Patterns of plasma sexual steroids in Duroc and Pietrain crossbred male pigs in organic farming and prediction of boar taint risk according to slaughter weight.

Animal 19 (2025) 101673 Contents lists available at ScienceDirect Animal The international journal of animal biosci ences Patterns of plasma sexual steroids in Duroc and Pietrain crossbred male pigs in organic farming and prediction of boar taint risk according to slaughter weight A. Prunier, PEGASE, INRAE, Institut Agro, 35590 Saint-Gilles, France B. Lebret ⁎ a r t i c l e i n f o Article history: Received 28 April 2025 Revised 23 September 2025 Accepted 23 September 2025 Available online 29 September 2025 Keywords: 17b–oestradiol Androstenone Boar Skatole Testosterone a b s t r a c t Boar taint risk in pork highly depends on fat tissue concentrations of androstenone and skatole, and indole to a lower extent. During pubertal development, androstenone is increasingly produced by testes and accumulates in fat tissues. Skatole and indole are produced and absorbed along in the colon and also accumulate in fat tissues. Skatole and indole are catabolised in the liver, but sexual steroids (including androstenone) inhibit their liver degradation. Therefore, slaughtering pigs before the pubertal peak of steroid production should reduce boar taint risk and thus make it possible to raise male pigs without inflicting detrimental castration. In the present experiment, non-castrated male pigs from two genotypes differing in their propensity to accumulate androstenone and skatole in backfat were compared: Large White × Duroc (D; n = 47) and Large White × Pietrain crossbreds (P; n = 34). Blood samples were drawn from the jugular vein two times during growth (at around 85 kg BW and 133 days of age, and around 105 kg BW and 153 days of age) and the day before slaughter (around 125 kg BW and 174 days of age). Plasma testosterone and 17b-oestradiol, whose concentrations increase during sexual development, were measured in all samples and correlated with backfat concentrations of androstenone, skatole and indole measured at slaughter. Plasma concentrations of both hormones increased (P < 0.05) from the first to the second, and from the second to the third sampling stage (P < 0.05). Plasma testosterone concentration did not differ between genotypes (P > 0.1) whereas plasma 17b-oestradiol was more than twice higher in D than in P pigs (P < 0.001) regardless of the age. Plasma 17b-oestradiol concentration measured the day before slaughter was highly correlated with backfat androstenone (r = 0.91, P < 0.001). Estimating backfat androstenone concentration from that of plasma 17b-oestradiol allowed to predict that reducing slaughter BW from more than 125 kg to less than 110 kg would reduce the percentage of carcasses with androstenone concentration above 1.7 l g/g liquid fat (severe threshold limit for boar taint) from 42 to 29% in D pigs and from 10 to 0% in P pigs, and with androstenone concentration above 3 l g/g (less severe limit for boar taint) from 19 to 7% in D pigs, whereas none of the P pigs would be above this limit regardless of the age. In conclusion, pig genotype and reducing slaughter BW (and age) are strong levers to avoid boar taint. © 2025 The Authors. Published by Elsevier B.V. on behalf of The animal Consortium. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Implications Avoiding surgical castration improves the welfare of male pigs but there is a risk of boar taint. This unpleasant odour and flavour are mainly due to androstenone and skatole that accumulate in fat along pubertal development. The plasma concentration of 17boestradiol is a good predictor of backfat androstenone. Thanks to blood samples at different ages/BWs, we showed that reducing age/BW at slaughter should be effective in reducing the boar taint risk related to androstenone in Duroc crossbred male pigs. In parallel, skatole should be controlled by husbandry practices. Both molecule s are less present in backfat of Pietrain crossbreds. ⁎ Corresponding author. E-mail addresses: [email protected] (A. Prunier), benedicte.lebret@inrae. fr (B. Lebret). https://doi.org/10.1016/j.animal.2025.101673 1751-7311/© 2025 The Authors. Published by Elsevier B.V. on behalf of The animal Consortium. This is an open access article under the CC BY licens e (http://creativecommons.org/licenses/by/4.0/). Introduction Surgical castration of male piglets without pain treatment is highly detrimental for animal