Change in fat skatole and indole content in lambs switched from a concentrate-based diet indoors to alfalfa grazing for various durations before slaughter

Sophie Prache¹, Lucille Rey-Cadilhac¹, Armelle Prunier²

¹Université Clermont Auvergne, INRAE, VetAgro Sup, UMR Herbivores, F-63122 St-Genès-Champanelle, France

²PEGASE, INRAE, Institut Agro, F-35590 St-Gilles, France

Corresponding author: Sophie Prache. Email: sophie.prache@inrae.fr

Abstract

Meat from lambs fattened on alfalfa is at risk of excessive pastoral flavours due to high levels of in-fat volatile indolic compounds (especially, skatole). Skatole has also been identified as a potential marker of interest for authenticating pasture-fed lamb meat. Here, we investigated the change in skatole and indole concentrations in kidney fat from lambs switched from an indoor-fed concentrate based diet to outdoor alfalfa grazing for various durations (0, 21, 42, 63 days) before slaughter. The study used a total of 219 lambs over 3 consecutive years. Kidney-fat skatole and indole concentrations increased from as early as 21 days on alfalfa, and then reached a plateau. Similarly, the proportion of lambs that had a kidney fat-skatole concentration above 0.15 µg/g liquid fat, a value that has been established as a sensory rejection threshold for pork, increased significantly from as early as 21 days on alfalfa and then
26 reached a plateau. This value was reached or exceeded in a significant
27 proportion of lambs fattened on alfalfa pastures (45.1%). However, skatole was
28 not detected in kidney fat from 20 out of 164 alfalfa-fattened lambs (i.e., 12.2%)
29 but was detected in 15 out of 55 concentrate-fed lambs (i.e., 27.3%). We thus
30 conclude that while skatole content in kidney fat can inform on dietary changes
31 made shortly before slaughter, it does not have the discrimination power
32 needed to reliably authenticate pasture-fed lamb meat, let alone duration of
33 finishing on pasture.
34

35 **Keywords:** authentication, flavour, indole, lucerne, pasture-feeding, skatole
36

37 1. Introduction
38

39 Meat from pasture-fed lambs has better nutritional properties than meat from
40 lambs raised indoors with a concentrate diet (Aurousseau et al., 2007; Gruffat et
41 al., 2020). However, meat from pasture-fed lambs is darker (Priolo et al., 2002)
42 and may have a stronger flavour (Resconi et al., 2009) or a higher intensity of
43 skatole-related odour and flavour (Rivaroli et al., 2019; Devincenzi et al., 2019)
44 than meat from lambs fed indoors a concentrate diet, which may be seen as a
45 defect. The stronger flavour has in fact been partly associated with higher
46 concentrations of volatile indolic compounds, especially 3-methylindole (or
47 skatole), in the fat of pasture-fed lambs compared to concentrate-fed lambs
48 (Young et al., 1997 and 2003).
49

50 Forage legumes offer many agro-environmental benefits and can play an
important role in sustainable sheep production, notably by reducing the level of
external inputs while improving animal productivity (Frank et al., 2016; Ponnampalam et al., 2022). These plants can improve soil fertility through their ability to fix atmospheric nitrogen. In addition, alfalfa (Medicago sativa) can improve soil structure due to its deep root system compared to grass, which also makes it resistant to dry conditions. Grazing alfalfa had also been shown to improve the nutritional quality of lamb meat compared to grazing perennial ryegrass (Lolium perenne) or feeding feedlot pellets (Ponnampalam et al., 2019; Gruffat et al., 2020). Nevertheless, pastures including alfalfa or white clover (Trifolium repens) may result in increased fat concentration of volatile indolic compounds in lamb meat (Schreurs et al., 2007a and b; Devincenzi et al., 2014), and thus in increased meat flavour intensity (Schreurs et al., 2007a; Devincenzi et al., 2014 and 2019; Rivaroli et al., 2019) and increased risk of undesirable pastoral flavours (Park et al., 1972 and 1975), especially for consumers accustomed to meat from grain-based farming systems. However, this has not always been observed (Schreurs et al., 2007b; de Brito et al., 2016; Frank et al., 2016), sometimes due to the small number of animals under study (Schreurs et al., 2007b). Note that alfalfa can be consumed in different forms, but only fresh forage or silage can lead to high levels of skatole and indole in the rumen and fat, as drying and dehydration greatly reduce protein degradation in the rumen (INRA, 2018), and thus skatole and indole formation in the rumen.

