The effects of short-chain fatty acids on androstenone metabolism in porcine hepatocytes

Abstract

The objective of this study was to assess the effects of short-chain fatty acids (SCFAs) on androstenone metabolism and associated gene expression in hepatocytes isolated from intact (boars) and castrated (barrows) male pigs. Isolated porcine hepatocytes were treated with the SCFAs acetate, propionate, and butyrate, both individually and in combination, and then incubated with androstenone. Androstenone metabolism and gene expression were subsequently assessed. Androstenone metabolism was significantly increased by propionate (P < 0.01) and all SCFA combination treatments (P < 0.0001) in hepatocytes from both boars and barrows, and by butyrate in hepatocytes from boars (P = 0.009). Several key genes were upregulated in hepatocytes from barrows; this included AKR1C1 following all SCFA combination treatments (P ≤ 0.01), UGT2A1 and SULT2A1 following treatment with the combination of propionate and butyrate (P < 0.05) and the combination of all three SCFAs (P < 0.05), and SULT1E1 following treatment with the combination of acetate and butyrate (P = 0.005). SCFA treatments did not alter gene expression in hepatocytes from boars; however, plasma levels of estrone-1-sulfate, an abundant testicular steroid in boars, were positively correlated (P < 0.05) with UGT1A1, SULT1E1, and UGT2A1 expression in response to treatment with different SCFAs. These results suggest that SCFA-induced effects on hepatic androstenone metabolism and gene expression depend on the specific SCFA profile and are influenced by testicular steroid hormones.

Short-chain fatty acids, acetate, propionate, and butyrate, which are produced through microbial metabolism of fermentable carbohydrates, increase androstenone metabolism in isolated hepatocytes from boars and barrows. These results provide a potential mechanism for the reductions in plasma androstenone levels that have been reported in boars after feeding fermentable carbohydrates.

Introduction

Boar taint is an undesirable odor and flavor in pork caused by the accumulation of androstenone and skatole in the adipose tissue of entire male pigs. Although castration of male piglets is effective for preventing boar taint, the negative effects of this procedure on animal welfare and production efficiency create a need for alternative control methods. Dietary supplementation with fermentable carbohydrates, such as raw potato starch and chicory root, has been shown to reduce plasma androstenone levels and skatole levels in both the plasma and fat of boars, suggesting this could be an effective alternative strategy to castration (Hansen et al. 2006; Chen et al. 2007). However, the physiological mechanisms responsible for these effects are not well understood, and the ability of fermentable carbohydrate treatments to reduce boar taint has been inconsistent (Byrne et al. 2008; Zammerini et al. 2012).

Short-chain fatty acids (SCFAs) are gut-derived metabolites produced from the breakdown of fermentable carbohydrates by anaerobic microbes, with acetate, propionate, and butyrate accounting for up to 95% of the total SCFA production (Mortensen and Clausen 1996; Nicholson et al. 2012). In humans and mice, SCFAs can influence hepatic gene expression by acting as histone deacetylase inhibitors (HDACi) (Waldecker et al. 2008), by transactivating peroxisome proliferator receptors (Alex et al. 2013), and acting as ligands for G-protein-coupled receptors (GPRs) (Caengprasath et al. 2020). These effects may also influence the expression of genes responsible for the hepatic metabolism of androstenone, which is an important two-phase process affecting boar taint development (Sinclair et al. 2005). During Phase I metabolism, androstenone is reduced by aldo-keto reductase (AKR) enzymes, AKR1C1, and AKR1C4, to 3β-androstenol and 3α-androstenol, respectively (Gower 1972; Endo et al. 2022). In Phase II metabolism, androstenone and both androstenol metabolites can either be conjugated to a sulfate group by the sulfotransferase (SULT) enzyme SULT2A1 or to a glucuronide group by uridine 5′-diphospho-glucuronosyltransferases (UGTs) (Laderoute et al. 2019). The resulting sulfate and glucuronide metabolites can then be excreted, helping to reduce androstenone accumulation in the fat. Androstenone metabolism may also influence skatole accumulation in fat, as androstenone has been shown to inhibit the protein expression of cytochrome P450 2E1 (CYP2E1), which is a key enzyme responsible for hepatic skatole metabolism (Doran et al. 2002).