welfare (Prunier et al., 2020). Techniques of anaesthesia and analgesia available for male piglet castration only partially relieve pain (Prunier et al., 2020), and animals lose their physical integrity, that can be considered non- ethical. Therefore, a better solution, from the animal welfare point of view, would be avoiding castration and raising non-castrated male pigs, even if welfare problems related to deleterious behaviours (mounting and aggressive behaviours) may be encountered (von Borell et al., 2020). Avoiding castration of male pigs also has the advantage of improving the efficiency of lean meat deposition (better feed conversion, greater protein retention) , which reduces the environmental impacts of pork production and increases the lean meat content of carcasses and therefore their commercial value (de Roest et al., 2009; Batorek et al., 2012a). However, the meat of non-castrated male pigs can present an unpleasant odour and flavour, known as boar taint (Lundström et al., 2009). It is mainly due to androstenone and skatole that are stored in adipose tissue since they are lipophilic molecules. Androstenone is a testicular steroid. Its synthesis and accumulation in adipose tissue increase during pubertal development. This period is marked by an increase in the synthesis and release in the general circulation of numerous testicular steroids such as androstenone, 17boestradiol, oestrone, testosterone and DHEA, and of their sulphated forms (Schwarzenberger et al., 1993; Sinclair et al., 2001b; Zamaratskaia et al., 2004). This increase starts between 3 and 5 months of age and plasma concentrations of testicular steroids peak around 7–8 months of age. Pigs raised for meat production are usually slaughtered around 5–6 months of age so that androstenone may have time to accumulate in fat (Zamaratskaia and Squires, 2009). Therefore, androstenone fat content depends on the stage of pubertal development reached at the slaughter stage and hence on pigs’ age/BW at slaughter as well as on environmental factors that may influence pubertal development (Parois et al., 2018). It is also largely under genetic influence with large differences among some breeds and a high heritability within breeds (Zamaratskaia and Squires, 2009; Parois et al., 2018). For example, fat androstenone has been shown to be higher in Duroc than in Landrace boars (Oskam et al., 2010). Finally, whatever the biological reason, high differences in fat androstenone concentr ation exist between pigs at a given age or BW (Zamaratskaia and Squires, 2009). Skatole is metabolised from tryptophan by gut bacteria and its content in adipose tissue is highly depende nt on health status, pig environmental or husbandry factors, especially stress before slaughter (Wesoly et al., 2015), diet composition (Wesoly and Weiler, 2012; Aluwé et al., 2017; Seoni et al., 2021), pen dirtiness and lack of ventilatio n (Hansen et al., 1994; Thomsen et al., 2015; Parois et al., 2017b). In addition, testicular steroids play an important role in the control of skatole accumulation since they reduce the liver catabolism of skatole, favouring its transfer in the general circulation and finally its accumulation in the adipose tissue (Zamaratskaia and Squires, 2009). A. Prunier and B. Lebret Animal 19 (2025) 101673 In order to reduce the risk of tainted meat, different levers can be activated at farm level: (a) using a pig genotype with slow sexual development allowing to slaughter the male pigs before accumulation of androstenone, (b) using a genotype with a low potential for androstenone accumulation even when sexually mature, (c) slaughter animals at a sufficiently early age/BW to precede androstenone accumulation. The strategy applied in pig farming and aiming at reducing boar taint can be focused on androstenone since its concentration may influence that of skatole due to their interrelated metabolism mentioned above and since levers related to pig feeding and, pen cleanliness and ventilation can be easily activated by farmers to reduce the risk for boar taint due to skatole. In the present experiment, we explored the lever of reducing age/BW at slaughter in two different genotypes of non-castrated male pigs, Large White × Pietrain and Large White × Duroc crosses, which may have different backfat androstenone concentrations (Oskam et al., 2010; Mathur et al., 2013). Our work is based on the animal experiment described in detail by Lebret et al. (2024), aiming at