Research has suggested that the detection threshold for skatole-related odour and flavour is about 0.05 µg/g fat in butter (Watkins et al., 2014) and up to 0.1 µg/g fat in pork (Banon et al., 2003; Lunde et al., 2010), and that the sensory rejection threshold for skatole is 0.15 µg/g fat in pork (Mörlein et al., 2012). This threshold can be considered as an indicator for the identification of carcasses
whose meat is likely to be rejected by consumers. However, consumer
acceptance of meat is highly dependent on how it is processed and consumed.
As skatole and indole are lipophilic and volatile molecules, their concentration is
much higher in fat than in lean tissue and their odour is more strongly perceived
when the meat product has been heated. Thus, the risk of consumer rejection is
higher for sausages or bacon than for rindless chops or ham (Parois et al.,
2018). Furthermore, because lamb meat is less processed than pork and other
volatile compounds may interfere with skatole and indole, such as
androstenone in pork (Parois et al., 2018) or branched chain fatty acids in lamb
meat (Young et al., 1997), generalization of this threshold established for pig
carcasses to lamb carcasses must be done with caution. However, in the
absence of comparable information for lamb carcasses, we used, in this study,
that established for pig carcasses. Finally, it should be noted that the number of
lambs used in previous studies was too small to enable proper assessment of
the proportion of lambs that are above this latter threshold (0.15 µg/g fat) based
on their diet.

Furthermore, recent studies in lambs switched to concentrate-feeding after a
period of alfalfa grazing demonstrated that the persistence of skatole and indole
in the fat was low (Gkarane et al., 2019; Eiras et al., 2022), but there is still a
lack of information on the time-related profile of these compounds in fat after a
switch to alfalfa grazing, including the time after which they become detectable
in fat after switching to alfalfa grazing.

Skatole in fat has also been investigated for its potential value in diet
authentication, particularly for authenticating pasture-fed meat (Priolo et al.,
2004; Vasta et al., 2006; Prache et al., 2020). However, the literature on this
issue is so far inconclusive, probably due to both the high variability in animal response and the low number of animals under study (Vasta et al., 2006; Prache et al., 2020).

The objective of the present study was to investigate the change in fat skatole and indole concentrations in lambs switched from concentrate-feeding indoors to alfalfa grazing for various durations before slaughter. We used a large number of animals over three consecutive years in order to provide information on the proportion of lambs at risk of developing excessive pastoral flavours, and whether fat skatole levels can be used for authentication purposes.

110

## 2. Material and methods

112

The experiment was conducted at the INRAE's 'Herbipôle' experimental unit.

The protocol was approved by the ethical committee of the French Ministry of Higher Education, Research and Innovation (committee approval 14289-2017042411419479V4).

117

### 2.1. Experimental design, animal management and diet regimens

119

The experiment compared four durations of grazing alfalfa before slaughter: 0 day (L0), 21 days (L21), 42 days (L42), and 63 days (L63). The experiment was replicated three times during three consecutive years. Nineteen Romane weaned male lambs were used each year for each treatment group, giving 228 Romane lambs used in total. The lambs were born within a few weeks (between 8 and 30 April in year 1, between 6 and 30 April in year 2, and between 29 April

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125
and 1 May in year 3). Slaughter dates were scheduled each year so that the lambs would be slaughtered at approximately 5 months old, an age that yields carcasses of commercially acceptable weight and degree of fatness, as shown in previous research using Romane lambs fattened on alfalfa pastures after weaning (Devincenzi et al., 2019; Rivaroli et al., 2019; Eiras et al., 2022). Dates for turning out to pasture were then calculated based on the planned slaughter dates. Figure 1 gives a schematic diagram of the experimental layout.

Lambs were randomly selected from the existing flock in the experimental unit, born during this 1-month period, and were randomly allocated to the treatment groups on the basis of live-weight (LW) at birth, birth date, and growth rate between birth and the beginning of the experiment. Mean lamb LW at birth was 4.0 kg (SD 0.84), and mean lamb age at slaughter was 143 days (SD 13).

