Histology and mRNA expression of unilateral testicular atrophy in three immunocastrated boars: a case report

Abstract

Background

This case study reports testicular asymmetry in entire male (EM) and immunocastrated pigs (IC), specifically focusing on histomorphometric and transcriptomic characteristics of testicular and epididymal tissue in three IC with pronounced unilateral testicular atrophy.

Case presentations

During post-mortem assessment of genital tract, three IC were identified with marked testicular asymmetry. Testes and cauda epididymides were collected from these animals for histomorphometric analysis and quantitative polymerase chain reaction (qPCR). In addition, percentage of testicular asymmetry was calculated in a broader cohort of EM and IC pigs to assess prevalence and degree of asymmetry.

Conclusion

The observed cases of severe unilateral atrophy did not appear to be systematically related to immunocastration, based on available data. In atrophic testes, there were distinct histological differences in testes and cauda epididymides, along with molecular changes, including downregulation of gonadotropin-releasing hormone receptor II (GnRHRII), estrogen receptor 1 (ESR1) and betaine homocysteine S-methyltransferase (BHMT), but no marked differences in plasma testosterone concentrations nor immunocastration efficacy. Although based on a limited number of cases, these findings provided the first combined histological and molecular characterization of unilateral testicular atrophy in immunocastrated boars.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12917-025-05243-4.

Background

Asymmetric testicular weights in humans [1] and animals [2–6] have been reported, but the degree of asymmetry is usually low (< 10%). According to European Association of Urology guidelines, a persistent small testis in humans is present when the percentage of testicular asymmetry (PTA) exceeds 20% [7], as in 14.8% of adolescent boys (n = 345; [1]). However, studies reporting incidence of testicular asymmetry in boars are scarce. A relatively high incidence of unilateral testicular asymmetry was reported in inbred Duroc (52%; n = 67; [3]) and culled boars (71.4%; n = 28; [4]), with PTA > 20% in 22.3 and 21.4%, respectively. A retrospective study on Göttingen minipigs (n = 104) reported a 74% incidence of spontaneous testicular atrophy [8], mostly bilateral (59%) and mild. Grade 1 lesions (< 5% of seminiferous tubules affected) were present in 71% of cases, with only a 1% average reduction in testicular weight. Moderate to marked changes (Grades 3 or 4) were present in 12% of cases, including epithelial vacuolization, multinucleated germ cells, and germ cell loss. Unilateral atrophy occurred in 11.4% of minipigs (severity not reported) [8].

Immunocastration, causing androgen deprivation by active immunization against hypothalamic gonadotropin-releasing hormone (GnRH) 1, has been widely studied as an alternative to surgical castration in piglets [9–11]. However, the prevalence of unilateral testicular atrophy in immunocastrated pigs (IC) or control boars (EM) has not been reported. Therefore, the present report aims to characterize three cases of severe unilateral testicular atrophy in IC.

Genital tract regression in IC occurs shortly after the second (booster) vaccination, albeit to varying extents, as evidenced by decreases in genital tract (GTI) and testes (TI) indices [12] and accessory sex gland weights [9, 12, 13]. In IC, testis regression is typically bilateral and immunocastration effectiveness can be assessed by testis weight, which is usually two- to three-fold lower than in EM [12]. Due to substantial decreases in testis size in IC pigs, visual scoring of testicular size can also be used to evaluate immunocastration effectiveness [14]. Documented changes in testes of IC at histomorphometric and transcriptional levels included: (i) reduced spermatogenesis [15–18]; (ii) Leydig cell atrophy, accompanied by increased nucleus to cytoplasm ratio (N:C ratio) [19]; and (iii) alterations in mRNA expression of several target transcripts, including: upregulation of androgen receptor (AR), follicle-stimulating hormone receptor (FSHR) and inhibin subunit beta (INHBA; a follicle-stimulating hormone (FSH) secretion inhibitor) [19], and downregulation of transcripts encoding for proteins involved in cholesterol transport in mitochondria [19], steroid biosynthesis [20] and Leydig cell functional/differentiation status [19, 20]. Furthermore, histomorphometric and transcriptomic differences were also observed between IC with varying responses to immunocastration [19] or due to recovery of Leydig cell function [21, 22].

To our knowledge, this is the first report describing three cases of severe unilateral testicular atrophy in IC, supported by histology and mRNA expression of selected genes in testicular tissue. Regardless of the efficacy of immunocastration (based on plasma testosterone concentrations), genital tract and testes indices, testes with marked PTA had distinct histomorphometric and transcriptomic profiles compared to their corresponding larger counterparts.