evaluating the effect of male pigs’ genotype: Large White × Duroc (D) vs Large White × Pietrain (P) on welfare indicators, carcass and pork quality traits in organic farming. We demonstrated that welfare indicators (skin scratches, tail lesions) showed less problems in D than P crossbreds, even though the occurrence of these problems was overall low. Growth rate and final BW (average of 127 kg) did not differ between genotypes, but D pigs had lower carcass lean meat content (Table S1) and higher backfat concentrations of androstenone, and of skatole to a lesser extent, than P pigs, suggesting a higher risk of rejection by consumers due to boar taint. In the present study, on the same D and P animals, we measured plasma concentrations of testosterone and 17b−oestradiol, two key testicular steroids, at three different physiological stages: early and mid-fattening periods and the day before slaughter, in order to predict backfat androstenone of male pigs at different stages of growth in both genotypes. Based on the fact that plasma testosterone and even more plasma 17b-oestradiol concentrations are highly linked to backfat androstenone concentration (Parois et al., 2015), we hypothesised that plasma concentration of these molecules could predict backfat androstenone and, consequently, boar taint risk. 2 Material and methods Animals and experimental design The pigs were raised at the INRAE Porganic (certified organic) experimental farm (INRAE GenESI, 86 480 Rouillé, France; https://doi.org/10.15454/1. 5572415481185847E12). The experimental protocol was approved by the local ethical committee and obtained the governmental authorisation (see Ethics approval section below). The experiment was carried out on a total of 81 non-castrated male pigs born from Large White sows (INRAE Porganic herd) inseminated with semen from either Duroc (D; Nucléus, 35 650 Le Rheu, France) or Pietrain NN boars (P; Nucléus, non-carriers of the halothane mutation at the RYR1 gene and chosen for their low risk for boar taint). The experimental protocol and housing environment were detailed in (Lebret et al., 2024). Briefly, pigs were produced in two successive batches (6 weeks apart between the two batches), including 30 D and 22 P pigs for the first batch, and 17 D and 12 P pigs for the second batch. Experimental pigs were selected on the basis of their BW at 70 days (average and SD of BW were balanced between genotypes) and raised by genotype in a collective pen on deep straw bedding (1.3 m 2 /pig) with fresh straw added weekly, and with free access to a covered outdoor area on a concrete floor (1.0 m 2 /pig). All animals were fed ad libitum the same organic growing (16.01% CP, 9.44 MJ NE/kg, from 10 until 16 weeks of age) and finishing diets (14.07% CP, 9.33 MJ NE/kg, from 16 weeks of age until slaughter), as detailed in Lebret et al. (2024). The diets were formulated in order to fulfil animal nutritional requirements and were offered as pellets. Pigs had permanent access to grassland hay distributed in a rack in each pen and had free access to water. Blood sampling and determination of plasma steroid concentratio ns Pigs were weighed individually at 2-week intervals. Three blood samples were scheduled per pig at around 85 (S1), 105 (S2) and 125 kg (S3) BW, with a minimum of two weeks between two successive blood samples, the last blood sample being taken the day before slaughter . In practice, the mean BW was 83 kg (n = 77 pigs), 103 kg (n = 78 pigs) and 127 BW (n = 80 pigs) for the 1st, 2nd and 3rd blood samples, respectively, and did not differ between genotypes (Table 1). Blood samples were drawn from the jugular vein. The stress associated with blood sampling was limited by using −−−−−− −−−−−− −−−−−− highly experienced technicians and by imposing a maximum duration of 2 min for the whole procedure, including catching the animal and applying the lasso for immobilisation. Blood was collected in tubes containing heparin (10 ml) and centrifuged (2 500 × g for 10 min at +4 °C), and the plasma was stored at −20 °C until analysis. The three blood samples were collected in all except three D pigs (two pigs with the first sample missing, one pig with the first and second samples missing). Plasma 17b-oestradiol and testosterone concentrations were measured using ELISA kits (ST AIAPack hsE2 and ST AIA-Pack Testosterone; Tosoh Corporation, Tokyo, Japan) developed for an automatic analyser (AIA 1800; Tosoh Corporation) as described by Parois et