Before grazing alfalfa, the L21, L42 and L63 lambs were kept indoors and-fed a high-concentrate diet. During the stall-feeding period, concentrate was given at a level adjusted to achieve similar mean patterns of growth between concentrate-fed lambs and pasture-fed lambs, and straw was given *ad libitum*.

Pasture-fed lambs from the different treatment groups co-grazed in a single group to limit potential confounding effects of sward characteristics, except during the first 5 days following the turn-out to pasture to allow for a dietary transition when switching from concentrate feeding to alfalfa grazing (Figure 1).

During this transition, lambs grazed a grass plot and were allowed access to an adjacent alfalfa plot for increasing time duration over the course of the 5 days.

Two dry ewes were added to this group of pasture-finished lambs to train them in pasture use. In each experimental year, lambs in the same treatment group were slaughtered on the same day, so that all the pasture-fed lambs in a given
treatment group could be turned out from stall to pasture at the same date, thus facilitating the 5-days diet transition. The plots were grazed rotationally, and lambs were moved to a new grazing break as pasture conditions dictated so as to ensure *ad libitum* availability of green alfalfa leaves. Pasture herbage was cut and the trimmings removed, one month prior to commencement of the experiment and after each grazing period, to ensure good-quality regrowths.

Pasture-fed lambs received drenches against digestive nematodes and *Moniezia* if necessary, based on regular measurements of faecal egg counts performed at monthly intervals after turn out to pasture.

When indoors, lambs were also managed in a single group. They were fed a commercial concentrate, the composition of which is given in Table 1, along with straw in collective feeding troughs and racks. The troughs and racks were long enough for all animals to have access to the feed at the same time. The concentrate was distributed every day at 9 a.m., after the previous concentrate refusals had been discarded. All lambs had *ad libitum* access to water and salt blocks.

The lambs were slaughtered in the INRAE's experimental slaughterhouse located next to the experimental site. On arrival, the lambs were immediately electrically stunned and slaughtered by throat cutting. Evisceration and skinning were performed according to standard commercial practices. The carcasses were then placed in a refrigerated room at 4°C until 24 h after slaughter.

## 2.2. Measurements

### 2.2.1. Lamb live-weight
176

177

Lambs were weighed at birth, then once weekly at the same time of the day throughout the experiment, and on the day of slaughter (full weight).

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179

180

### 2.2.2. Carcass characteristics and fat sampling

181

182

After chilling for 24 h at 4°C, the carcasses were weighed, graded for conformation and assessed for fatness using a 15-point scale (Eiras et al., 2022). Kidney fat weight and dorsal fat thickness over the last thoracic rib were measured. A sample of kidney fat of approximately 30 g was then taken, wrapped and vacuum-packed in a sealable polyamide bag, frozen, and stored at -20° C until chemical analysis.

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### 2.2.3. Fat skatole and indole concentration analysis

190

191

Kidney fat skatole and indole concentrations were determined by HPLC using the procedure described by Batorek et al. (2012) as follows: fat samples were liquefied in a microwave oven for 2 x 1 min at 350 W. The liquefied lipids were centrifuged for 20 min at 11,200 g at 20°C. After centrifugation, the fat was heated to 50°C and 0.5 ± 0.01 g of water-free liquid fat was transferred into 2.5 mL Eppendorf tubes with 1 mL methanol containing 0.050 mg/L 2 of 3-methylindole (internal standard). After stirring for 30 s, the tubes were incubated for 5 min at 30°C in an ultrasonic water bath, put on ice for 20 min, and centrifuged for 20 min at 11,200 g at 4°C. For skatole and indole determinations, 20 µL of supernatant was injected into an HPLC column, and

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200
fluorescence was detected (excitation at 285 nm and emission at 340 nm) using
a HP1200 system (Agilent Technologies, Waldbronn, Germany).
Concentrations were expressed in µg per gram of lipid fraction from adipose
tissue. The limit of detection was 0.03 µg/g liquid fat.

**2.3. Data analyses**

The data for lamb LW and LW gain, carcass weight, conformation, degree of fatness, and dorsal fat thickness were analysed by ANOVA using a mixed model, with treatment set as a fixed factor and year set as a random factor (SAS Institute Inc, 2014), and using the Tukey test for pair-wise comparisons.