Testicular sex steroids have been shown to affect the expression of key genes involved in the Phase II metabolism of androstenone. For example, expression levels of UGT1A1, UGT1A6, UGT2A1, and UGT2B31 were significantly higher in boars with high serum androgen levels compared to both females and boars with low levels of serum androgens, while expression of SULT2A1 was lower in boars that had high levels of serum androgens (Moe et al. 2008; Kojima and Degawa 2014). The hepatic expression of UGT1A6, UGT1A1, UGT2B31, and UGT2A1 was also found to be positively correlated with plasma levels of estrone-1-sulfate (E₁S), which is one of the most abundant steroids produced by slaughter-weight boars and serves as an indicator of overall testicular steroid hormone levels (Schwarzenberger et al. 1993; Bone and Squires 2024). This suggests that the effects of SCFAs on hepatic androstenone metabolism may be influenced by testicular sex steroids.

Therefore, the objectives of this study were to assess the effects of SCFAs on androstenone metabolism and associated gene expression in isolated porcine hepatocytes and to determine whether SCFA-induced effects differ between hepatocytes isolated from boars and castrated male pigs (barrows) to investigate the influence of testicular steroids. We hypothesized that SCFA treatment would increase hepatic androstenone metabolism by altering the expression of key Phase I and II metabolic genes and that these effects would differ in hepatocytes from boars and barrows.

Materials and methods

Animals and sample collection

Animals were used in accordance with the guidelines set out by the University of Guelph Animal Care Policy and the Canadian Council of Animal Care (CCAC, 2009; Animal utilization protocol #4600). Boar (n = 7) and barrow (n = 6) three-way commercial crosses ([Yorkshire × Landrace] × Duroc) from Pig Improvement Company were used for this trial. Boars and barrows were slaughtered at 160.5 ± 3.5 d of age (mean ± SE) at body weights of approximately 140 to 160 kg. Liver lobes were collected from each animal at the time of slaughter for the isolation of hepatocytes described below. Blood was also collected from boars at slaughter and centrifuged at 1789 × g for 15 min at 4 °C to obtain plasma. Plasma E₁S levels were subsequently quantified by radioimmunoassay as described by Raeside et al. (1988).

Isolation of hepatocytes

Hepatocytes were isolated and cultured as previously described by Bone and Squires (2024). Briefly, the liver lobe was blanched for 10 min at 25 mL/min with Hank’s balanced salt solution (10×; without Ca²⁺, Mg²⁺, HCO³⁻, and phenol red; containing 10 mM HEPES and 1 mM ethylene glycol tetraacetic acid), and subsequently rinsed for 10 min with Hank’s balanced salt solution containing 10 mM HEPES. The lobe was then digested with Williams’ Medium E containing 10 mM HEPES and 0.71 mg/mL collagenase Type I for 30 min at a flow rate of 20 mL/min and dissected. Liberated hepatocytes were collected in attachment media (Williams’ Medium E containing 10 mM HEPES, 12.1 nM insulin from bovine pancreas, 10% fetal bovine serum, and 1% penicillin/streptomycin), filtered through a 255 µm nylon mesh, and centrifuged at 100 × g for 3 min. The resulting cell pellet was rinsed twice with fresh attachment media, and cell viability was assessed using a 0.04% trypan blue exclusion test. Hepatocytes were then plated in 24-well standard surface-treated polystyrene tissue culture plates (Fisher Scientific, Toronto, ON, Canada) at a density of 0.5 million cells/well in 0.5 mL of attachment media. The cells were placed in an incubator set to 37 °C, humidified to 95% air and 5% CO₂ for 4 h to allow hepatocytes to attach to the plate.

Treatment of hepatocytes

Acetic acid (acetate; ≥99.7% purity; Fisher Scientific, Toronto, ON, Canada), propionic acid (propionate; ≥99% purity; Fisher Scientific, Toronto, ON, Canada), and butyric acid (butyrate; ≥99% purity; Sigma, Oakville, ON, Canada) were used as SCFA treatments for hepatocytes. Baseline SCFA treatment concentrations (900 µM acetate, 300 µM propionate, and 160 µM butyrate) were selected to reflect predicted portal vein levels in pigs fed a sugar beet pulp‑based diet, which has been commonly evaluated as a dietary fermentable carbohydrate treatment for boar taint. These concentrations were estimated by applying published differences between ileal and portal vein SCFA concentrations measured in pigs fed a wheat‑based diet to reported ileal SCFA concentrations from pigs fed a sugar beet pulp-based diet (Knudsen et al. 2005; Zhao et al. 2019).

