted with 2004. Dist. Solstice = Distance to Solstice, the absolute difference in days between the sampling date and June 21 st (used to capture seasonal variation). SVL = Snout-Vent Length. doi:10.1371/journal.pone.0028444.t002

			Standard Error
	Predictor 1	Coefficient	Coefficient	Z	P	Odds Ratio	Lower 95% CI	Upper 95% CI
	Constant	21.02834	2.18103	20.47	0.637
	Water Temp	20.188474	0.0424268	24.44	0.000	0.83	0.76	0.90
	Dist Solstice	0.0648780	0.0155885	4.16	0.000	1.07	1.03	1.10
	SVL (mm)	20.0240867	0.0188217	21.28	0.201	0.98	0.94	1.01
	Year
	2009	0.310455	0.579158	0.54	0.592	1.36	0.44	4.24
	2010	23.80550	0.865731	24.40	0.000	0.02	0.00	0.12
	Log-Likelihood = 270.328.
	Test that all slopes are zero: G = 83,12, DF = 5, P-Value = 0.000.

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December 2011 | Volume 6 | Issue 12 | e28444

PLoS ONE | www.plosone.org

Acknowledgments

We thank M. Sredl and Arizona Game and Fish for permission to conduct this work (permits SP634342, SP782209 and SP805477). Site access was provided by D. Turner, R. Burton, K. Uschyk, D. Wolgast, B. Rogers, M. Haberstich, S. Walker, C. Andresen (The Nature Conservancy), and H. Blasius and J. Crooks (Bureau of Land Management). Special thanks to S. Marcum for assistance in the field, The Nature Conservancy for support, and to T. Edwards (University of AZ) and J. Wood (Pisces Molecular) for assistance with PCR materials and analyses. E. Sandoval, J. Brunner and M. Levey assisted with figures and GIS work. This work greatly benefitted from advice and reviews by B. Han, R. Norris, J. Leichter, J. Jaeger, M. Edwards, K. Zamudio, and an anonymous reviewer.

Partial funding for this work was provided to MF by the Halliday Student Field Research Award from the Scripps Institution of Oceanography, and from the Integrative Graduate Education and Research Training Mini-grant. No additional external funding received for this study. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Materials and Methods

The research presented here was conducted in accordance with State University of New York, College of Environmental Science and Forestry Institutional Animal Care and Use Committee (IACUC) permit 2009-4 (amended), with Dr. Martin Schlaepfer as the Principal Investigator. The IACUC at the University of California San Diego also approved our Animal Use Protocol (Protocol Number S11013) on 1/31/2011, with Dr. Gregory Rouse as Principal Investigator.

We sampled amphibians from twelve sites in Arizona in 2004, 2009, and 2010 (Table 1). Seven of the sites were influenced by geothermal springs or wells, while five were not. We searched for frogs at night (except at Markham Creek, which was sampled by day). At each site, we captured as many individuals as possible by hand, using a new pair of disposable Nitrile gloves to capture and handle each animal. Each individual was retained in a new, closable plastic bag (ZiplocH) until all sampling was completed to ensure that animals were only sampled once, and to prevent crosscontamination. In order to minimize storage times, frogs were processed in the order of capture, generally within 30-90 min. No individuals showed obvious signs of stress, and all animals swam or hopped away immediately upon release.

Bd is transmitted aquatically [START_REF] Kriger | Large-scale seasonal variation in the prevalence and severity of chytridiomycosis[END_REF], [START_REF] Woodhams | Ecology of chytridiomycosis in rainforest stream frog assemblages of tropical Queensland[END_REF], [START_REF] Stuart | Threatened Amphibians of the World[END_REF]; therefore we used the water temperature at the place and time of each capture as an independent variable. This approach was intended to capture microhabitat (spatial) variation in water temperature, rather than relying on daily or monthly air temperatures at sites. It also has the benefit of being measured at the same time as the frog was sampled for Bd. When the captured frogs were partially or fully immersed in water, temperature was measured at the point of capture using a digital thermometer (CDNH Model Q2-450; accuracy 60.5uC). Six frogs were captured on the banks (within less than 2 m of water), in which case the temperature was measured at the nearest water point. Samples from 2004 represent a subset of records from a previous survey of Bd in Arizona [START_REF] Schlaepfer | High prevalence of Batrachochytrium dendrobatidis in wild populations of lowland leopard frogs Rana yavapaiensis in Arizona[END_REF] that also included water temperature at the point of capture. We determined the sex (male, female, or juvenile) of captured frogs, and measured snout-vent length (SVL) and mass before releasing at the point of capture. We defined juveniles as individuals shorter than 50 mm SVL; the approximate size at which ranids in this group of species (''pipiens'') become sexually mature [START_REF] Wright | Handbook of Frogs and Toads of the United States and Canada[END_REF].

Methods for detecting the presence of Bd followed standard procedures. In 2009 and 2010 we used a Sterile Omni Swab (WhatmanH WB100035) to sample skin cells from each animal's venter, flanks, and groin. We swabbed each amphibian a total of 25 times using the applicator, which was then ejected into in a 2ml sterile tube filled with a buffer solution containing 50 mM Tris, pH 8, 50 mM EDTA, 25 mM Sucrose, 100 mM NaCl, and 1% SDS. In 2004, frogs were scraped 25 times in the same body locations using a wood applicator, which was then placed in 70% ethanol [START_REF] Schlaepfer | High prevalence of Batrachochytrium dendrobatidis in wild populations of lowland leopard frogs Rana yavapaiensis in Arizona[END_REF]. All samples were assayed within one month of being collected for the presence of Bd using Polymerase Chain Reaction amplification by a commercial lab (Pisces Molecular, Boulder, CO), following the methods from Annis et al [START_REF] Annis | A DNA-based assay identifies Batrachochytrium dendrobatidis in amphibians[END_REF] with modifications to increase sensitivity and specificity (J. Wood personal communication 2009). Experiments comparing skin scrapes versus skin swabs demonstrated that the ability to detect Bd-positive animals did not differ significantly between the methods [START_REF] Livo | Evaluation of techniques for detecting Batrachochytrium dendrobatidis from amphibians for PCR testing[END_REF]. Moreover, the Qiagen DNA spin column procedure that we used for DNA extraction is not inhibited by tannins or other compounds found in wood, unlike other DNA extraction methods (J. Wood, pers. comm., 2011).

We restricted our statistical analyses to the most common amphibian species, the lowland leopard frog (Rana [Lithobates] yavapaiensis) (Fig. 4). A binomial logistic regression using a logit link function tested for an association between the response variable (Bd presence/absence) and several predictor variables: Snout-Vent Length (SVL) of individual, Year, Distance to Solstice (absolute difference in days between the sampling date and June 21 st used to capture seasonal variation), Site, and water temperature. Twosample t-tests, Fisher's exact, and chi-square tests were used to test for differences in distributions. Analyses were conducted in Minitab (vers. 15), and results are reported as means and standard errors.