Differences between treatment groups in the distribution of lambs in the different classes of fat skatole and indole concentrations (i) below or above the limit of detection, i.e., 0.03 µg/g liquid fat, and (ii) below or above 0.15 µg/g liquid fat, the value for backfat skatole concentration identified as causing sensory rejection in pork (Mörlein et al., 2012) were analysed using a Chi-square test. As the variance for kidney fat weight and skatole and indole concentrations differed between treatment groups and was not stabilized using log-transformed data, these variables were analysed using non-parametric statistics (Kruskal-Wallis test, pair-wise comparisons being performed with a Bonferroni correction).

**3. Results and discussion**

**3.1. Animal performance and carcass characteristics**
226

227 Four lambs had to be removed from the experiment due to injury or sickness, and six others died during the experiment. A final total of 55, 55, 56 and 52 lambs completed the experiment in the L0, L21, L42 and L63 treatment groups, respectively. Lamb LW at the beginning of the grazing period and LW gain during the grazing period are given in Table 2.

232

233 There were treatment effects on carcass weight, degree of carcass fatness, dorsal fat thickness and kidney fat weight (*P* < 0.001 for all variables, Table 2 and Figure 2). Carcass weight and degree of fatness were lower in L21 and L42 lambs than in L0 and L63 lambs (*P* < 0.05 to *P* < 0.001), but not significantly different between L21 and L42 lambs and between L0 and L63 lambs. Dorsal fat thickness was higher in L0 lambs than in the other treatment groups (*P* < 0.001), which did not differ. Kidney fat weight was lower in L21 than in L0 (*P* < 0.01) and L63 (*P* < 0.01) lambs, with no other significant differences in pairwise comparisons (Figure 2). Carcass conformation score averaged 6.72 (which corresponds to R- in the EUROP classification) and showed no between-group differences (Table 2).

244

245 The LW change during the 3 weeks after the switch from stall to pasture was in the order L63 (4.15 kg, range: -1.3 to 8.0 kg) > L42 (1.48 kg, range: -2.3 to 4.6 kg) > L21 lambs (0.01 kg, range: -6.0 to 6.0 kg), with a high variability between animals in a same feeding treatment. This abrupt change in diet and living conditions, which was dictated by the objectives of the study, caused some stress to some animals, particularly in the L21 and, to a lesser extent, L42
treatment groups, as had already been observed by Huang et al. (2015) and Eiras et al. (2022) for the reverse pattern of change in diet, i.e., switching from pasture-feeding to concentrate-feeding indoors. We chose to co-graze all pasture-fed lambs together in order to avoid confounding effects of sward characteristics (which could have induced undesirable differences in the quantity and quality of the herbage ingested and therefore, potentially, in the fat skatole and indole concentrations). However, co-grazing did not avoid unpredictable variability in animal performance, and resulted in lower carcass weight and degree of fatness in L21 and L42 lambs compared to L0 and L63 lambs. These differences were, however, relatively unsubstantial.

261

## 3.2. Fat skatole and indole concentrations

263

In this study, skatole and indole concentrations were measured in kidney fat to ensure that all lambs had sufficient fat for sampling. Previous studies in sheep have measured skatole and indole concentrations in kidney fat (Young et al., 2003; Girard et al., 2016), subcutaneous fat (Young et al., 1997; Priolo et al., 2004 and 2009), or both these fat depots (Devincenzi et al., 2014 and 2019; Rivaroli et al., 2019; Eiras et al., 2022). Devincenzi et al. (2014) observed a good correlation between these two fat depots for skatole concentration ($r^2$ = 0.78); such an analysis was not possible for indole concentration due to a large number of samples below the detection limit. Moreover, the effect of various treatments on fat skatole and indole concentrations showed high similarity between the two fat depots (Rivaroli et al., 2019; Devincenzi et al., 2019; Eiras et al., 2022). Skatole and indole, which are lipophilic compounds, are also
expected to be present in intramuscular fat, and the corresponding analysis would be of great interest. However, this analysis poses technical difficulties because of the low level of intramuscular fat and thus the low concentrations of skatole and indole in the lean tissue. To our knowledge, no such data is available for lamb, but Devincenzi et al. (2014), in lambs fed various dietary proportions of fresh alfalfa forage showed that there was substantial similarity between the lean part of the chop and the fat part of the chop in the change in intensity of the skatole-related odour and flavour. Some studies on pork showed a significant correlation between subcutaneous fat and lean tissue for skatole and indole concentrations, and, as expected, lower concentrations in lean tissue than in subcutaneous fat (Rius and Garcia-Regueiro, 2001; Wauters et al., 2016; Meinert et al., 2017).