Case presentations

This retrospective study used samples collected during previous studies on the effects of standard and alternative immunocastration protocols in boars [12, 19, 22]. Immunocastration was done with Improvac^® (Zoetis; vaccination and booster, 2 mL subcutaneous).

To assess the extent of testes asymmetry (i.e., difference in size between the two testes) in EM (n = 91) and IC (n = 151), PTA was calculated as follows:

All EMs and 132 ICs were commercial crosses (average slaughter age, 26 weeks) [12]. In contrast, 19 of 151 ICs were mature purebred Duroc, Pietrain, or Large White with an average slaughter age of 69 weeks [19]. A non-parametric test (pairwise Wilcoxon rank-sum test with Bonferroni adjustment) was used to assess differences in PTA between EMs and ICs. Statistical analyses were performed using R statistical software (Version 4.4.1) and data presented as medians with interquartile ranges.

Mean PTA was not different between IC and EM (p = 0.58; Supplementary Figure) and amounted to 6.1% (average weight difference, 16.5 ± 27.7 g; n = 151) and 5.6% (average weight difference, 19.6 ± 18.1 g; n = 91) in IC and EM, respectively. In our cohort, the prevalence of PTA > 10% was 16.5 and 15.9% in IC and EM, whereas the prevalence of PTA > 20%, considered the threshold for a persistent small testis, was 2.0 and 1.1% in IC (n = 3) and EM (n = 1).

Three cases of severe testicular asymmetry (PTA > 50%) in IC were identified (Supplementary Figure) and all corresponding pairs of testes and cauda epididymides were collected for further analyses. Two tissue samples (~ 1 cm³) of testicular parenchyma were excised from the area between the tunica albuginea and the mediastinum testis of the left and right testis, as well as tissue samples (~ 1 cm³) of left and right cauda epididymis within 20 min after slaughter and fixed in Bouin’s solution for histological and histomorphometric analyses. Additional samples of testicular parenchyma from left and right testis were either stored in RNAlater (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) or snap-frozen in liquid nitrogen for subsequent quantitative PCR (qPCR) analyses. Unfortunately, one additional case, identified by the PTA calculation in EM, that slightly exceeded the threshold of 20% for PTA [7], was overlooked at the processing line and was not sampled for further analyses.

Individual biometric data of three identified ICs with severe PTA are in Table 1. Case 1 was a young IC slaughtered 8 weeks after booster vaccination at heavy weight (142 kg and 228 days of age, Large White x Landrace crossbreed) with a PTA of 78.2%. This animal had relatively high GTI and TI values (0.630 and 0.654, respectively) and high testosterone concentration (9.95 ng/ml plasma), suggesting either a weak response to vaccination or alternatively a recovery of Leydig cell function due to the prolonged interval between booster vaccination and slaughter [22].

Table 1.

Biometric data of immunocastrated male pigs with unilateral testicular atrophy

Parameter	Case 1	Case 2	Case 3
Age at first vaccinationa (days)	147	424	42
Age at second vaccinationa (days)	157	452	70
Age at third vaccinationa (days)	−	-	133
Age at slaughter (days)	228	480	182
Genital tract weightb (g)	643	959	364
Genital tract index (%)c	0.630	0.480	0.485
Testis index (%)d	0.654	0.311	0.501
Right testis weighte (g)	119	427	322
Left testis weighte (g)	548	194	54
PTA (%)f	78.2	54.6	83.2
Testosterone concentration (ng/mL plasma)	9.95	0.55	5.6

^aVaccination with Improvac (Zoetis; 2 mL, s.c.)

^bWeight of the pelvic part of the genital tract, accessory glands, and emptied bladder

^cCalculated as the genital tract weight (weight of the pelvic part of the genital tract, accessory glands, and emptied bladder) divided by warm carcass weight x 100

^dCalculated as the testis weight (weight of both testes, with epididymides included) divided by warm carcass weight x 100

^eWeight of the right/left testes with epididymis included

^fCalculated as the [(weight of larger testis with epididymis – weight of smaller testis with epididymis)/weight of larger testis with epididymis] x 100. PTA; percentage of testicular asymmetry

Case 2, a mature Duroc boar immunocastrated after being culled from an artificial insemination service (due to low sperm quality) at the age of 480 days, had a PTA of 54.6%. Slaughter occurred 4 weeks after the booster. This animal had a low GTI (0.480), TI (0.311) and plasma testosterone concentration (0.55 ng/ml), indicating successful immunocastration [12, 19].