Annabelle Dubost

Didier Micol

Bruno Meunier

Claire Lethias

Anne Listrat
email: anne.listrat@clermont.inra.fr

Relationships between structural characteristics of bovine intramuscular connective tissue assessed by image analysis and collagen and proteoglycan content

Keywords:

Three muscles (Longissimus thoracis, Semimembranosus, Biceps femoris) of 40 young bulls from 3 breeds were used to quantify structural characteristics of bovine connective tissue by image analysis, with both macroscopic and microscopic approaches. Collagen and proteoglycan content was also investigated. Perimysium occupied a greater area (8 vs 6%), and was wider (42 vs 2 μm) and shorter per unit area (1.9 vs 30 mm mm -2 ) than endomysium. Perimysium and endomysium from Longissimus were thinner, less ramified than in Biceps. Longissimus showed less total collagen and cross-linking and more proteoglycans (P b 0.0001) than Biceps muscle. Blond d'Aquitaine perimysium occupied less area, was more ramified and muscles contained less collagen, cross-linking and more proteoglycans than Angus (P b 0.001). Limousin was intermediate. High proteoglycan content in muscle containing less total collagen suggested a complementarity between these molecules. They might influence mechanical properties of intramuscular connective tissue. This was especially true given that proteoglycans and total collagen were negatively and positively linked with structural parameters, respectively.

Introduction

Connective tissue in skeletal muscle is divided into endomysium, surrounding each muscle fibre individually, perimysium surrounding groups of muscle fibres and epimysium surrounding the muscle as a whole. Intramuscular connective tissue (IMCT) (endomysium and perimysium) is a three-dimensional network whose morphology and composition varies from one type (species, gender, age) of animal to another and from one muscle to another according to muscle position and function, while having a complex link with meat tenderness [START_REF] Purslow | Intramuscular connective tissue and its role in meat quality[END_REF].

The first structural descriptions of IMCT were done manually and subjectively, by projection of stained muscle sections using an overhead projector (for large sections and muscle fibre bundle studies) or by microscopic observation (small sections and study of microscopic structures) [START_REF] Dumont | Carcass composition and muscle structure in hypertrophied animals[END_REF][START_REF] Dumont | Conséquences de l'hypertrophie musculaire héréditaire sur la trame conjonctive du muscle de bovin[END_REF][START_REF] Schmitt | Etude morphoanatomique du périmysium (méthodes de description et d'évaluation)[END_REF][START_REF] Schmitt | Méthodes d'analyse de la structure musculaire[END_REF]. More recently, image analysis associated with histological methods have been developed. Except [START_REF] Sifre-Maunier | A global unimodal thresholding based on probabilistic reference maps for the segmentation of muscle images[END_REF] and Del [START_REF] Del Moral | Image analysis application for automatic quantification of intramuscular connective tissue in meat[END_REF], authors have rarely described their method accurately. Image analysis has been used to study the structural characteristics of perimysium thickness [START_REF] An | Effect of myofiber characteristics and thickness of perimysium and endomysium on meat tenderness of chickens[END_REF][START_REF] Brooks | Perimysium thickness as an indicator of beef tenderness[END_REF][START_REF] Das | Collagen content and architecture of the pectoralis muscle in male chicks and broilers reared under various nutritional conditions[END_REF][START_REF] Fang | Relationship between development of intramuscular connective tissue and toughness of pork during growth of pigs[END_REF][START_REF] Lachowicz | Comparison of the texture and structure of selected muscles of piglets and wild boar juveniles[END_REF][START_REF] Nishimura | Relationships between physical and structural properties of intramuscular connective tissue and toughness of raw pork[END_REF][START_REF] Sifre-Maunier | A global unimodal thresholding based on probabilistic reference maps for the segmentation of muscle images[END_REF][START_REF] Torrescano | Determination of perimysium and endomysium thickness in bovine, ovine and caprine semimembranosus and semitendinosus muscles by video image analysis[END_REF] or muscle fibre bundles, but few authors have been interested in endomysium [START_REF] An | Effect of myofiber characteristics and thickness of perimysium and endomysium on meat tenderness of chickens[END_REF][START_REF] Lachowicz | Comparison of the texture and structure of selected muscles of piglets and wild boar juveniles[END_REF][START_REF] Torrescano | Determination of perimysium and endomysium thickness in bovine, ovine and caprine semimembranosus and semitendinosus muscles by video image analysis[END_REF]. This may be explained by the fact that the major part of the IMCT is perimysium [START_REF] Light | Characterization of muscle epimysium, perimysium and endomysium collagens[END_REF][START_REF] Light | The role of epimysial, perimysial and endomysial collagen in determining texture in 6 bovine muscles[END_REF]. Nevertheless, a negative correlation has been found between endomysium thickness and meat tenderness in breast muscle (Pectoralis superficialis and Pectoralis profundus) of chicken [START_REF] An | Effect of myofiber characteristics and thickness of perimysium and endomysium on meat tenderness of chickens[END_REF] and a positive correlation has been found in pork (Biceps femoris, Semimembranosus, Quadriceps femoris, Longissimus dorsi, all muscles combined) [START_REF] Lachowicz | Comparison of the texture and structure of selected muscles of piglets and wild boar juveniles[END_REF]. Therefore, the study of perimysium in conjunction with endomysium would seem important. We have developed an image analysis method and we have worked at two scales, macroscopic (magnification ×5) and microscopic (magnification ×100).