In the present study, there were treatment effects on kidney-fat skatole ($P < 0.001$, Figure 3) and indole concentrations ($P < 0.001$, Figure 4). Kidney-fat skatole concentration was lower in L0 lambs than in L21, L42 and L63 lambs ($P < 0.001$ for all comparisons), but not significantly different between L21, L42 and L63 lambs. A new finding of this study was thus that skatole concentration in kidney fat was significantly increased from as early as 21 days of alfalfa grazing, and then stabilized for longer periods of fattening on alfalfa.

Given that there was a treatment effect on kidney fat weight (Figure 2), it is questionable whether kidney fat weight may have been a confounding factor. There is no scientific literature on this kind of potential dilution effect. However, there was no significant correlation between kidney fat skatole content and kidney fat weight for either pasture-fed or concentrate-fed lambs. Moreover, the
301 kidney fat skatole concentration found here was fairly similar to that found by
302 Eiras et al. (2022), Rivaroli et al. (2019) and Devincenzi et al. (2019) in Romane
303 lambs grazing alfalfa that had much higher kidney fat weights. This casts doubt
304 on a possible dilution effect of kidney fat skatole content related to kidney fat
305 weight. Here, kidney fat skatole concentration in the L63 lambs ranged from
306 0.01 to 0.54 µg/g liquid fat, whereas it ranged from 0.08 to 0.41 µg/g in Eiras et
307 al. (2022), from 0.03 to 0.41 µg/g in Rivaroli et al. (2019), and from 0.09 to 0.56
308 µg/g in Devincenzi et al. (2019).

309 Kidney-fat indole concentration was lower in L0 lambs than in L21, L42 and L63
310 lambs (P < 0.001 for all comparisons). Kidney-fat indole concentration was also
311 lower in L42 lambs than in L21 lambs (P < 0.05) (Figure 4). There were no other
312 significant differences in pairwise comparisons. Kidney fat indole concentration
313 was thus significantly increased from as early as 21 days of alfalfa grazing and
314 was similar between L21 and L63 lambs (0.12 and 0.11 µg/g, respectively), but
315 it was lower in L42 lambs than in L21 lambs (0.08 vs 0.11 µg/g). This latter
316 difference may be related to a slight change in grazing conditions at the end of
317 the fattening period. Actually, even though pasture-fed lambs co-grazed as a
318 single group, they were slaughtered on different days to accommodate a
319 feeding transition (when turned out to alfalfa pasture). This difference in indole
320 concentration is however of little importance for meat quality, as indole is much
321 less odorous than skatole.

322

323 **3.3. Proportion of lambs in which skatole concentration was above 0.15**
324 **µg/g liquid fat**

325
There was a treatment effect on the proportion of lambs in which skatole concentration was above 0.15 µg/g liquid fat (*P* < 0.001). This proportion was lower in L0 lambs than in the other treatment groups (3.6% in L0 lambs vs. 50.0%, 46.4% and 38.5% in L21, L42 and L63 lambs, respectively; *P* < 0.001 for all pairwise comparisons), without significant differences between L 21, L42 and L63 lambs. The proportion of lambs for which skatole concentration in kidney fat was above 0.15 µg/g liquid fat was thus significantly increased from as early as 21 days of alfalfa grazing, and then stabilized for longer periods of fattening on alfalfa. It should be noted, however, that this sensory rejection threshold was established for pork and could be different for lamb meat, which has a different composition of odorant molecules, resulting in a different bouquet in which skatole could be less identifiable or a source of rejection or, on the contrary, interact with other odorant molecules (Prache et al., 2022).