Case 3 was a young IC slaughtered 7 weeks after second booster at a standard weight (93 kg and 182 days of age, Large White x Landrace crossbreed) with PTA of 83.2% and indices (GTI; 0.485, TI; 0.501 and plasma testosterone concentration; 5.6 ng/mL) indicating a weak response to vaccination/recovery of Leydig cell function.

Histology and histomorphometry

After 72 h of fixation in Bouin’s solution, testis and cauda epididymis samples were processed using standard paraffin-embedding protocols with a tissue processor (Leica, Nussloch, Germany) and the Tissue-Tek^® TEC™ 5 Tissue Embedding Console System (Sakura Finetek Europe HQ, The Netherlands), as described [19]. Subsequently, 5-µm-thick sections were cut using a Leica SM2000R microtome (Nussloch, Germany), stained with hematoxylin and eosin (HE), mounted, and cover-slipped using the Gemini AS slide stainer and ClearVue coverslipper (Thermo Fisher Scientific, Cheshire, United Kingdom). Images were captured using a Nikon Eclipse Ni-UM light microscope equipped with a DS-Fi1 camera and histomorphometric analysis conducted using NIS Elements BR 4.6 imaging software (Nikon Instruments Europe B.V., Badhoevedorp, Netherlands). Quantitative histomorphometric image analysis followed recommendations for testicular tissue [23], with assessment of the following: (i) seminiferous tubules diameter and germinal epithelium height; (ii) germinal epithelium area ratio (%) calculated as [germinal epithelium area/seminiferous tubules area × 100]; (iii) Leydig cell N:C ratio calculated as [nucleus area/(cell area – nucleus area)]; (iv) epididymal duct diameter, epididymal duct epithelial height and periductal muscle layer thickness; and (v) epididymal duct area ratio (%) calculated as [(epididymal duct area – lumen area)/epididymal duct area × 100]. Representative photomicrographs of HE-stained testis sections of testes and cauda epididymis sections associated with larger and smaller testes are presented in Figs. 1 and 2, respectively, and derived histomorphometric data are summarized in Table 2.

Fig. 1.

Representative photomicrographs (20× objective) of testis cross-sections from Cases 1–3 showing seminiferous tubules and interstitial Leydig cells in larger (panels A, C, E) and smaller testes (panels B, D, F). Note marked differences between seminiferous tubules of larger and smaller testes (black double-headed arrows), including disintegration of the germinal epithelium with exfoliated germ cells/debris, presence of multinucleated giant cells indicating tubular degeneration (black asterisks), small and partially luminized tubules with occasional basally located gonocytes (Case 3, panel F), and retention of spermatid heads deep within the seminiferous epithelium (black arrowheads; Case 2, panel C). Differences in interstitial Leydig cell morphology and number are also evident (black arrows) and lymphocyte infiltration (white asterisks; Case 1, panel B). Hematoxylin and eosin staining; scale bar = 200 μm. sc, Sertoli cell; ps, primary spermatocytes; rs, round spermatids; es, elongated spermatids; g gonocyte

Fig. 2.

Representative photomicrographs (4× objective) of cauda epididymis cross-sections associated with larger (LT) and smaller (ST) testes from Cases 1–3 showing epididymis duct profiles. Note the difference in sperm cell density in the lumen of duct profiles between epididymides associated with larger testes (LT, panels A, C, E) and smaller testes (ST, panels B, D, F), diameter of epididymis duct profiles (black double-headed arrows; Case 1 and 3), peritubular musculature thickness (black arrows; Case 1 and 2) and presence of cribriform changes characteristic of reduced luminal content (asterisks) Case 1 (panel B) and Case 2 (panels C, D). Hematoxylin and eosin staining, scale bar = 1000 μm

Table 2.