Initial attachment media was replaced with 0.5 mL of serum-free media (10 mM HEPES, 10 mM pyruvate, 12.1 nM insulin from bovine pancreas, 0.35 mM L-proline, and 1% penicillin/streptomycin; final pH 7.25) containing fivefold (5×) baseline concentrations of individual SCFAs (4.5 mM acetate, 1.5 mM propionate, and 0.8 mM butyrate), SCFA combinations, or serum-free media without SCFAs as a negative control. All treatments were applied in triplicate to hepatocyte cultures from each animal, and the cells were subsequently incubated for 21 h. Media was then replaced with serum-free media containing radiolabeled [³H]-androstenone (20 µM, 3.56 µCi/µmol, 0.1% ethanol, Moravek Biochemicals Inc., Brea, CA, USA), with incubations terminated at 1.5 h following androstenone treatment.

Analysis of androstenone metabolism and metabolite production

Culture media collected from each incubation was diluted 1:1 (v/v) with 100% acetonitrile, centrifuged at 8000 x g for 15 min, and filtered through a 0.2 µm nylon syringe filter. High-performance liquid chromatography was then performed to quantify androstenone metabolism and corresponding metabolite production by hepatocytes using a Luna 5 µm C18 HPLC column (250 × 4.60 mm; Phenomenex, Torrance, CA, USA). The HPLC profile, previously described by Laderoute et al. (2018), was as follows: an isocratic flow with acetonitrile and water (33:67, v/v) for 8 min, a linear gradient from 33% to 60% acetonitrile for the next 17 min, an isocratic flow with 100% acetonitrile for the next 5 min, and an isocratic flow with acetonitrile and water (33:67, v/v) for the remaining 10 min. A β-RAM model 2 radioisotope detector (IN/US Systems, Tampa, FL, USA) was used to monitor the elution of free radiolabeled androstenone and its respective metabolites. Free androstenone had a retention time of 34 min, while 16-androstene glucuronide metabolites were detected at 3 min (Laderoute et al. 2018; Bone and Squires 2024).

For each SCFA treatment and untreated control incubation, absolute androstenone metabolism was quantified from the disappearance of androstenone from the culture media and expressed as a percentage of the amount that was initially added. Androstenone metabolism was also expressed as a percentage relative to the amount quantified in untreated control incubations.

Gene expression analysis

Hepatocytes incubated for 21 h with or without SCFA treatments in the absence of androstenone were collected from culture plates into 350 µL of lysis buffer for gene expression analysis. Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Santa Clarita, CA, USA), treated with RNase-free DNase I (Fisher Scientific, Toronto, ON, Canada), and converted to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Fisher Scientific, Toronto, ON, Canada), according to the manufacturers’ instructions. The resulting cDNA was amplified by real-time PCR, as previously described by Bone and Squires (2021), to assess the expression of each gene of interest (Table 1). Reactions were performed in duplicate using the forward and reverse primer sequences listed in Table 2. Gene expression values were normalized to β‑actin, expressed relative to the untreated control, and standardized across biological replicates to reduce variability, as described by Willems et al. (2008).

Table 1.

Abbreviations, names, functions, and NCBI reference sequences (RefSeq ID) for genes of interest.

Gene abbreviation	Gene name	Function	RefSeq ID	Reference
AKR1C1	Aldo-keto reductase 1C1	Phase I androstenone metabolism	NM_001044618	Endo et al. (2022)
SULT1E1	Sulfotransferase 1E1	Phase II sulfoconjugation of estrogens	NM_213992.1	Mueller et al. (2015)
SULT2A1	Sulfotransferase 2A1	Phase II sulfoconjugation of androstenone	NM_001037150	Laderoute et al. (2018)
UGT1A1	UDP glucuronosyltransferase 1A1	Phase II glucuronidation	KJ922612.1	Jarrar and Lee (2021)
UGT1A6	UDP glucuronosyltransferase 1A6	Phase II glucuronidation	NM_001278750.1	Jarrar and Lee (2021)
UGT2A1	UDP glucuronosyltransferase 2A1	Phase II glucuronidation	XM_003356958.4	Moe et al. (2008)
UGT2B31	UDP glucuronosyltransferase 2B31	Phase II glucuronidation	NM_001244124.1	Jang et al. (2023)
β-Actin	Beta-Actin	Housekeeping gene	XM_003357928	Bunnell et al. (2011)

Table 2.

Forward and reverse primer sequences for genes of interest.