The skatole concentration in kidney fat varied widely between lambs in a same treatment group, in line with previous studies (Young et al., 2003; Schreurs et al., 2008; Devincenzi et al., 2019; Rivaroli et al., 2019; Eiras et al., 2022). Six out of 52 concentrate-fed (L0) lambs had high levels of kidney fat skatole and were considered outliers. High skatole concentrations in kidney fat are rare in concentrate-fed lambs but have been observed in previous studies (Devincenzi et al., 2019; Eiras et al., 2022). Despite these empirical evidences, the underlying reasons remain poorly understood. Conversely, skatole and indole were not detected in a number of lambs fattened on alfalfa, as already observed in previous studies (Rivaroli et al., 2019; Eiras et al., 2022). This high variability between animals suggests that future research could be directed at assessing the content of these off-flavour-causing compounds in the live animal (e.g. in
the blood), as proposed by Eiras et al. (2022), in order to furnish estimates of
between-animal variation in fat skatole concentration and adapt the nutritional
strategy to mitigate over-intense flavours if necessary.

Figure 5 gives the lamb distribution stratified by kidney fat skatole concentration
classes, with data for the L0 and L63 lambs studied here pooled with data from
three previous experiments on lambs concentrate-fed indoors or fattened for at
least two months on alfalfa without any supplementation (Rivaroli et al., 2019;
Devincenzi et al., 2019; Eiras et al., 2022) (n = 94 and 93 for each type of
lambs, in 6 experimental years). Based on these data, 4.2% of concentrate-fed
lambs were above the target value of 0.15 µg/g liquid fat that Mörlein et al.
(2012) identified as causing sensory rejection in pork, whereas 45.9% of lambs
fattened for at least two months on alfalfa were above this target value. As there
is still insufficient knowledge (and therefore uncertainty) regarding the threshold
value for fat skatole concentration causing sensory rejection in lamb, we have
presented all the individual data in Figure 5. This could allow to better define in
the future the proportion of lambs finished on alfalfa that are at risk of
developing excessive pastoral flavours, if scientific studies better define this
threshold and complete this first database. It would actually be worthwhile to
study the relationship between fat skatole concentration and the intensity of
skatole-related odour and flavour on a large data set. The difficulty lies in the
fact that (i) odour and flavour appreciation and liking vary considerably across
consumer groups (Sanudo et al, 2007; Font-i-Furnols et al., 2009), (ii) there are
between-studies methodological differences in the qualifiers used in sensory
tests (Watkins et al., 2013) and in the method used for analysing skatole and
375 indole concentrations, (iii) as discussed above, other compounds affect meat
376 odour and flavour and may interact with skatole.

377
378 **3.4. Proportion of lambs in which skatole was detected (authentication**
379 **purpose)**

380
381 There was a treatment effect on proportion of lambs in which skatole was
detected in kidney fat (P < 0.001); this proportion was lower in L0 lambs than in
382 the other treatment groups (27.3 %, 96.4 %, 96.4 % and 69.2 % in L0, L21, L42
383 and L63 lambs, respectively; P < 0.001 for all pairwise comparisons). Analysis
384 with the alfalfa-finished lambs (L21, L42 and L63 lambs) pooled together
385 showed that the proportion of alfalfa-finished lambs in which skatole was
detected in kidney fat was higher than the proportion of L0 lambs (concentrate-
386 fed lambs indoors) in which skatole was detected in kidney fat (87.8% vs
387 27.3%, P < 0.001).

390 Thus, another new finding from this study is that, even though skatole content in
391 kidney fat can inform on dietary changes made shortly before slaughter (i.e., as
392 early as 21 days of grazing), it does not have the discriminatory power to
393 reliably authenticate pasture-fed lambs. Even though alfalfa is known to
394 promote high levels of in-fat skatole, skatole was not detected in kidney fat from
395 12.2% of alfalfa-grazed lambs, but was detected in kidney fat from 27.3% of
396 concentrate-fed lambs. Furthermore, the reliability of the discrimination between
397 concentrate-fed and pasture-fed lambs based on kidney fat skatole content is
398 likely lower when lambs graze grass pastures or legumes containing condensed
399 tannins (such as sainfoin, sulla, or birdsfoot, for example), which are known to
400 lead to lower fat skatole concentration (Schreurs et al., 2008; Girard et al., 2016; Rivaroli et al., 2019). This means that fat skatole concentration has to be combined with other tissue composition measures, preferably showing different pattern of change with duration of pasture-feeding before slaughter, to increase the reliability of diet authentication. A candidate compositional variable for this purpose could be meat n-3 PUFA content, as Noci et al. (2005) observed a linear increase in n-3 PUFA content in beef as a function of pasture-feeding duration before slaughter. Spectral methods that incorporate diet-related differences in tissue composition can take advantage of these different patterns of change with duration of pasture-finishing (Prache et al., 2020).