Histomorphometric data of testis and epididymis from immunocastrated male pigs with unilateral testis atrophy

		Case 1		Case 2		Case 3
Parameters		Larger testis	Smaller testis	Larger testis	Smaller testis	Larger testis	Smaller testis
Testis
Leydig cell N:C ratioa		0.192	0.360	0.416	0.433	0.337	0.574
Seminiferous tubules diameterb (µm)		193	159	159	129	208	87
Germinal epithelium heightc (µm)		65.6	39.1	46.6	21.9	65.3	29.5
Germinal epithelium area ratiod (%)		89.4	74.0	82.7	56.6	85.8	89.4
Cauda epididymis
Epididymis ducts diameterb (µm)		1094	807	1398	1433	1194	475
Epithelium height (µm)e		45.9	60.3	51.9	83.9	37.7	77.5
Muscularis thickness (µm)e		44.9	83.7	102.7	361.7	65.1	63.2
Epididymis wall area ratiof (%)		30.8	57.9	39.5	85.4	32.8	82.9

Data are reported as mean values derived from individual animals for each of the three IC cases

^aLeydig cell nucleus-to-cytoplasm (N:C) ratio calculated as: [nucleus area/(cell area – nucleus area)]; a total of 100 Leydig cells were randomly selected and measured

^bEquivalent diameters were derived from the surface areas of 50–100 seminiferous tubule and epididymal duct profiles, selected based on circularity > 0.90

^cAverage germinal epithelium height calculated as: [(seminiferous tubule diameter – lumen diameter)/2]

^dCalculated as: [(germinal epithelium area/seminiferous tubule area) × 100]

^eEpididymal duct epithelial height and peritubular muscularis thickness were indirectly assessed using the equivalent diameter, based on analysis of 20–30 cross-sectional duct profiles per section with circularity > 0.90

^fCalculated as: [(epididymal duct area – lumen area)/epididymal duct area × 100]

In HE-stained histologic sections of testis samples, marked differences in morphology of seminiferous tubules and surrounding interstitial tissue were observed, with some case-specific changes. In Case 1, spermatogenesis was retained in the seminiferous tubules’ epithelium of the larger testis and surrounding interstitial compartment was predominantly occupied by Leydig cells (Fig. 1A). In contrast, seminiferous tubules of the smaller testis had disintegration of germinal epithelium, their lumina were enlarged and contained exfoliated germ cells/cell debris and multinucleated giant cells (signs of tubular degeneration and atrophy [24]). Surrounding interstitial tissue had inconspicuous Leydig cells and lymphocyte infiltration, indicating inflammation (Fig. 1B). The PTA in Case 2 was from a successfully immunocastrated mature boar (Table 1); histologic signs suggestive of androgen deficiency were observed in the larger testis, including spermatids near the basal compartment of the seminiferous tubule epithelium, germ cell detachment and marked Leydig cell atrophy (Fig. 1C). In addition, histomorphology of the smaller testis was comparable to Case 1, without signs of inflammatory cell infiltration (Fig. 1D).

In Case 3, histology of the seminiferous tubules in the larger testis (Fig. 1E) was comparable to that observed in Case 1. However, interstitial Leydig cells appeared smaller. Interestingly, histological features of the smaller testis (Fig. 1F) resembled those of sexually immature animals; seminiferous tubules were small and partially luminized, gonocytes were occasionally present in the basal compartment of the tubules, and the interstitium contained abundant but small Leydig-like cells.

The epididymis of both testes was also examined, as it can provide information about the testis [25]. Photomicrographs of cauda epididymis cross-sections associated with larger testes (Fig. 2, panels A, C and E) had larger profiles with abundant luminal contents, whereas the latter was less evident in Case 2. Hyperplastic changes of the epithelium, so-called cribriform changes, were mainly observed in Case 2 (Fig. 2C). In contrast, cauda epididymis cross-sections associated with smaller testes (Fig. 2, panels B, D and F) had smaller profiles with markedly reduced luminal content. In Cases 1 and 2, cribriform changes in the epithelium were observed, with a marked increase in thickness of peritubular musculature (Fig. 2B and D). Based on a semi-quantitative 5-grade method for evaluating microscopic changes in testis and epididymis [8], observed changes in smaller testes and associated epididymides were classified as Grade 5 in all three PTA cases, as > 75% seminiferous tubules/epididymal duct profiles were affected.

Histomorphometric analyses further supported observed histological differences between testes and their corresponding cauda epididymides (Table 2). In all three cases, the larger testes had larger diameter of the seminiferous tubules and an increased height of germinal epithelium. In contrast, the corresponding cauda epididymides had reduced epithelial height and a lower epididymis wall area ratio. The Leydig cell N:C ratio was lower in the larger testes of Cases 1 and 3 (decreased 47 and 41%, respectively), but not in Case 2, consistent with its low plasma testosterone (0.55 ng/mL). Increased germinal epithelium area ratio was also observed in the larger testis of Case 2. Epididymal ducts in larger testes tended to be larger, except in Case 2, whereas peritubular musculature thickness was generally thinner, except in Case 3 (Table 2).