Gene	Forward primer sequence	Reverse primer sequence	Amplification efficiency, %	Product size, base pairs
AKR1C1	5′-GGAGGACTTTTTCCCAAAGG-3′	5′-TCCCTCGTTCTTGCACTTCT-3′	91.7	100
SULT1E1	5′-GCATCAGATGAGCTTGTGGA-3′	5′-AGTCTCCTGCAATCCCCTTT-3′	93.6	148
SULT2A1	5′-ACACGAGAAGCGCCGTAGAG-3′	5′-TGGACATGTTGTTTTCTTTCATGA-3′	96.9	120
UGT1A1	5′-ATAATTACCCGAGGCCCATC-3′	5′-CCCCAAAGAGAAAACCACAA-3′	99.0	140
UGT1A6	5′-TGCTTTGGGCAAAATACCTC-3′	5′-CTTTGGGTGACCAAGCAGAT-3′	100.6	124
UGT2A1	5′-TGCACGTTACTGAAAATGCAAG-3′	5′-TTGTAAAAGCCAGAGCACATCA-3′	91.1	122
UGT2B31	5′-TTTGAGACAATGGGGAAAGC-3′	5′-AGGTAGGGGTTTTGCAGGTT-3′	98.3	129
β-Actin	5′-CGTGGACATCAGGAAGGAC-3′	5′-TCTGCTGGAAGGTGGACAG-3′	101.8	206

Statistical analysis

Statistical analysis was conducted using SAS 9.4 (SAS Institute, Cary, NC, USA). For each analysis, P < 0.05 was considered statistically significant. The effects of SCFA treatments on androstenone metabolism or gene expression were evaluated using the PROC MIXED procedure with the following model:

$$Y_{ij}=\mu +A_i+B_j+{ϵ}_{ij}$$

where Y_ij represents androstenone metabolism or gene expression in hepatocytes isolated from either boars or barrows, µ is the overall mean, A_i is the fixed effect of SCFA treatments, B_j is the random effect of animal, and ϵ_ij is the experimental error. Treatment means were compared with the corresponding untreated control using a Dunnett’s test.

Student’s t-tests were performed to evaluate differences in SCFA-induced effects on androstenone metabolism or gene expression, as well as basal gene expression levels, between hepatocytes isolated from boars and barrows. Additionally, Pearson correlation coefficients were calculated to assess the relationship between plasma E₁S levels and the expression of key genes involved in androstenone metabolism in hepatocytes from boars. Androstenone metabolism (%) and basal gene expression (2^−ΔCt) data are presented as means ± SE, while gene expression data are presented as mean standardized fold change (2^−ΔΔCt) with 95% confidence intervals.

Results

Determination of SCFA dose and incubation time

We first conducted a dose-response experiment to determine the optimal dosage for the SCFA treatments using 1-, 2.5-, 5-, 10-, and 20-fold baseline concentrations of 900, 300, and 160 µM, for acetate, propionate, and butyrate, respectively. Following SCFA treatment, hepatocytes were incubated for 3 h with androstenone based on the results of Bone and Squires (2024). Androstenone metabolism exhibited a robust response to 5× baseline SCFA concentrations (Figure S1A). A time course was then performed using the 5× baseline concentrations to determine the optimal incubation time with androstenone. An incubation time of 1.5 h was selected, as androstenone metabolism remained within a linear range at this time point (Figure S1B).

Effects of SCFAs on androstenone metabolism in hepatocytes from boars and barrows

The effects of SCFA treatment on androstenone metabolism relative to untreated control incubations in hepatocytes isolated from boars and barrows are shown in Figure 1A and B, respectively. Phase II 16-androstene glucuronides were the only metabolites of androstenone produced within this system. In hepatocytes from boars, treatment with propionate (P < 0.0001), and butyrate (P = 0.009) increased androstenone metabolism relative to the untreated control. Androstenone metabolism was also significantly increased by combinations of SCFA treatments, including acetate and propionate (P < 0.0001), acetate and butyrate (P < 0.0001), butyrate and propionate (P < 0.0001), and all three SCFAs (P < 0.0001).

Figure 1.

Androstenone metabolism (%) following SCFA treatment in hepatocytes from boars (A) and barrows (B), expressed relative to the untreated control, and as absolute androstenone metabolism for boars and barrows (C). Values are presented as the mean ± SE (n = 7 boars, n = 6 barrows). Significant differences between SCFA treatments and the untreated control (A and B), or between boars and barrows for a given SCFA treatment (C), are indicated by *P < 0.05, **P < 0.01, and ***P < 0.001. Ace, acetate; Prop, propionate; But, butyrate; APB, acetate + propionate + butyrate.

Similar effects were observed in hepatocytes from barrows. Relative to the untreated control, androstenone metabolism was significantly increased by propionate treatment (P = 0.004), as well as by combinations of acetate and propionate (P < 0.0001), acetate and butyrate (P = 0.0005), propionate and butyrate (P < 0.0001), and all three SCFAs (P < 0.0001). However, butyrate treatment alone did not affect androstenone metabolism in hepatocytes from barrows, and acetate treatment had no effect in either boars or barrows.