410

411 **Conclusion**

412

413 The time to appearance of volatile indolic compounds in kidney fat after lambs were switched from a concentrate-based diet indoors to alfalfa grazing was short. Kidney fat skatole and indole concentrations were sharply increased from as early as 21 days of alfalfa grazing, and their concentration then stabilized for longer durations on alfalfa onwards. With respect to authentication, the results of the present study show that, while skatole content in kidney fat can inform on dietary changes made shortly before slaughter, it does not have the discrimination power needed to reliably authenticate pasture-feeding in lambs, let alone the duration of finishing at pasture.

422

423 **Ethics approval**

424
425 All procedures received ethical approval from Ethical Review Committee of the
426 French Ministry of Higher Education, Research and Innovation (committee
427 approval 14289-2017042411419479V4).

428

429 **Data and model availability statement**

430

431 None of the data used were deposited in an official repository

432

433 **Author ORCIDs**

434

435 **S. Prache**: https://orcid.org/0000-0003-1660-5058

436 **L. Rey-Cadilhac**: https://orcid.org/0000-0002-4554-7662

437 **A. Prunier**: https://orcid.org/0000-0003-3070-6613

438

439 **Author contributions**

440

441 **Sophie Prache**: conceptualization, investigation, writing, review and editing,
442 project administration, funding acquisition, supervision. **Lucille Rey-Cadilhac**:
443 investigation, statistical analysis, writing original draft. **Armelle Prunier**:
444 conceptualization, fat skatole and indole analysis, investigation, review. All
445 authors have read and agreed to the manuscript.

446

447 **Declaration of competing interest**

448

449 None.
450

451 **Data availability**

452

453 Data will be made available on request

454

455 **Acknowledgements**

456

457 The authors thank L. Lavelle, C. Coustet and the staff of the INRAE Herbipôle

458 Experimental Unit, S. Faure and C. Labonne from UMR Herbivores for their

459 technical support at the slaughterhouse, L. Lenormand from UMR PEGASE for

460 analyzing the fat indole and skatole concentrations, and A. Imbert for her advice

461 on the experimental design and the corresponding statistics. We also thank V.

462 Bellet from Institut de l'Elevage and Catherine Experton from Institut Technique

463 de l'Agriculture Biologique for the implementation and animation of the

464 REVABIO project.

465

466 **Funding sources**

467

468 This work was supported by the Special Assignment Account for "Agricultural

469 and Rural Development" (CASDAR) of the French Ministry of Agriculture,

470 Agrifood and Forestry, under project 19AIP5912 - REVABIO.

471

472

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650
<figure><img src="image_1.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_2.png" /><figcaption>Figure 2</figcaption></figure>
668

<figure><img src="image_3.png" /></figure>

669

670

<caption>Figure 3</caption>

672

673

674

675

676

677
<figure><img src="image_4.png" /><figcaption>Figure 4</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 5</figcaption></figure>
Table 1 Composition of the concentrate given to lambs when raised indoors (g/kg). The net energy for meat production and the crude protein content were 0.93 Unités Fourragères Viande/kg DM and 133 g/kg DM, respectively.