IMCT is mainly composed of fibrillar collagens embedded in a matrix of proteoglycans (PGs). These two classes of molecules form a continuous network inside perimysium but also between perimysium, endomysium and muscle fibres [START_REF] Voermans | Clinical and molecular overlap between myopathies and inherited connective tissue diseases[END_REF]. Fibrillar collagens make up the structure of IMCT and are linked between each other by different chemical cross-links. The main cross-links in muscle are the pyridinolines (hydroxylysylpyridinoline and dehydroxylysylpyridinoline). They are trivalent and thermo-stable. PGs play an important role in the stabilization of the intramuscular collagen network [START_REF] Nishiumi | Isolation and characterization of a small proteoglycan associated with porcine intramuscular connective tissue[END_REF], are sensitive to physiological changes (growth, age) [START_REF] Nakano | Changes in the chemical composition of the bovine temporomandibular joint disc with age[END_REF][START_REF] Rooney | Inverse relationship between hyaluronan and collagens in development and angiogenesis[END_REF] and are quickly degraded by proteases during post-mortem storage [START_REF] Nishimura | Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef[END_REF]. The degradation of PGs during post-mortem ageing would expose collagen to collagenases and facilitate its degradation [START_REF] Nishimura | Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef[END_REF]. Thus, PGs could influence the mechanical properties of IMCT and tenderness. With regard to beef meat tenderness, proteoglycans have been little considered while collagen and its cross-links have been extensively studied and compared between muscles [START_REF] Rhee | Variation in palatability and biochemical traits within and among eleven beef muscles[END_REF][START_REF] Torrescano | Shear values of raw samples of 14 bovine muscles and their relation to muscle collagen characteristics[END_REF], but much less data exist between breeds. Nevertheless, it has been shown that muscularity affects the connective tissue structural parameters and its composition in collagen [START_REF] Dumont | Carcass composition and muscle structure in hypertrophied animals[END_REF][START_REF] Listrat | Grass valorisation and muscular characteristics of blonde d'aquitaine steers[END_REF]. In addition, late-maturing cattle breeds (as Belgian blue, Limousin and Blond d'Aquitaine) grow more slowly and deposit more muscles and less fat, compared to early-maturing cattle breeds (as Angus and Japanese Black cattle) which tend to have muscles richer in collagen with a higher proportion of insoluble collagen [START_REF] Campo | Assessment of breed type and ageing time effects on beef meat quality using two different texture devices[END_REF][START_REF] Christensen | Relationship between collagen characteristics, lipid content and raw and cooked texture of meat from young bulls of fifteen european breeds[END_REF][START_REF] Jurie | Muscle characteristics from young bulls of different beef breeds[END_REF]Blanco et al., in press;[START_REF] Monson | Influence of cattle breed and ageing time on textural meat quality[END_REF].

The aim of the present study was to i) quantify structural characteristics of bovine IMCT (perimysium and endomysium) by image analysis, ii) show whether there was a relationship between these parameters and the biochemical composition of IMCT (collagen and PGs), and iii) determine which parameters best discriminate muscles and breeds.

Materials and methods

This study was carried out in compliance with the French recommendations and those of the Animal Care and Use Committee of the National Institute for Agricultural Research (INRA, Institut National de la Recherche Agronomique) of Clermont-Ferrand/Theix, France for the use of experimental animals including animal welfare.

Beef production and muscle sampling

The experiment was performed on 40 young entire males of Aberdeen Angus (AA) (n=12), Limousin (LI) (n = 14) and Blond d'Aquitaine (BA) (n=14) pure breeds. Animals (12 month-old) were assigned for a 100 day finishing period. The animals were housed in straw pens, individually fed and weighed every 2 weeks. Diets consisted of concentrate (75%) and straw (25%). Animals were slaughtered at the same age (around 17 months) and final live weight (around 665 kg) (Table 1) in order to avoid weight and age effects on IMCT characteristics. They were slaughtered at the experimental slaughter-house of the INRA Research centre. For the other parameters (average daily gain, muscle and fat carcass characteristics), AA was significantly different to LI and BA, which were similar. AA had 17% higher daily gain, 34% higher fatty tissue weight and 19% lower carcass muscle weight than LI and BA.

Three muscles were taken for each animal: Longissimus thoracis (LT), Semimembranosus (SM) and Biceps femoris (BF). For each muscle and each analysis, samples were taken at the same location. Muscle samples were removed from the 9th rib for LT and from the centre of the muscle for SM and BF.

For histological and PG measurements, muscle samples were taken 15 min after exsanguination. For histology, muscle samples of 1.5 × 1.5 × 1 cm were taken and for PG measurement, muscle samples (60-80 g) were cut up into small pieces. The two sets of samples were stored at -80 °C until preparation for analysis. For histology, samples were initially frozen in isopentane chilled in liquid nitrogen.

The carcasses were chilled in a cold room (+ 2 °C) and muscle samples were taken at 24 h post-mortem for collagen and cross-link measurements. Muscle samples (about 150 g) were cut into pieces of 1 cm cross-section, sealed under vacuum in plastic bags and stored at -20 °C until preparation for analysis.

Histology associated with image analysis

Muscle blocks (1.5 x 1.5 x 1 cm) were fixed with Tissue-Tek® OCT TM (Sakura) in a cryostat (HM 500 M, Microm, France) maintained at -25 °C, and cross-sections of 10 μm thickness were prepared. For each animal, the three muscle sections were placed on the same slide. A total of 40 slides were stained. For each staining bath, the three breeds were mixed to limit staining differences between two staining baths. The staining was carried out according the Picro-Sirius red method previously described by Flint et al. [START_REF] Flint | Demonstration of collagen in meat-products by an improved picro-sirius red polarization method[END_REF] with slight modifications. Briefly, after acetone fixation (60 min), cross-sections were fixed 10 min in Picro-Formalin solution, rinsed in 90% ethanol (1 min), then in distilled water (10 min) and immersed in Sirius red staining solution (60 min) before washing in 0.01 M hydrochloric acid (5 min). After rinsing in distilled water (1 min), cross-sections were dehydrated successively in 95% ethanol and then twice in 100% ethanol. Stained sections were cleared in Safesolvant (Labonord) and mounted with Safemount (Labonord). IMCT (perimysium and endomysium) was stained red and muscle fibres yellow (dark grey and light grey, respectively, in Fig. 1). The aim was to create a contrast between IMCT and muscle fibres in order to develop an image analysis method to study perimysium, endomysium and muscle fibre morphology, on the same section, in an automatic and objective manner.

Image acquisitions

2.2.1.1. Perimysium studymacroscopy. Each stained muscle section (1.5 × 1.5 cm) was scanned entirely in transmission mode using an EPSON Expression 10000XL PRO A3 scanner (resolution of 2400 dpi, corresponding to a spatial resolution of 10.58 μm/pixel).

2.2.1.2. Endomysium studymicroscopy. Stained sections were visualized in bright field mode with an Olympus BX51 microscope (Olympus, Tokyo, Japan) using a 10x objective (NA = 0.3). Five images were acquired randomly from the stained sections of 1.5× 1.5 cm, used for perimysium study, with a DP-72 colour camera and Cell-D image Both image acquisition strategies allowed a reliable control of geometrical (square pixel), colourimetric (white balance) and lighting (flatness and intensity) conditions. All colour images were saved in the uncompressed RGB-TIFF format.

Image processing and analysis

Image processing was realised under the Visilog 6.7 Professional Software (Noesis, Gif-sur-Yvette, France). This software provides fullaccess to a wide range of low-level precompiled image processing procedures. Indeed, it allows developing visual basic for application (VBA) programs to execute semi-automatically a specific algorithm, through a personalized user interface.

Two different VBA programs were developed: i) Perimysium Analyzer to study perimysium, and ii) Endomysium Fibre Analyzer to study endomysium and muscle fibres.