Effects of SCFAs on gene expression in hepatocytes from boars and barrows

Gene expression in hepatocytes from boars and barrows was analyzed following SCFA treatments to assess how SCFAs affected the expression of key genes regulating androstenone metabolism. Relative to the untreated control, gene expression in hepatocytes from boars was unaffected by SCFA treatments (Table 3A), while hepatocytes from barrows displayed increased expression of some key genes following treatment with combinations of SCFAs (Table 3B). Specifically, acetate and propionate treatment increased expression of AKR1C1 (P = 0.003), acetate and butyrate increased expression of AKR1C1 (P = 0.01) and SULT1E1 (P = 0.005), and propionate and butyrate increased expression of AKR1C1 (P < 0.0001), SULT2A1 (P = 0.004), and UGT2A1 (P = 0.03). Additionally, treatment with all three SCFAs increased expression of AKR1C1 (P = 0.001), SULT2A1 (P = 0.009), and UGT2A1 (P = 0.03). The expression of UGT1A1, UGT1A6, and UGT2B31 was not affected by SCFA treatment.

Table 3.

Gene expression levels in hepatocytes from (A) boars (n = 7) and (B) barrows (n = 6) following different SCFA treatments.

A. Boar hepatocyte gene expression relative to control
Gene	Treatment2
	Acetate	Propionate	Butyrate	Ace + Prop	Ace + But	Prop + But	APB
AKR1C1	1.15 (0.51, 2.56)	0.94 (0.69, 1.29)	1.01 (0.71, 1.42)	1.01 (0.64, 1.58)	1.44 (0.84, 2.46)	1.07 (0.80, 1.43)1	1.57 (0.95, 2.61)
SULT1E1	1.14 (0.57, 2.30)	0.86 (0.72, 1.04)	1.00 (0.64, 1.57)	0.88 (0.44, 1.74)	1.28 (0.63, 2.58)	0.78 (0.60, 1.01)	1.22 (0.64, 2.33)
SULT2A1	1.18 (0.77, 1.80)	1.23 (0.94, 1.60)	1.17 (0.84, 1.62)	1.23 (0.90, 1.67)	1.57 (1.06, 2.32)	1.03 (0.81, 1.30)1	1.19 (0.89, 1.59)
UGT1A1	0.95 (0.72, 1.25)	0.91 (0.67, 1.22)	0.99 (0.72, 1.35)	0.81 (0.54, 1.23)	0.97 (0.76, 1.24)	0.89 (0.70, 1.19)	0.87 (0.61, 1.23)
UGT1A6	0.92 (0.66, 1.30)	1.08 (0.80, 1.45)	1.19 (1.02, 1.40)1	0.93 (0.72, 1.21)	1.12 (0.77, 1.63)	1.04 (0.81, 1.35)	0.99 (0.71, 1.40)
UGT2A1	1.13 (0.59, 2.16)	1.18 (0.78, 1.78)	1.49 (1.01, 2.20)	1.36 (0.83, 2.21)	1.70 (1.24, 2.32)	1.43 (0.97, 2.09)	1.72 (1.16, 2.54)
UGT2B31	1.02 (0.73, 1.42)	0.98 (0.70, 1.36)	0.89 (0.69, 1.14)	1.11 (0.86, 1.42)	0.94 (0.71, 1.24)	1.16 (0.83, 1.62)	1.15 (0.83, 1.60)
B. Barrow hepatocyte gene expression relative to control
Gene	Treatment2
	Acetate	Propionate	Butyrate	Ace + Prop	Ace + But	Prop + But	APB
AKR1C1	0.83 (0.68, 0.94)	1.27 (1.06, 1.53)	1.10 (0.68, 1.83)	1.75 (1.43, 2.15)**	1.60 (1.36, 1.88)*	2.12 (1.71, 2.63)***1	1.82 (1.37, 2.44)**
SULT1E1	1.28 (1.05, 1.55)	1.19 (0.88, 1.62)	1.11 (0.81, 1.53)	1.41 (1.02, 1.95)	1.89 (1.30, 2.75)**	1.33 (1.03, 1.71)	1.46 (1.02, 2.11)
SULT2A1	0.96 (0.77, 1.21)	1.34 (1.17, 1.53)	1.13 (0.74, 1.71)	1.47 (1.04, 2.07)	1.46 (1.07, 2.00)	1.72 (1.59, 1.8)**1	1.65 (1.32, 2.05)**
UGT1A1	0.87 (0.64, 1.18)	1.07 (0.84, 1.37)	0.78 (0.59, 1.03)	0.88 (0.63, 1.23)	1.04 (0.92, 1.18)	1.12 (0.91, 1.37)	1.12 (0.83, 1.50)
UGT1A6	0.95 (0.83, 1.09)	1.06 (0.85, 1.33)	0.92 (0.80, 1.06)1	1.07 (0.89, 1.30)	1.19 (1.03, 1.37)	1.09 (1.00, 1.19)	1.23 (1.11, 1.36)
UGT2A1	0.84 (0.65, 1.08)	1.09 (0.85, 1.40)	0.94 (0.62, 1.43)	1.64 (1.33, 2.02)	1.28 (0.96, 1.71)	1.71 (1.33, 2.20)*	1.73 (1.10, 2.74)*
UGT2B31	1.12 (0.85, 1.47)	1.25 (0.89, 1.76)	1.12 (0.74, 1.70)	1.55 (1.21, 1.99)	1.26 (0.99, 1.60)	1.54 (1.13, 2.10)	1.53 (1.22, 1.90)