al. (2017a). As described by the manufacturer for the 17b−oestradiol assay, cross−reactivity with the sulphate form was low (0.03%). The detection limits were respectively 3 pg/ml of plasma for 17boestradiol and 0.01 ng/ml for testosterone. For 17b-oestradiol, inter-assay CVs were 4.5% and 3.4%, and intra-assay CVs were 3.2 and 1.4% at 67 and 764 pg/ml, respectively. For testosterone, inter-assay CVs were 6.0 and 2.5% at 0.09 and 1.45 ng/ml, respectively, and intra-assay CVs were 5.3and 1.8% at 0.05 and 1.50 ng/ ml, respectively. A. Prunier and B. Lebret Animal 19 (2025) 101673 Table 1 Influence of genotype and stage at blood sampling on age, BW, plasma testosterone and 17 b-oestradiol concentrations and backfat concentrations of androstenone, skatole and indole in non-castrated male pigs. First stage of blood sampling Second stage of blood sampling Third stage of blood sampling Duroc crossbred Pietrain crossbreds P-value 1 77 pigs 78 pigs 80 pigs 43 to 46 pigs 34 pigs Item lsmean SE lsmean SE lsmean SE lsmean SE lsmean SE Stage Genotype Batch Age at sampling, days 133 a 1.1 153 b 1.1 174 c 1.1 154 1.4 153 1.7 <0.001 0.59 0.93 BW at sampling, kg 83 a 1.2 103 b 1.2 127 c 1.2 104 1.5 107 1.7 <0.001 0.73 0.24 Plasma testosterone, ng/ml 1.3 a 0.12 2.0 b 0.19 2.9 c 0.27 2.1 0.19 1.7 0.18 <0.001 0.14 <0.001 Plasma 17b−oestradiol, pg/ml 14.0 a 1.64 20.1 b 1.96 26.4 c 2.24 28.8 2.79 12.6 8.68 <0.001 <0.001 0.002 1.32 (0.29, 3.16) 0.60 (0.17,1.40) Backfat androstenone, l g/g liquid fat 2,3 −0.002 0.004 0.02 (0.02,0.14) 0.02 (0.02,0.03) Backfat skatole, l g/g liquid fat 2,3 −0.05 0.75 0.02 (0.02, 0.06) 0.02 (0.02, 0.02) Backfat indole, l g/g liquid fat 2,3 −0.03 0.08 Abbreviation: lsmean = least-square mean. a, b, c lsmeans followed by different superscripts differed significantly (P < 0.01). 1 For age, BW, testosterone and 17b−oestradiol, P-values of the fixed effects of stage at blood sampling, genotype and batch, obtained from analyses of variance applied to data (the interaction Genotype × Stage was removed in all models since it was never significant with P > 0.1). For androstenone, skatole and indole, the batch and genotype effects were analysed by a non-parametric test as indicated in Lebret et al. (2024). 2 Backfat was sampled on carcasses of pigs slaughtered the day after the 3rd blood sampling. 3 Median, and first and last decile values between brackets (data from Lebret et al., 2024). Slaughter In order to limit variability in BW at slaughter, pigs were slaughtered in two different series for each batch, separated by two (batch 1) or three weeks (batch 2). Slaughter was performed in a commercial abattoir (Cooperl, 79 800 Sainte-Eanne) at a mean BW of 125 kg (range 96–163 kg) and a mean age of 176 days (range 165–191 days). The heaviest half of the pigs from each genotype were slaughtered in the first series, and the other half remained in their original pen until the second series of slaughter. The day before slaughter, pigs were sampled for blood, individually weighed, and grouped within genotype in two different pens from a roofed platform. No feed was available but pigs had free access to water. During the following night, all pigs were loaded into the same truck and transported without non-experimental pigs to the slaughterhouse (maximum duration of 30 min). In the slaughterhouse, they were all placed in a single pen with free water available (still without mixing with non-experimental pigs) for 50 min to 2 h. Pigs were slaughtered in the early morning using electrical stunning at high voltage, followed by exsanguination. 3 Backfat sampling and biochemical analyses of boar taint components Twenty-four hours after slaughter, a piece of backfat (whole thickness, i.e. including both fat layers) was collected at the neck level, vacuum packaged and stored at −20 °C until analysis of androstenone, skatole and indole concentrations by HPLC, as previously described (Batorek et al., 2012b; Van Baelen et al., 2024). Backfat sample was missing for one Duroc pig, and this pig was removed from the statistical analyses. Briefly, backfat samples were melted in a microwave and centrifuged and the liquid phase was removed and stored at −20° C for 2 weeks. All samples were analysed by HPLC (Agilent Technologies, 1200 series, Santa Clara, CA, USA) with a C18 column (waters sunfire, 3.5 l m, 4.6 × 75 mm, USA). Flow rate was 1.2 ml/min for skatole and 1.0 ml/min for androstenone. The detection limits were 0.08 l g/g of liquid