The different steps of the Perimysium Analyzer procedure are summarized in the flow chart (Fig. 2,A). Briefly, the colour image (Fig. 2B) was loaded and converted into greyscale RGB (Red, Green, Blue) and HLS (Hue, Luminance, Saturation) components. The more contrasted greyscale was selected for the following segmentation step. The Green component (Fig. 2C) was used for perimysium detection. Thin perimysium was identified by morphological tophat filtering and thick perimysium was segmented after manual thresholding. The saturation component (Fig. 2D) was used for sample detection (identification of muscle cells and perimysium from background) by automatic segmentation. These two segmentation steps resulted in binary image of perimysium (Fig. 2E) and of sample (Fig. 2F). After visual control of perimysium and sample segmentation, image analysis was initiated. Data of estimated parameters outlined below were stored in .txt and could be exported for further analysis.

The different steps of the Endomysium Fibre Analyzer procedure are summarized in a flow chart (Fig. 3A). The colour image (Fig. 3B) was loaded and converted into greyscale HLS components. The H component (Fig. 3C) was used for endomysial segmentation using fixed thresholding followed by watershed lines algorithm to close the network. This last step resulted in a separation of objects which correspond to the muscle fibres. We thus obtained the binary image of the endomysium (Fig. 3D) and the binary image of the muscle fibres (Fig. 3E). The binary image of the endomysium was skeletonised (Fig. 3F) and the binary image of fibres was labelled and filtered so that uncompleted fibres were automatically discarded (Fig. 3G). After visual control of endomysium segmentation, image analysis was initiated. Data of estimated parameters outlined below for endomysium and muscle fibre were stored in .txt and could be exported for further analysis.

For perimysium and endomysium study, the binarised images were used to determine the area of the perimysium and endomysium (both expressed as % of the total image area). The skeletonised image was used to determine total length of perimysium and endomysium (both expressed in mm mm -2 ) and triple points (named branch points for perimysium and connection points for endomysium, expressed as number cm -2 and as number mm -2 , respectively). Indeed, two parameters were calculated for perimysium and endomysium: width (area/ total length, determined above for perimysium and endomysium respectively and expressed in μm for both) and length between 2 triple points (total length/triple point number, named unit length and expressed in mm for perimysium and in μm for endomysium). So, for all variables above, the comparison between perimysium and endomysium was relevant. For the muscle fibre study, the labelled images were used to determine the muscle fibre area (mean fibre area, expressed in μm 2 ) and the number of muscle fibres per mm 2 . The processing algorithm classified fibres according to the number of sides. Three groups were formed; fibres with 3-4 sides, fibres with 5-6 sides and fibres with 7 sides or more, expressed in %. The number of sides per muscle fibre was determined according to the connection points previously identified by algorithm (1 side was delineated by 2 connection points).

For endomysium and muscle fibre study, parameters obtained for the 5 acquired images of a muscle section were averaged. For the perimysium study, the algorithm did not differentiate the different perimysium levels which surround different muscle fibre bundle levels. In our study, perimysium included 3 levels, from the perimysium surrounding primary fibre bundles to perimysium surrounding tertiary bundles (shown in Fig. 1). Moreover, lipids or holes (in white on Fig. 1) were not stained. So, during image processing they were not considered as belonging to the studied image.

Biochemical characteristics of IMCT

For total and insoluble collagen contents and cross-link measurements, frozen muscle was homogenized in a household cutter (Robot coupe®, R2), freeze-dried for 48 h, pulverized in a horizontal blade mill (Moulinex, type M CUA1) and stored at +4 °C in stopper plastic flasks until analyses. For PG measurement, frozen muscles were powdered in liquid nitrogen and stored at -80 °C until analyses.

Collagen and cross-link measurements

For total collagen (TC) and cross-links, about 250 mg of muscle powder were weighed, acid hydrolysed with 10 mL of 6 N HCl, over-night at 110 °C in a screw-capped glass tube. For TC, the acid hydrolysate was diluted 5 times in 6 N HCl and the subsequent procedure used was that previously described and updated by Listrat et al. [START_REF] Listrat | Analytical limits of total and insoluble collagen content measurements and of type i and iii collagen analysis by electrophoresis in bovine muscles[END_REF][START_REF] Listrat | Effect of the type of diet on muscle characteristics and meat palatability of growing salers bulls[END_REF]. For cross-links, 1 ml of the 6 N acid hydrolysate was centrifuged at 16,000 ×g for 5 min at+ 4 °C. For insoluble collagen (IC), muscle powder was solubilised according to the method of [START_REF] Hill | The solubility of intramuscular collagen in meat animals of various ages[END_REF] as follows. Lyophilised muscle powder was rehydrated for one hour with 1X solubilization buffer (0.23 M NaCl, 25 mM Tris-HCl, pH 7.4) and heated in a water bath at 75 °C for one hour. The insoluble fraction was dissociated from the soluble fraction by filtration (pleated filters in cotton cellulose, VWR 512-0206) and hydrolysed according to the same method as for TC. Both for TC and IC, each sample was weighed and measured in triplicate and data were expressed in mg of collagen per g of dry matter (mg g -1 DM), assuming that collagen corresponds to 7.14× hydroxyproline content [START_REF] Ngapo | Perimysial collagen crosslinking in belgian blue double-muscled cattle[END_REF].

For cross-links, 600 μL of 6 N NaOH and 600 μL of 1 M Tris were added to 600 μL of acid supernatant. Final pH was adjusted between 7 and 8 with a few microliters of 6 N HCl or NaOH. Pyridinoline cross-links (pyridinoline+ deoxypyridinoline) were determined by the enzyme-linked immunoassay Metra Pyd EIA kit (Quidel Corporation, USA) according to the manufacturer. Previous assays had indicated no interfering effect of the 1/10 diluted muscle extract on the pyridinoline cross-link determination (results not shown). Results were expressed in nM of pyridinoline per g of dry matter (nM pyr g -1 DM) and per mole of collagen (mM pyr M -1 collagen), assuming that collagen had a molecular weight of 300 kDa) [START_REF] Ngapo | Perimysial collagen crosslinking in belgian blue double-muscled cattle[END_REF].