¹Indicates significant differences in expression of a given gene between boars and barrows (P < 0.05).

²Values are expressed as the mean standardized fold change with 95% confidence intervals relative to the untreated control. Ace, acetate; Prop, propionate; But, butyrate; APB, acetate + propionate + butyrate.

*,**,***Indicates significant differences in expression between a given SCFA treatment and the untreated control

^* P < 0.05,

^** P < 0.01, and

^*** P < 0.001).

Differences in androstenone metabolism and gene expression between boars and barrows

We next examined whether SCFA-induced effects on hepatic androstenone metabolism and gene expression differed in hepatocytes from boars and barrows. Absolute androstenone metabolism in response to SCFA treatments is shown for hepatocytes from boars and barrows in Figure 1C, with corresponding differences in gene expression presented in Table 3A and 3B. Relative to barrows, androstenone metabolism in hepatocytes from boars was greater following butyrate treatment (P = 0.02) and combined acetate and butyrate treatment (P = 0.05). In contrast, rates of androstenone metabolism following all other SCFA treatments were not different between boars and barrows. Butyrate treatment also significantly increased UGT1A6 expression in hepatocytes from boars relative to barrows (P = 0.009), while combined treatment with butyrate and propionate resulted in significantly higher expression of SULT2A1 (P = 0.004) and AKR1C1 (P = 0.007) in barrows compared to boars. These SCFA‑induced differences in gene expression were not due to differences in basal gene expression quantified in untreated hepatocytes from boars and barrows (Table 4), as only SULT1E1 showed higher basal expression in boars compared with barrows (P = 0.02).

Table 4.

Basal expression levels of genes of interest in untreated hepatocytes from boars (n = 7) and barrows (n = 6).

Gene	Basal gene expression, 2−ΔCt × 10−3		P-value
	Boar	Barrow
AKR1C1	0.17 ± 0.044	0.20 ± 0.037	0.6
SULT1E1	6.11 ± 1.50	1.63 ± 0.41	0.02
SULT2A1	0.58 ± 0.17	0.47 ± 0.093	0.6
UGT1A1	1.62 ± 0.38	1.18 ± 0.20	0.3
UGT1A6	11.89 ± 2.46	8.02 ± 0.93	0.2
UGT2A1	0.015 ± 0.0048	0.0071 ± 0.0024	0.2
UGT2B31	1.55 ± 0.30	1.68 ± 0.28	0.8

Values are presented as the mean ± SE and are expressed as fold change relative to β-actin.

Relationship between plasma E₁S levels and SCFA-induced effects on gene expression

To assess whether SCFA-induced effects are influenced by circulating testicular steroid levels, plasma E₁S concentrations measured in boars at slaughter were correlated with corresponding gene expression levels in hepatocytes (Figure S2). Across boars, plasma E₁S levels averaged 39.12 ± 7.59 ng/mL but varied considerably between individual animals, ranging from 9.86 to 71.33 ng/mL. Plasma E₁S levels were positively correlated with UGT1A1 (r = 0.77, P = 0.04) in untreated hepatocytes, as well as following treatment with acetate (r = 0.82, P = 0.03) and with all three SCFAs combined (r = 0.77, P = 0.04). Positive correlations were also identified between plasma E₁S levels and SULT1E1 expression in hepatocytes treated with propionate (r = 0.83, P = 0.02), propionate and butyrate (r = 0.84, P = 0.02), or all three SCFAs combined (r = 0.76, P = 0.05), and with UGT2A1 expression following acetate treatment (r = 0.90, P = 0.006).