fat for androstenone and 0.02 l g/g of liquid fat for skatole and indole, and these values were assigned to pigs with concentrations below those limits. Statistical a nalyses Statistical analyses were performed with the R software (version 4.3.3, The R Core Team, 2024). Age, BW, plasma testosterone and plasma 17b-oestradiol were analysed using an analysis of variance (ANOVA) including the genotype (n = 2), the batch (n = 2) and the sampling stage (n = 3 per pig except for two pigs with one sample and one pig with two samples), the interaction between sampling stage and genotype as fixed effects and the animal (n = 80) as a random effect (lme procedure from the nlme package and Anova procedure of the car package). When the interaction was not significant, it was removed from the model. The normality of the distribution of the residuals was checked for each variable, and least square means as well as the SEs of the lsmeans were calculated using the lsmeans procedure of the emmeans package. A square-root transformation was applied to plasma 17b-oestradiol and to plasma testosterone to reach a normal distribution of the residuals. For these transformed data, least square means and SE were back calculated specifying ‘‘type = response” in the lsmeans procedure. Spearman correlations between backfat androstenone, skatole and indole and plasma 17b-oestradiol and testosterone concentrations were calculated using raw data. A. Prunier and B. Lebret Animal 19 (2025) 101673 The influence of BW on the risk of boar taint was assessed based on the prediction of backfat androstenone concentration from plasma 17b-oestradiol concentration. First, we checked that the relationship between fat androstenone and plasma 17boestradiol at S3 was not influenced by the genotype by including the genotype, plasma 17b-oestradiol at S3 and their interaction as explanatory factors for fat androstenone using the lm procedure. The analysis was performed on the whole set of 80 pigs after square root transformation of both concentrations to normalise the data. Second, the parameters of the linear regression between plasma 17b-oestradiol at S3 and fat androstenone were calculated. Third, fat androstenone was estimated from plasma 17boestradiol. Fourth, individual estimations of fat androstenone concentrations were allocated to three classes of pigs according to their BW determined at blood sampling: 87 to 110 kg BW (n = 9 P plus 14 D pigs), 110 to 125 kg BW (n = 15 P plus 18 D pigs), and 125 to 163 kg BW (n = 21 P + 26 D pigs). In each class, a given pig was present only once. A total of 23 pigs were present in two different classes. Results and discussion Age, BW, plasma testosterone and 17 b-oestradiol at sampling As shown in Table 1, age and BW increased from the first to the second, and from the second to the third blood sampling stage (P < 0.001). Age and BW did not differ according to genotype or batch (P > 0.1). Plasma 17b-oestradiol and testosterone also increased between the first and the second, and between the second and the third blood sampling stages ( P < 0.001). This is due to the pig pubertal development in agreement with previous studies (Zamaratskaia et al., 2004; Prunier et al., 2013). Fig. 1. Influence of genotype (Large White × Duroc: D crossbreds vs Large White × Pietrain: P crossbreds) in non-castrated male pigs on plasma concentrations (adjusted means ± SE) of 17b-oestradiol and testosterone measured at three stages during growth (Stage 1: around 133 days of age 83 kg of BW, Stage 2: around 153 days of age and 103 kg of BW, Stage 3: around 74 days of age and 127 kg of BW). Data were analysed by ANOVA with genotype and stage of sampling as main effects and pig as a random effect (the interaction Genotype × Stage was removed in all models since it was never significant with P > 0.1). Data are represented as boxplots after square root transformation, as the statistical analyses were performed after square root transformation. 4 Plasma testosterone was overall higher in the first (2.9 ± 0.24 ng/ml, adjusted mean ± SE) than in the second batch (1.26 ± 0.14 ng/ml, P < 0.001), and this was also the case for plasma 17boestradiol (first batch: 25.9 ± 2.46 pg/ml, second batch: 14.6 ± 2. 