Total proteoglycan content

Muscle powder was incubated 24 h at + 4 °C with extraction buffer (6 M Urea, 1 M NaCl, 2% CHAPS and protease inhibitor cocktail [Complete,Roche Diagnostics GmbH,ref. 11 836 145 001]). The solid to liquid ratio was 100 mg of muscle powder to 1 mL of extraction buffer. The next day, samples were centrifuged 40 min at + 4 °C, 15,000 ×g. Supernatant (muscle extract) was recovered and used to determine proteoglycan content according to a modified [START_REF] Barbosa | Improved and simple micro assay for sulfated glycosaminoglycans quantification in biological extracts and its use in skin and muscle tissue studies[END_REF] Farndale method [START_REF] Farndale | A direct spectrophotometric micro-assay for sulfated glycoaminoglycans in cartilage cultures[END_REF]. This assay was based on the ability of sulphated glycosaminoglycans (GAGs) to bind the cationic dye 1,9-dimethylmethylene blue (DMMB). Briefly, 1 mL of DMMB solution was added in excess to 100 μL of muscle extract and shaken 30 min at room temperature. After centrifugation 15 min, 12,000 ×g, supernatant (DMMB excess) was removed. One mL of 50 mM sodium acetate buffer solution was added to the residue and shaken 30 min. Absorbance was measured at 656 nm, with a micro-plate reader (TECAN Infinite® M200). Concentrations were determined by comparison with a standard curve of chondroitine-4-sulfate (C4S), ranging from 0 to 2.5 μg of C4S. Each sample was measured twice and data were expressed in μg of C4S-GAGs equivalents per g of dry matter (μg C4S-GAGs g -1 DM) and in mg of C4S-GAGs per g of collagen (mg C4S-GAGs g -1 collagen).

Statistical analysis

Pearson correlation matrix was performed using XLSTAT-Pro (v. 2011.3.01) in order to study relationships between structural and biochemical variables. Then, data were subjected to analysis of variance using the SAS GLM procedure (Cary, NC: SAS Institute Inc.,). The fixed effects of muscle and breed type and muscle effect nested within animals were included in the model. Effect of muscle × breed interaction was also included in the model. The percentage of variance explained by this model was nearly the same as that of the model without muscle × breed interaction. The effects were mainly due to the muscle effect. So, we retained the model without muscle × breed interaction. Difference was claimed significant for P b 0.05. All results were presented as LSmeans with appropriate standard error of the mean (SEM) and the level of significance.

Results

Perimysium and endomysium distribution in muscle

For all structural parameters in Table 2, the comparison between perimysium and endomysium was relevant because for both, the parameters were expressed in comparable units. Perimysium occupied 8% of the total image area and endomysium occupied 6%. Perimysium Table 2 Means, standard deviations (SD), coefficients of variation (CV), minimum and maximum of structural and biochemical parameters of IMCT and of muscle fibre (n=120), across the three breeds and three muscles. had an area 1.4 times higher than endomysium. However, per mm 2 , perimysium was 15 times shorter than endomysium (1.9 vs 30 mm) and 22 times wider (42.4 vs 1.9 μm). Per mm 2 , endomysium had 603 connection points. The length (unit length) between two points was on average 50 μm. Per cm 2 , perimysium network had 91 branch points. The distance between two branch points was on average 2.1 mm (unit length). Our results also showed 308 muscle fibres per mm 2 with an average cross-sectional area of 3,336 μm 2 (equivalent to a circle with a diameter of 65.2 μm). Muscle had a majority of fibres with 5-6 sides (54%). Table 3 summarizes the correlations between the structural and biochemical parameter studied. The structural parameters were positively correlated to each other. Correlations between area and total length or width for perimysium and endomysium, respectively, were particularly significant (ranged from 0.50 to 0.97, P b 0.05). The structural parameters were also positively correlated with total and insoluble collagen content and cross-link (significant correlations ranged from 0.19 to 0.56). The above parameters were negatively correlated with PGs (significant correlations of -0.48 with total collagen and crosslinks and -0.50 with perimysium area and length). Fibre number, endomysium length and connection points were negatively correlated with fibre area (r= -0.98, -0.41 and -0.44, respectively). Within muscles or breeds, correlations similar to those in Table 3 between structural and biochemical variables were found (results not shown).

Mean

Table 2 showed that, with the exception of perimysium width (CV = 8%) and total endomysium length (8.4% of variation), all parameters were highly variable, probably due to muscle and breed variations. CVs of structural parameters ranged from 11.2 to 34.5%. CVs of biochemical parameters ranged from 16.4 to 58%.

Muscle effect on structural and biochemical parameters

Table 4 showed that 21 of 22 parameters (except perimysium unit length) had a P-value of 0.001 or less. Parameters which differed significantly between the 3 muscles were, among structural parameters, perimysium area (expressed in %) and width, and among biochemical parameters, total and insoluble collagen, cross-links expressed per g of DM and PGs expressed per g of collagen. The other parameters (endomysium and muscle fibre) differed significantly between LT, SM and BF. LT showed respectively 30 and 35% less perimysium area than SM and BF muscles. The same pattern was observed in the endomysium area, LT endomysium area was 26% lower than for SM and BF muscles. LT perimysium was 25% shorter and 10% narrower than SM and BF, whereas endomysium was 4% shorter and 24% narrower. BF perimysium showed significantly more branch points than LT (98 vs 84 branch points cm -2 ) and SM was intermediate.

Length between two branch points was not significantly different (2 μm on average). These two parameters were relative to degree of ramification. LT had lower muscle fibre density (288 fibres mm -2 ) and fibres had cross-sectional area 11% greater than BF and SM muscle fibres. It had a higher proportion of fibres with 5-6 sides and a Total proteoglycan (mg C4S-GAGs g -1 collagen)

22.6 a 10.9 b 8.9 c 0.7 b0.0001 lower proportion of fibres with 3-4 and 7 sides. The average length of LT fibre sides was longer (53.4 μm) than that of SM and BF muscles (48.6 and 49.5 μm respectively). LT muscle had 24 and 42% less total collagen content than SM and BF, respectively. Insoluble collagen content was lower in LT muscle than in SM and BF (17 and 35% less,respectively). Cross-link content was 41% lower in LT than in SM and BF and PG content was 33% greater in LT than in SM and BF. Expressed relative to total collagen, LT cross-link content was only 10% lower and PG content 57% greater than in SM and BF.

Breed effect on structural and biochemical parameters

Table 5 showed that 11 of 23 parameters had a P-value for breed effects of at least≤0.001. Several perimysium characteristics (area, total length and branch point number) and the proportion of muscle fibres with 5-6 sides differed significantly between the breeds. Percentage of fibres with 3-4 sides discriminated AA from the others breeds, while percentage of fibres with 7 sides differed between BA and the others breeds.

The other structural parameters did not allow differentiating breeds. AA perimysium area (9.5% of the total image area) was 17 and 29% higher than in LI and BA (7.9 and 6.7% respectively). In AA, endomysium had an area 14% higher than BA (LI was not significantly different from AA and BA). AA showed 14% longer perimysium and 5% longer endomysium than BA. Perimysium of LI was different from AA and BA and endomysium was similar to AA and BA. AA perimysium was more ramified than BA (95 vs 69 branch points) but they had the same unit length (2.5 mm). LI had more branch points (109) than AA and BA and a shorter distance between 2 points (1.8 mm).