Discussion

Dietary fermentable carbohydrates increase the production of the SCFAs acetate, propionate, and butyrate by gut microbes and have been shown to decrease androstenone levels in plasma when fed to boars (Chen et al. 2007; Bai et al. 2020). While the mechanisms responsible for these changes in plasma androstenone are not well understood, the rate of hepatic androstenone metabolism is important for regulating circulating androstenone levels and subsequent boar taint development (Squires et al. 2020). Studies in humans and rodents have demonstrated that SCFAs produce diverse effects on hepatic metabolism by altering gene expression and activating signaling pathways involved in energy expenditure (Gao et al. 2009), lipid metabolism (Shimizu et al. 2019), and bile acid homeostasis (Zhao et al. 2017). Therefore, the present study evaluated the effects of SCFAs on androstenone metabolism and associated gene expression in isolated porcine hepatocytes and assessed the influence of testicular steroids by comparing SCFA-induced responses in hepatocytes from boars and barrows.

SCFAs act through several signaling mechanisms that differ in responsiveness to individual SCFAs. For example, they are well-established ligands for both GPR41 and GPR43, but differences in carbon chain length result in acetate preferentially activating GPR43, butyrate preferentially activating GPR41, and propionate acting on both receptors (Brown et al. 2003; Liu et al. 2023; Zheng et al. 2023; Lee et al. 2024). Butyrate can also activate GPR109A, and both butyrate and propionate can regulate gene expression via HDACi activity (Hinnebusch et al. 2002; Singh et al. 2014). Consistent with this, we showed that individual SCFAs influenced hepatic androstenone metabolism differently. Androstenone metabolism was significantly increased by propionate in hepatocytes from both boars and barrows, and by butyrate in only hepatocytes from boars, but was unaffected by acetate. In contrast, treatment with all combinations of SCFAs consistently increased androstenone metabolism in both boars and barrows, suggesting that interactions among individual SCFAs may also influence hepatic androstenone metabolism. Although the extent of androstenone metabolism differed between hepatocytes from boars and barrows and in response to SCFA treatment, Phase II glucuronides were the only metabolites produced within this system. This is consistent with previous work by Bone and Squires (2024), which identified Phase II glucuronides are the major metabolites of hepatic androstenone metabolism and suggested that Phase I androstenols function as metabolic intermediates.

Interestingly, SCFA-induced increases in androstenone metabolism occurred without corresponding changes in the expression of related genes in boars. However, several key genes involved in Phase I and II androstenone metabolism were upregulated in barrows, including AKR1C1 by all SCFA combination treatments, along with UGT2A1 and SULT2A1 in response to the combination of propionate and butyrate and the combination of all three SCFAs. Treatment with the combination of acetate and butyrate also upregulated expression of SULT1E1, which is involved in Phase II estrogen metabolism (Mueller et al. 2015). These results suggest that SCFAs influence hepatic androstenone metabolism through both genomic and non-genomic mechanisms that differ between boars and barrows.

In line with our results, SCFAs have been shown to activate AMP-activated protein kinase (AMPK) within the liver, which regulates the activity of metabolic enzymes and influences gene expression by activating transcription factors and transcriptional coactivators (Den Besten et al. 2015; Joo et al. 2016; Herzig and Shaw 2018; Mihaylov et al. 2023). Several transcription factors regulated by AMPK signaling, such as nuclear factor erythroid 2-related factor 2 (NRF2), hepatocyte nuclear factor 4-α (HNF4α), and proliferator-activated receptor α (PPARα), act to increase the expression of key Phase I and II metabolic genes, including AKR1C1, SULT2A1, and SULT1E1 (Fang et al. 2005, 2007; Jung et al. 2013; Guo et al. 2015). Importantly, castration has been reported to reduce hepatic AMPK phosphorylation and alter chromatin accessibility at regions enriched for HNF4α- and PPARα-binding motifs (Sakr et al. 2018; Chan et al. 2024). The effects of SCFAs may also be mediated through post-transcriptional and post-translational regulatory mechanisms, as HNF4α has been shown to increase expression of the liver-specific microRNA-122 and GPR43 signaling can activate protein kinase C, which influences UGT activity through phosphorylation (Volak and Court 2010; Li et al. 2011; Ahmad et al. 2026). Therefore, future research should investigate the involvement of these different signaling mechanisms as potential mediators of SCFA-induced effects on androstenone metabolism.