46 pg/ml, P < 0.002). This effect could be explained by the influence of the season and especially of the light duration. Indeed, pigs from the first batch were sampled between mid-December and early February under shorter days than those from the second batch that were sampled between late January and late March. Even though domestic pigs are not purely seasonal breeders, they can be influenced by the season, with a tendency for plasma testosterone to increase in male pigs subjected to a reduction in daily light duration (Weiler et al., 1996; Andersson et al., 1998; Prunier et al., 2013). Alternatively, the difference between batches may be explained by individual differences (Zamaratskaia and Squires, 2009; Parois et al., 2018). Indeed, male pigs from the two batches were born from different sows and different boars (only two out of five Duroc boars were similar between the two batches). Differences in hereditary propensity to deposit fat androstenone between sires could therefore also contribute to explain the batch effect on fat androstenone concentration. Genotype had a strong influence on plasma 17b-oestradiol with higher values in D than P pigs ( P < 0.001), but not on plasma testosterone concentration (P > 0.10) (Table 1). The interaction between sampling stage and genotype was not significant for the two hormones (P > 0.1). The difference in plasma 17b-oestradiol between genotypes was already present at the first sampling stage, i.e. around 133 days of age and 83 kg of BW (Table 1; Fig. 1). Similar plasma testosterone but higher plasma 17b-oestradiol concentrations in Duroc crossbred than Pietrain crossbred male pigs were also observed at an earlier stage, i.e. about 50 kg BW and 100 days of age by Heyrman et al. (2019). At about 80 kg BW and 120 days of age, close to our first sampling stage, these authors observed again higher plasma 17b-oestradiol in Duroc than Pietrain crossbred male pigs but the difference was no more significant. In parallel to plasma 17b-oestradiol, backfat androstenone concentration in our study was higher in the Duroc than Pietrain crossbred pigs (Table 1). This difference between the two genotypes which is in agreement with the literature (Werner et al., 2020) was already discussed in Lebret et al. (2024). A. Prunier and B. Lebret Animal 19 (2025) 101673 Across genotypes, fat androstenone concentration was very highly correlated with plasma 17b-oestradiol concentration and significantly correlated with plasma testosterone concentration (Table 2, Fig. 2). Fat skatole was significantly correlated only with plasma 17b-oestradiol concentration but the correlation coefficient was much lower than for androstenone (Table 2, Fig. 2). Fat indole was significantly correlated with both plasma steroid concentrations and fat androstenone, but the values of correlations were moderate (Table 2). Much higher correlation between fat androstenone and plasma 17b-oestradiol than with plasma testosterone was already observed in pure Pietrain or Pietrain crossbred male pigs (Parois et al., 2015; Prunier et al., 2016). The high correlation between fat androstenone and plasma 17b-oestradiol concentration suggests that this hormone could be a good estimator of the androstenone content in fat tissue, as previously found (Parois et al., 2015) but not of fat skatole. Measuring oestrone sulphate in plasma instead of 17b-oestradiol, previous studies have also shown closer relationships between fat androstenone and oestrogen than with testosterone, and closer relationships between plasma steroids and fat androstenone than with fat skatole (Squires and Lundstrom, 1997; Babol et al., 1999; Sinclair et al., 2001a; Chen et al., 2006). Prediction of boar taint risk according to pig genotype and slaughter BW The statistical model explaining fat androstenone by plasma 17b-oestradiol at S3, genotype and their interaction indicated non-significant effects for the genotype (P = 0.96) and the interaction (P = 0.45), contrarily to 17b−oestradiol concentration (P < 0.0001). Comparison with a statistical model including only plasma 17 b−oestradiol as explanatory variable indicated that the two models were not statistically different (P = 0.19). Therefore, it can be concluded that the relationship between plasma 17b −oestradiol and fat androstenone is similar in both genotypes, as illustrated in Fig. 3, and that the same coefficients of linear regression can be used in both genotypes to predict fat androstenone from plasma 17b−oestradiol concentration. Since the relationship was established in pigs submitted to a blood sample between 164 and 190 days of age and since it was not possible to demonstrate that it can be applied to younger pigs, the estimation of