Among biochemical parameters, PGs (expressed per g of collagen) distinguished the 3 breeds from each other while collagen characteristics (total, insoluble and cross-links in nM pyr g -1 DM) demonstrated that AA was different from LI and BA. Cross-link content (expressed per g of DM) was 22% higher in AA than in LI and BA. Reported to the total collagen content, cross-link content was not significantly different between the 3 breeds. PG content was 17% higher for BA than AA and LI. Reported to the total collagen content, BA had 14 and 42% higher PG content than LI and AA, respectively.

Discussion

In image analysis, two constraints emerged. The first one concerned the sampling representativeness linked to the field of view and the second the detailed characterisation linked to the resolution. To take into account perimysium heterogeneity, we chose a macroscopic approach by scanning the whole section (1.5 x 1.5 cm). For accurate characterisation of endomysium and muscle fibre, we needed for a microscopic approach. This two scale approach proved efficient for an automatic and accurate characterisation of IMCT components. Ideally, it would be interesting to contain all information in a unique image. To this end, a mosaic image containing several hundred megabytes could be acquired under the microscope. Unfortunately, these bulky images are still impossible to automatically analyze and visualize today.

Several authors have attempted to characterize perimysium but, up to now only its area and width have been studied in several species. Table 6 summarizes the characteristics of intramuscular connective tissue previously studied. In pork, Del [START_REF] Del Moral | Image analysis application for automatic quantification of intramuscular connective tissue in meat[END_REF] showed a LT perimysium area of 22.6 and 9.9% (% of the total image area) respectively for Large White crossbreed and Iberian pigs and LT perimysium area ranged from 3% for Bos Taurus Rubia Gallega to 5.8% for Kobe beef. The same authors [START_REF] Del Moral | Image analysis application for automatic quantification of intramuscular connective tissue in meat[END_REF] also showed for sheep a perimysium area of 13. 8 and 20.7% (% of the total image area) respectively for Triceps brachii and LT. [START_REF] Sifre-Maunier | A global unimodal thresholding based on probabilistic reference maps for the segmentation of muscle images[END_REF] showed, for 5-year-old Charolais cull cows, a perimysium area ranging from 3.1 to 14% in LT muscle, for a threshold confidence level of 0.80 and 0.55 (the former allowing the thicker perimysium detection and the latter allowing overall perimysium detection), respectively. BF perimysium area ranged from 4.9 to 13.8%. For perimysium width, [START_REF] Torrescano | Determination of perimysium and endomysium thickness in bovine, ovine and caprine semimembranosus and semitendinosus muscles by video image analysis[END_REF] determined the perimysium thickness surrounding the first and the second level of muscle fibre bundles. In cattle, data ranged from 30 to 120 μm, in sheep from 10 to 30 μm and in goat from 10 to 20 μm. [START_REF] Sifre-Maunier | A global unimodal thresholding based on probabilistic reference maps for the segmentation of muscle images[END_REF] determined a cattle perimysium width ranged from 58 to 74 μm for LT and reached 82 μm for BF bovine muscle. In pork, [START_REF] Lachowicz | Comparison of the texture and structure of selected muscles of piglets and wild boar juveniles[END_REF] determined an average perimysium width (average of LT, SM and BF) of 15 and 20 μm respectively for domestic piglets and wild boar juveniles. Results obtained for Landrace pigs [START_REF] Fang | Relationship between development of intramuscular connective tissue and toughness of pork during growth of pigs[END_REF][START_REF] Nishimura | Relationships between physical and structural properties of intramuscular connective tissue and toughness of raw pork[END_REF] showed an average perimysium width (perimysium surrounding secondary bundles) of 24 μm for 6 month pigs. In chicken, [START_REF] Das | Collagen content and architecture of the pectoralis muscle in male chicks and broilers reared under various nutritional conditions[END_REF] showed a perimysium width for broilers (8 weeks) ranging from 30 μm (thick perimysium) to 10 μm (thin perimysium). [START_REF] An | Effect of myofiber characteristics and thickness of perimysium and endomysium on meat tenderness of chickens[END_REF] determined a perimysium width for chicks (6 to 18 weeks) ranging from 14 μm to 39 μm. Finally, [START_REF] Torrescano | Determination of perimysium and endomysium thickness in bovine, ovine and caprine semimembranosus and semitendinosus muscles by video image analysis[END_REF] determined an endomysial thickness of 10 μm in goat and sheep and of 15 μm in cattle. [START_REF] Lachowicz | Comparison of the texture and structure of selected muscles of piglets and wild boar juveniles[END_REF] determined a width of 1.7 μm in piglets and 2.3 μm in wild juvenile boars (average of LT, SM and BF muscles). [START_REF] An | Effect of myofiber characteristics and thickness of perimysium and endomysium on meat tenderness of chickens[END_REF] showed a width ranging from 5 to 8 μm for 6-18 week chicks.

On average in this study, perimysium area was 8% and perimysium width was 42 μm. Our data are consistent with cattle results above taking into account that observation scales are different from one study to another and that for perimysium thickness it is sometimes difficult to determine which level was considered. However, cattle perimysial area appears to be lower than perimysial area in pig and sheep. Perimysial thickness appears to be greater in cattle than in pig and chicken maybe because of the higher level of organization in the perimysium of cattle. Except perimysium width, total length of endomysium, the mean values of all structural and biochemical descriptors presented a high variation. This probably reflects the functional differences of the muscles, since LT is a positional muscle and SM and BF are locomotion muscles. According to the functional requirements of muscle (contraction, elongation), quantity, spatial distribution and composition of IMCT vary in order to coordinate force transmission in muscle [START_REF] Purslow | Muscle fascia and force transmission[END_REF]. During locomotion, functional effort is greater than during posture resulting in different perimysium and endomysium characteristics for the different muscle types. Thus, LT muscle perimysium was less ramified (number of branch points), shorter and thinner than BF muscle perimysium which had a higher perimysium area, total length and was more ramified. The same tendency was found for LT muscle endomysium, which was narrower, shorter and had fewer connection points than BF muscle.

LT muscle contained less collagen than BF, and SM was intermediate. These results are in agreement with those of others authors [START_REF] Rhee | Variation in palatability and biochemical traits within and among eleven beef muscles[END_REF][START_REF] Stolowski | Factors influencing the variation in tenderness of seven major beef muscles from three angus and brahman breed crosses[END_REF][START_REF] Torrescano | Shear values of raw samples of 14 bovine muscles and their relation to muscle collagen characteristics[END_REF]. One of the other main components of muscle is proteoglycans (PGs). The LT muscle contained the most PGs. LT perimysium area was lower than BF perimysium area. Collagen fibrils are used as a scaffold for the anchorage of PGs which link collagen fibrils together in order to stabilize the collagenous network and to maintain space between fibrils [START_REF] Eggen | Structure and solubility of collagen and glycosaminoglycans in two bovine muscles with different textural properties[END_REF]. So, more PG in the IMCT could result in a greater space between collagen fibrils, and of a dilution by PGs.