<table><tr><td>Barley</td><td>302</td></tr><tr><td>Wheat</td><td>228</td></tr><tr><td>Sugar beet pulp</td><td>142</td></tr><tr><td>Rapeseed meal</td><td>110</td></tr><tr><td>Sunflower meal</td><td>76</td></tr><tr><td>Wheat red shorts</td><td>37</td></tr><tr><td>Wheat bran</td><td>30</td></tr><tr><td>Corn grain</td><td>20</td></tr><tr><td>Sugarcane molasses</td><td>15</td></tr><tr><td>Calcium carbonate</td><td>10</td></tr><tr><td>Palm oil</td><td>10</td></tr><tr><td>Vegetable extracts to prevent renal calculus</td><td>6</td></tr><tr><td>Trace elements and vitamins</td><td>10.5</td></tr><tr><td>Salt</td><td>3</td></tr></table>
693 **Table 2** Carcass characteristics of lambs fattened on alfalfa pasture for various

694 durations before slaughter

<table><thead><tr><th>Treatment group<sup>(1)</sup></th><th>L0</th><th>L21</th><th>L42</th><th>L63</th><th>SEM<sup>(2)</sup></th><th>P-value</th></tr></thead><tbody><tr><td>Number of lambs</td><td>52</td><td>56</td><td>55</td><td>55</td><td></td><td></td></tr><tr><td>LW<sup>(3)</sup> at the beginning of the grazing period (kg)</td><td>-</td><td>37.3a</td><td>31.9b</td><td>27.0c</td><td>2.82</td><td>P < 0.001</td></tr><tr><td>LW<sup>(3)</sup> gain during the grazing period</td><td>-</td><td>0.01a</td><td>7.71b</td><td>15.26c</td><td>1.048</td><td>P < 0.001</td></tr><tr><td>Carcass weight (kg)</td><td>18.75a</td><td>17.29b</td><td>17.60b</td><td>19.41a</td><td>1.518</td><td>P < 0.001</td></tr><tr><td>Carcass conformation</td><td>6.83</td><td>6.54</td><td>6.60</td><td>6.89</td><td>0.292</td><td>P = 0.41</td></tr><tr><td>Carcass fatness</td><td>7.44a</td><td>6.14b</td><td>6.33b</td><td>6.99a</td><td>0.515</td><td>P < 0.001</td></tr><tr><td>Dorsal fat thickness (mm)</td><td>3.2b</td><td>2.6a</td><td>2.6a</td><td>2.9a</td><td>0.291</td><td>P < 0.001</td></tr></tbody></table>

695 Means with unlike letters are significantly different.

696 (1) L0, L21, L42 and L63: lambs fattened on alfalfa pasture for 0 day, 21 days, 42

697 days and 63 days before slaughter, respectively.

698 (2) SEM: standard error of the mean.

699 (3) Live-weight
701

702 **Figure captions**

703

704 **Figure 1** Overview of the course of the experiment. Red and green refer to indoor feeding with a concentrate-based diet and alfalfa grazing periods, respectively.
705
706

707

708 **Figure 2** Box-plot representation of kidney-fat weight in lambs that have grazed lucerne for 0, 21, 42 or 63 days before slaughter (n = 55, 55, 56 and 52 lambs, respectively). The upper edge of the box indicates the 75th percentile of the data-set, the bold line in the box indicates the median value, and the lower edge indicates the 25th percentile of the data-set. The black circles represent the outliers. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.
709
710
711
712
713

714

715 **Figure 3** Box plot representation of kidney fat skatole concentration in lambs that have grazed lucerne for 0, 21, 42 or 63 days before slaughter (n = 55, 55, 56 and 52 lambs, respectively). The upper edge of the box indicates the 75th percentile of the data-set, the bold line in the box indicates the median value, and the lower edge indicates the 25th percentile of the data-set. The black circles represent the outliers. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001.
716
717
718
719
720
721

722

723 **Figure 4** Box plot representation of the kidney fat indole concentration in lambs that have grazed lucerne for 0, 21, 42 or 63 days before slaughter (n = 55, 55, 56 and 52 lambs, respectively). The upper edge of the box indicates the 75th
percentile of the data-set, the bold line in the box indicates the median value,
and the lower edge indicates the 25th percentile of the data-set. The black
circles represent the outliers. *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P <
0.0001.

730

Figure 5 Distribution of lambs fattened for at least two months on alfalfa pastures without any supplementation (solid symbols) and lambs fed indoors a concentrate-based diet (open symbols), stratified into classes of kidney fat skatole concentration. The data (n = 93 and 94 for lambs fattened for at least two months on alfalfa pastures and lambs fed indoors a concentrate-based diet, respectively) come from four experiments (this study, plus Rivaroli et al., 2019, Devincenzi et al., 2019 and Eiras et al., 2022) conducted during 6 consecutive years.

739