androstenone was limited to blood samples collected at a minimum of 164 days of age and hence to only 103 measures (80 at S3 and 23 at S2). Thereafter, the percentages of pigs above 1.7 l g androstenone/g of liquid fat or above 3.0 l g androstenone/g of liqTable 2 Spearman’s correlation coefficients and their P −values in brackets between concentrations of plasma hormones measured the day before slaughter, concentrations of boar taint compounds in backfat collected the day after slaughter, age and BW the day before slaughter. Data were calculat ed on the whole data set (n = 80 non-castrated male pigs). Plasma testosterone Fat androstenone Fat skatole Fat indole Age at sampling BW at sampling Plasma 17b-oestradiol 0.63 (<0.001) 0.91 (<0.001) 0.28 (0.012) 0.41 (<0.001) −0.02 (<0.85) 0.11 ( <0.34) Plasma testosterone 1.00 0.58 (<0.001) 0.03 (0.765) 0.36 (<0.001) −0.09 (<0.42) −0.08 (<0.48) Fat androstenone 1.00 0.22 (0.049) 0.40 (<0.001) −0.08 (<0.48) 0.11 (<0.35) Fat skatole 1.00 0.35 (<0.002) −0.07 (<0.54) 0.09 (<0.46) Fat indole 1.00 −0.05 (<0.65) 0.28 ( <0.02) Age at sampling 1.00 −0.11 (<0.32) 5 Fig. 2. Relationship between plasma 17b-oestradiol concentration (A) or plasma testosterone (B) and backfat androstenone concentration as well as between plasma 17b-oestradiol concentration and backfat skatole concentration (C) in Large White × Duroc (D crossbreds) or Large White × Pietrain (P crossbreds) noncastrated male pigs. Blood was collected at an average of 174 days of age and 127 kg of BW. Backfat was collected on carcasses from pigs slaughtered the day after blood collection. (Bonneau and Chevillon, 2012). However, it should be mentioned that this limit was defined for pork with low fat concentratio n of skatole and hence may not be applied when this condition is not satisfied. A. Prunier and B. Lebret Animal 19 (2025) 101673 Fig. 3. Relationship between plasma 17b−oestradiol concentration (square-root transformed) and backfat androstenone concentration in Large White × Duroc (D crossbreds) or Large White × Pietrain (P crossbreds). Animals were classified according to genotype (A) or BW (B). Blood was collected at an average of 174 days of age and 127 kg of BW. Backfat was collected on carcasses from pigs slaughtered the day after blood collection. 0 10 20 30 40 50 [87-110] ]110-125] ]125-163] %,sessacracfoegatnecreP Estimated androstenone > 1.7 µg/g liquid fat Dcrossbreds P crossbreds 0 10 20 30 40 50 [87-110] ]110-125] ]125-163] %,sess acracfoegatnecreP Estimated androstenone > 3 .0 µg/g liquid fat Fig. 4. Percentages of carcasses with estimated backfat androstenone concentration above 1.7 l g/g liquid fat (top) or 3.0 l g/g liquid fat (bottom) in Large White × Duroc (D crossbreds) or Large White × Pietrain (P crossbreds) non-castrated male pigs, according to their BW (87 to 110 kg: n = 14 D and 9 P, 110 to 125 kg: n = 18 D and 15 P, 125 to 163 kg, n = 26 D and 21 P). These two concentrations correspond to two thresholds of backfat androstenone concentration for consumer rejection of pork due to boar taint, proposed in the literature (Lundström et al., 2009; Bonneau and Chevillon, 2012). The absence of bar indicates that the percentage is zero. uid fat were calculated within genotype for three classes of BW (Fig. 4). These two limits for androstenone concentration were retained since a unique threshold level of androstenone for consumers’ rejection of pork due to boar taint is still under debate (Bee et al., 2015). The lower limit (1.7 l g/g of liquid fat) corresponds to the limit of 1 ppm often cited in the literature (Lundström et al., 2009) after conversion of the unit from ppm in backfat to l g/g liquid fat as described by (Pauly et al., 2008). Indeed, our androstenone concentrations were determined in liquid fat. The upper limit (3.0 l g/g of liquid fat) is derived from the study of Bonneau and Chevillon (2012) on the consumers’ evaluation of odour and taste of pork originating from carcasses measured for backfat androstenone and skatole concentrations with exactly the same techniqu e as used in the present study 6 In Pietrain crossbred male pigs, we predicted that the percentage of pigs above both threshold limits would be very low until 125 kg BW and close to 10% above 125 kg BW but only when considering the more severe (i.e. lower backfat concentration) limit (Fig. 4). For the Duroc crossbred male pigs, we observed an increase in the boar taint risk, from nearly 10% below 110 kg BW up to nearly 