The development of boar taint varies considerably between animals with high and low capacities for testicular steroidogenesis (Zamaratskaia and Squires 2009; Squires et al. 2020; Squires and Bone 2025). In slaughter-weight boars, steroidogenic capacity is classified according to plasma E₁S concentrations and influences the expression of key genes involved in hepatic androstenone metabolism (Kojima and Degawa 2014; Bone and Squires 2024). Our results suggest that steroidogenic capacity may also influence SCFA-induced effects on androstenone metabolism, as plasma E₁S levels measured in boars at slaughter were positively correlated with UGT1A1, SULT1E1, and UGT2A1 expression in response to different SCFA treatments. Thus, the absence of testicular steroids in barrows, but not boars, may alter hepatic responsiveness to SCFAs, contributing to the differential gene expression patterns observed. Consistent with these results, testicular steroid production at puberty is known to promote male-specific growth hormone secretion patterns to masculinize hepatic gene expression, whereas castration results in feminization of hepatic gene expression (Lai et al. 2016; Oshida et al. 2016a, 2016b). Given the significant variation in plasma E₁S levels observed in the present study, SCFA-induced effects on hepatic androstenone metabolism may also differ between individual boars with high and low steroidogenic capacities. Similar effects of steroidogenic capacity on response to dietary biochar treatment for boar taint were recently described, where biochar consistently prevented the development of boar taint in low but not high steroidogenic capacity boars (Parent et al. 2026). Therefore, future research evaluating fermentable carbohydrates as dietary treatments for boar taint should consider differences in steroidogenic capacity when assessing treatment response.

The rate of hepatic androstenone metabolism varies considerably between different breeds of boars and among individuals within a breed, and this variation has been shown to contribute to differences in the response to treatments intended to increase androstenone metabolism (Doran et al. 2004; Bone and Squires 2025). Thus, while hepatocytes in the present study were isolated from three-way commercial crossbred boars and barrows to reflect the typical genetic background of pigs raised for pork production, the genetic heterogeneity of these animals may have influenced the observed effects of SCFAs on androstenone metabolism. The applicability of our results to other breeds and genetic lines of boars also remains unclear and should be investigated in future studies with larger sample sizes.

In summary, our results demonstrate that SCFAs increase hepatic androstenone metabolism, providing a potential mechanism for the decreased plasma levels of free androstenone that have been reported from dietary supplementation with fermentable carbohydrates (Hansen et al. 2006; Chen et al. 2007). Additionally, we showed that the effects of SCFAs on hepatic androstenone metabolism and associated gene expression differed by SCFA type and between boars and barrows. While additional research is needed to determine whether the effects of SCFAs on hepatic androstenone metabolism result in meaningful reductions in fat androstenone levels in vivo, our results suggest that both the specific SCFA profile and the steroidogenic capacity of the animal are important considerations when evaluating fermentable carbohydrates as dietary treatments for boar taint, which may partially explain why androstenone levels are not consistently reduced across different studies (Byrne et al. 2008; Zammerini et al. 2012). Since dietary fermentable carbohydrates have also been shown to reduce plasma and fat levels of skatole (Hansen et al. 2006; Chen et al. 2007; Øverland et al. 2011), which is another compound that contributes to boar taint, additional research is needed to determine whether SCFAs similarly increase hepatic skatole metabolism either directly or indirectly through increased androstenone metabolism and reduced inhibition of CYP2E1 protein expression (Doran et al. 2002).

Glossary

Abbreviations

AKR

aldo-keto reductase

AMPK

AMP-activated protein kinase

CYP2E1

cytochrome P450 2E1

E₁S

estrone-1-sulfate

GPR

G-protein-coupled receptor

HDACi

histone deacetylase inhibitors

HNF4α

hepatocyte nuclear factor 4-α

NRF2

nuclear factor erythroid 2-related factor 2

PPARα

proliferator-activated receptor α

SCFAs

short-chain fatty acids

SULT

sulfotransferase

UGT

uridine 5'-diphospho-glucuronosyltransferase

Author contributions

Austin Lawson (Investigation, Methodology, Validation, Writing—original draft), Christine Bone (Conceptualization, Funding acquisition, Methodology, Supervision, Validation, Writing—review & editing), Melissa Parent (Data curation, Visualization, Writing—review & editing), and James Squires (Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Validation, Writing—review & editing)

Supplementary data

Supplementary data is available at Journal of Animal Science online.

Conflict of interest statement. The authors declare no conflicts of interest.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.