In meat tenderness context, PGs are not often considered. However, they seem to play an important role in the process of meat ageing. [START_REF] Nishimura | Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef[END_REF] have shown that PGs are degraded during post-mortem ageing of beef. This degradation involves the separation of collagen fibrils and muscle fibres from endomysium and perimysium. In addition, microscopic studies have shown that a fracture line first appears between the collagen fibres at the level of some collagen-PG interactions when tension is applied perpendicularly to the myofibres in meat [START_REF] Purslow | The physical basis of meat texture: Observations on the fracture behaviour of cooked bovine m. Semitendinosus[END_REF]. PGs are sensitive to enzymatic digestion resulting in an exposure of collagen fibrils to degradation by collagenases during ageing and therefore to an IMCT disorganization [START_REF] Nishimura | The role of intramuscular connective tissue in meat texture[END_REF]. LT muscle had a PG/collagen ratio (total PGs expressed in mg C4S-GAGs g -1 collagen) higher than that of BF muscle. These results are in accordance with the studies of [START_REF] Pedersen | Sulfated glycosaminoglycans and collagen in two bovine muscles (m-semitendinosus and m-psoas major) differing in texture[END_REF] and [START_REF] Eggen | Structure and solubility of collagen and glycosaminoglycans in two bovine muscles with different textural properties[END_REF] which showed that Psoas major muscle, a muscle very poor in collagen [START_REF] Torrescano | Shear values of raw samples of 14 bovine muscles and their relation to muscle collagen characteristics[END_REF], had a higher PG/collagen ratio than Semitendinosus, a muscle rich in collagen [START_REF] Torrescano | Shear values of raw samples of 14 bovine muscles and their relation to muscle collagen characteristics[END_REF]. In addition, it has been suggested that with fewer PGs available to form a network around the collagen fibres, the formation of cross-linkages in collagen could increase [START_REF] Pedersen | Sulfated glycosaminoglycans and collagen in two bovine muscles (m-semitendinosus and m-psoas major) differing in texture[END_REF]. Our results confirmed this suggestion since we showed greater cross-link content in BF muscle containing fewer PGs, compared to LT muscle, and a negative relationship between PG content and insoluble collagen and cross-link contents. Our results are in agreement with those of the other authors and suggest that there may be a balance between PG, collagen and cross-link contents, which could affect meat tenderness during ageing.

The cattle breed might also impact the deviation shown on almost all the descriptors, but breed effect was lower than muscle effect, as already shown [START_REF] Schreurs | Meta-analysis of the effect of animal maturity on muscle characteristics in different muscles, breeds, and sexes of cattle[END_REF]. The BA breed had a less ramified and shorter perimysium and muscles had fewer total and insoluble collagen. On the contrary, the Angus breed had a more ramified and longer perimysium with a higher area, and higher total and insoluble collagen content. For structural parameters LI were intermediate, while for total and insoluble collagen, they were not different from BA. This difference of content in total collagen between Limousin and Angus has already been shown [START_REF] Christensen | Relationship between collagen characteristics, lipid content and raw and cooked texture of meat from young bulls of fifteen european breeds[END_REF][START_REF] Jurie | Muscle characteristics from young bulls of different beef breeds[END_REF]. In a previous study, we showed that BA had a high muscularity [START_REF] Listrat | Grass valorisation and muscular characteristics of blonde d'aquitaine steers[END_REF]. This particularity is often associated with a modification of structural characteristics [START_REF] Dumont | Carcass composition and muscle structure in hypertrophied animals[END_REF] and of content in total and insoluble collagen [START_REF] Christensen | Relationship between collagen characteristics, lipid content and raw and cooked texture of meat from young bulls of fifteen european breeds[END_REF][START_REF] Jurie | Muscle characteristics from young bulls of different beef breeds[END_REF][START_REF] Monson | Influence of cattle breed and ageing time on textural meat quality[END_REF][START_REF] Oliván | Effect of muscular hypertrophy on physico-chemical, biochemical and texture traits of meat from yearling bulls[END_REF].

The BA animals showed also more PGs (relative to the total collagen) than LI and AA, and the three breeds were the same cross-link content, expressed per mole of collagen. The assumption that there is a balance between PG, collagen and cross-link contents seems to be confirmed for the breed. Nonetheless, the relationship between PGs and cross-links (more PG and less cross-link) is less clear, because cross-link content was not significantly different between the three breeds.

Conclusion

The image analysis method developed here allowed to quickly quantifying intramuscular connective tissue characteristics at different muscle levels. The positive correlations between structural IMCT descriptors and collagen content suggested that histology associated with image analysis could replace biochemical measurements, which are long and sometimes tedious to implement.

The high PG content in muscle containing less total collagen suggested a complementarity between these molecules and they might play a role on the mechanical properties of IMCT and perhaps on meat quality. This was especially true given that PGs and total collagen were negatively and positively link with structural parameters, respectively.

Fig. 1 .Fig. 2 .

 12 Fig.1. Histological section of BF muscle stained according to the Picro-Sirius red method. Intramuscular connective tissue (P: perimysium and E: endomysium) appeared in dark grey and muscle fibres (MF) in light grey. Adipocytes (Ad) or holes (H) were not stained and appeared in white. The wall of blood vessels (BV) appeared in an intermediate level of grey.

Fig. 3 .

 3 Fig. 3. Flow chart of the Endomysium Fibre Analyzer (A) image processing application dedicated to the endomysium and muscle fibre characterisation associated with resulting images. After loading of the colour image (B), it was converted into HLS (Hue, Saturation, Luminance) components in greyscale. The H component (C) (the more contrasted components of the image) was used for segmentation. Binary image of endomysium (D) and muscle fibre (E) were obtained and skeletonised (F) and labelled (G), respectively. These images (D to G) were used for image analysis step, after visual control by the user. Data were stored in .txt and could be exported for further analysis.

a- c

 c Within a row, means without a common superscript letter differ (P b 0.05). Means with no superscript letters are not significantly different.d LT = Longissimus thoracis, SM=Semimembranosus, BF=Biceps femoris.

Table 3

 3 Correlation coefficients a between structural and biochemical parameters b (n = 120), across the three breeds and three muscles.