20% above 125 kg BW with the less severe (i.e., higher backfat concentration) limit, and from nearly 30% to more than 40% with the more severe limit (Fig. 4). It is worth predicting the concentration of androstenone in fat based on the plasma concentration of 17b-oestradiol in pigs less than 164 days old in order to assess the influence on boar taint risk of a marked reduction in slaughter age/live weight. To do this, the equation obtained in the present study would need to be validated by measuring the concentration of both molecules in pigs aged between 140 and 164 days. Two options are possible: develop an analytical technique using a very small amount of fat in order to perform a painless biopsy of very small diameter in live pigs, or slaughter the animals at the desired ages in order to collect the necessary quantity of fat from the carcass. We did not evaluate the risk for boar taint due to skatole and indole since the correlations with plasma 17b-oestradiol or plasma testosterone were quite low (0.03 ≤r≤0.41, Table 2). In addition, based on the literature, one can assume that the risk for boar taint due to skatole and indole would most likely be independent of the slaughter BW (Aluwé et al., 2011; Prunier et al., 2013) and can be largely controlled by nutritional and/or environmental conditions (Hansen et al., 1994; Wesoly and Weiler, 2012; Thomsen et al., 2015; Parois et al., 2018). Conclusion Our data clearly indicate that the pubertal development of the male pigs, as indicated by the concentrations in plasma sex steroids, gradually continues from about 83 kg BW/133 days of age to 103 kg BW/153 days and to 127 kg BW/174 days. We also showed that there is a marked difference between Duroc and Pietrain crossbreds for plasma 17b-oestradiol concentration, with higher values for the D pigs, whatever the sampling stage. Indeed, plasma 17b-oestradiol was already high in Duroc crossbred pigs at the first sampling stage, with a value similar to that measured in Pietrain crossbreds at the third sampling stage. Contrarily, plasma testosterone was similar in both breeds. Plasma 17b-oestradiol appears as a good estimator of backfat androstenone concentration and therefore of boar taint risk due to this molecule. Our results indicate an increasing risk for boar taint due to androstenone with increasing slaughter BW from less than 110 to more than 125 kg BW, especially in Duroc crossbred male pigs. A. Prunier and B. Lebret Animal 19 (2025) 101673 Supplementary material Supplementary Material for this article (https://doi.org/10. 1016/j.animal.2025.101673) can be found at the foot of the online page, in the Appendix section. Ethics approval All procedures contributing to this work complied with the French legislation on animal experimentation and were approved by the local Committee for Consideration of Ethics in Animal Experimentation. On these bases, the present animal experimentation was authorised by the French Ministry of Higher Education, Research and Innovation (Authorisation: APAFIS#30357202103041121621 v4 delivered on July 2, 2021). Data and model availability statement Data set and list of variables have been deposited in the national repository Recherche Data Gouv: https://entrepot.recherche.data.- gouv.fr/dataset.xhtml?persistentId=https://doi.org/10.57745/ SCSMVX. Information can be made available from the authors upon request. Declaration of generative AI and AI-assisted technologies in the writing process During the preparation of this work the author(s) did not use any AI and AI-assisted technologie s. Author ORCIDs A. Prunier: https://orcid.org/0000-0003-3070-6613. B. Lebret: https://orcid.org/0000-0001-5435-0389. CRediT authorship contribution statement A. Prunier: Writing – review & editing, Writing – original draft, Visualisation, Validation, Methodology, Investigation, Formal analysis, Conceptualisation. B. Lebret: Writing – review & editing, Writing – original draft, Visualisation, Validation, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Conceptualisation. Declaration of interest None. Acknowledgements The authors gratefully acknowledge the staff of INRAE experimental farm (D. Grivault, T. Terrasson, S. Moreau; GenESI, Porganic, 86480 Rouillé, France) and laboratories (R. Comte, L. Le Normand; PEGASE, 35590 Saint-Gilles, France) for their excellent assistance. They also thank S. Ferchaud for coordinating the work in the experimental farm. 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