		Area_P	Lgth_P	Wdth_P	BP	Area_E	Lgth_E	Wdth_E	CP	FbreNber	Area_F	CL_DM	TC	IC	PG_DM
	Area_P	1
	Lgth_P	0.97	1
	Wdth_P	0.50	0.26	1
	BP	0.33	0.37	-0.01	1
	Area_E	0.50	0.45	0.35	0.16	1
	Lgth_E	0.37	0.36	0.15	0.14	0.45	1
	Wdth_E	0.51	0.47	0.34	0.06	0.61	0.14	1
	CP	0.39	0.36	0.23	0.13	0.51	0.93	0.22	1
	FbreNber	0.24	0.21	0.18	0.004	0.41	0.41	0.29	0.43	1
	Area_F	-0.24	-0.22	-0.19	-0.003	-0.41	-0.41	-0.26	-0.44	-0.98	1
	CL_DM	0.54	0.47	0.45	0.19	0.56	0.18	0.51	0.25	0.23	-0.26	1
	TC	0.55	0.46	0.50	0.25	0.53	0.18	0.49	0.25	0.17	-0.18	0.77	1
	IC	0.45	0.39	0.38	0.18	0.41	0.10	0.44	0.17	0.14	-0.13	0.65	0.81	1
	PG_DM	-0.50	-0.50	-0.27	-0.25	-0.40	-0.25	-0.36	-0.30	-0.19	0.22	-0.48	-0.48	-0.41	1

a Correlation coefficient values in bold are significant for P b 0.05. b Area_P, Area_E: perimysium and endomysium area; Lgth_P, Lgth_E: perimysium and endomysium length; Wdth_P, Wdth_E: perimysium and endomysium width; BP: branch points; CP: connection points; TC: total collagen; IC: insoluble collagen; CL_DM: cross-link expressed per g of dry matter; PG_DM: proteoglycan content expressed per g of dry matter; Area_F: mean fibre area; FbreNber: fibre number per mm 2 .

Table 4

 4 Muscle effect on structural (perimysium, endomysium, muscle fibre) and biochemical (collagen, cross-links, proteoglycans) characteristics of muscles.

		Muscle d
		LT	SM	BF	SEM	P-value
		(n = 40)	(n = 40)	(n = 40)
	Perimysium
	Area (%)	6.0 c	8.6 b	9.2 a	0.3	b0.0001
	Total length (mm mm -2 )	1.5 b	2.0 a	2.1 a	0.1	b0.0001
	Width (μm)	39.4 c	43.0 b	44.8 a	0.4	b0.0001
	Number of branch points cm -2 84 b	90 ab	98 a	5	b0.0001
	Unit length (mm)	1.8	2.2	2.1	0.1	0.07
	Endomysium
	Area (%)	4.7 b	6.3 a	6.4 a	0.2	b0.0001
	Total length (mm mm -2 )	29.1 b	30.5 a	30.3 a	0.4	b0.0001
	Width (μm)	1.6 b	2.1 a	2.1 a	0.04 b0.0001
	Number of connection points	545 b	630 a	619 a	15	b0.0001
	mm -2
	Unit length (μm)	53.4 a	48.6 b	49.5 b	0.8	b0.0001
	Muscle fibre
	Fibre number mm -2	288 b	324 a	314 a	8	0.0006
	Mean fibre area (μm 2 )	3591 a	3164 b	3253 b	84	0.0002
	Fibres with 3-4 sides (%)	16.2 b	19.2 a	20.1 a	0.6	b0.0001
	Fibres with 5-6 sides (%)	59.2 a	52. 6 b	50.7 b	0.8	b0.0001
	Fibres with 7 sides (%)	24.6 b	28.2 a	29.2 a	0.7	b0.0001
	Collagen
	Total collagen (mg g -1 DM)	29.6 c	39.1 b	50.6 a	1.2	b0.0001
	Insoluble collagen	21.6 c	25.6 b	32.9 a	0.9	b0.0001
	(mg g -1 DM)
	Cross-linking
	Cross-links (nM pyr g -1 DM)	19.0 c	28.2 b	36.6 a	1.0	b0.0001
	Cross-links (mM pyr M -1	195 b	218 a	216 a	5	0.001
	collagen)
	Proteoglycans
	Total proteoglycan	629 a	416 b	429 b	17	b0.0001
	(μg C4S-GAGs g -1 DM)

Table 5

 5 Breed effect on structural (perimysium, endomysium, muscle fibre) and biochemical (collagen, cross-links, proteoglycans) characteristics of muscle (n = 120).

		Breed d
		AA	LI	BA	SEM P-value
		(n = 36)	(n = 42)	(n = 42)
	Perimysium
	Area (%)	9.5 a	7.9 b	6.7 c	0.3 b0.0001
	Total length (mm mm -2 )	2.2 a	1.9 b	1.6 c	0.1 b0.0001
	Width (μm)	42.2	41.7	43.3	0.5	0.07
	Number of branch points cm -2 95 b	109 a	69 c	4	b0.0001
	Unit length (mm)	2.5 a	1.8 b	2.5 a	0.1	0.0004
	Endomysium
	Area (%)	6.4 a	5.7 ab	5. 5 b	0.2	0.01
	Total length (mm mm -2 )	30.7 a	30.1 ab	29.1 b	0.4	0.04
	Width (μm)	2.0	1.9	1.9	0.1	0.16
	Number of connection points	630	590	579	16	0.14
	mm -2
	Unit length (μm)	49.3	51.7	50.3	0.9	0.10
	Muscle fibre
	Fibre number mm -2	317	308	301	8	0.53
	Mean fibre area (μm 2 )	3244	3336	3416	89	0.53
	Fibres with 3-4 sides (%)	15.4 b	18.8 a	21.3 a	0. 9	0.0003
	Fibres with 5-6 sides (%)	58.2 a	54.8 b	49.4 c	0.9 b0.0001
	Fibres with 7 sides (%)	26.4 b	26.3 b	29.4 a	0.9	0.04
	Collagen
	Total collagen (mg g -1 DM)	46.9 a	37.3 b	36.1 b	1.7 b0.0001
	Insoluble collagen	31.1 a	25.2 b	24.5 b	1.1 b0.0001
	(mg g -1 DM)
	Cross-linking
	Cross-links (nM pyr g -1 DM)	33.0 a	26.5 b	25.0 b	1.5	0.0002
	Cross-links	208	214	207	6	0.68
	(mM pyr M -1 collagen)
	Proteoglycans
	Total proteoglycan	437 b	478 b	552 a	22	0.001
	(μg C4S-GAGs g -1 DM)
	Total proteoglycan	10 c	14.6 b	17.2 a	1.1 b0.0001
	(mg C4S-GAGs g -1 collagen)

a-c

Within a row, means without a common superscript letter differ (P b 0.05). Means with no superscript letters are not significantly different.

d AA = Aberdeen Angus, LI = Limousin, BA = Blond d'Aquitaine.

Table 6

 6 Overview of characteristics of perimysium and endomysium measured by image analysis.

A. Dubost et al. / Meat Science 93 (2013) 378-386

Acknowledgements

The authors express their thanks to the staff of INRA Experimental Unit (UERT) for animal management and slaughtering, and the staff of the 'Animal, Muscle, Viande' team for technical assistance, particularly Jean-Luc Montel and David Chadeyron for the collagen and cross-link content measurements, and Christiane Barboiron for her pertinent advice for the PG study.