Gene expression analysis

Next, qPCR was performed to validate potential effects of testis atrophy on testicular mRNA expression of selected genes related to control of testicular function, steroidogenesis and Leydig cell differentiation/functional status. Total RNA was extracted from testicular tissue samples, as described [19, 22], using an RNeasy Mini Kit (Qiagen, Hilden, Germany; catalog number: 74104) following manufacturer’s protocol, plus an on-column DNase digestion step with RNase-Free DNase Set (Qiagen). To assess RNA purity, 260/280 and 260/230 absorbance ratios were determined using a UV-VIS Lambda 25 spectrophotometer (Perkin Elmer, Waltham, MA, USA). Integrity and quality of the RNA samples were monitored by Qubit RNA IQ assay using Invitrogen™ Qubit™ 4 Fluorometer. cDNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Scientific GmbH, Vienna, Austria; catalog number: 4368814) using 1.0 µg of each RNA sample, with 260/280 and 260/230 ratios close to 2.0 and the RNA integrity number > 8. A QuantStudio™ 5 Real-Time PCR System (Applied Biosystems, Thermo Scientific GmbH) was used for qPCR. Primers and fluorescent 6-FAM dye-labelled minor-groove-binder probes/predesigned assays (Applied Biosystems (Thermo Scientific GmbH) are listed in Table 3. Beta-2-microglobulin (B-2-M) and eukaryotic ribosomal (r) 18 s RNA (18s rRNA) were used as endogenous controls [26, 27]. Quantitative PCRs were performed in a final volume of 10 µL containing 4.5 µL of each RT sample (diluted 10-fold), 5 µL of TaqMan Universal Master Mix II, and 0.5 µL of TaqMan Gene Expression Assay, under the following conditions: one cycle of 50 °C for 2 min and one cycle of 95 °C for 10 min, followed by 45 cycles of 15 s at 95 °C and 1 min at 60 °C. Each reaction was performed in triplicate. Results were calculated from the threshold cycle (Ct) that was fixed at 0.10, with a Ct value > 35 regarded as the cutoff. The Ct values for B-2-M and 18 s RNA were used to normalize Ct values of evaluated preselected target transcripts. Delta Ct (ΔCt) values were calculated using the comparative Ct method (ΔCt = Ct target transcript – Ct geometric mean of controls). Relative changes in expression of studied target transcripts (fold changes in expression) were compared between larger vs. smaller testis using the 2^−ΔΔCt method [28]. Relative Quantification Analysis Module, Version 3.9 (Thermo Fisher Scientific, Applied Biosystems) was used for data analyses and results presented in Table 4.

Table 3.

List of predesigned TaqMan gene expression assays used for quantitative PCR

Full gene name	Gene	Amplicon length	Assay ID	Function/use
Estrogen receptor 1	ESR1	70	Ss03383398_u1	Mediates estrogenic effects
Estrogen receptor 2	ESR2	84	Ss03391479_m1	Mediates estrogenic effects
Follicle-stimulating hormone receptor	FSHR	99	Ss03384581_u1	Control of gonadal function and reproduction
Luteinizing hormone/choriogonadotropin receptor	LHCGR	64	Ss03384991_u1	Control of gonadal function and reproduction
Inhibin subunit beta A	INHBA	90	Ss03393536_s1	Forms part of inhibin A, pituitary FSH secretion inhibitor
Inhibin subunit alpha	INHA	76	Ss03383260_u1	Forms part of inhibin A and B, pituitary FSH secretion inhibitor
Gonadotropin-releasing hormone receptor II	GNRHRII	63	Ss03391559_m1	Control of gonadal function and reproduction
Androgen receptor	AR	86	Ss03822350_s1	Mediates androgenic effects
Steroidogenic acute regulatory protein	STAR	73	Ss03381250_u1	Transfer of cholesterol into the mitochondria
Hydroxysteroid 17-beta dehydrogenase 7	HSD17β7	61	Ss04246893_m1	Biosynthesis of steroid hormones from cholesterol
Betaine Homocysteine S-Methyltransferase	BHMT	83	Ss03374598_m1	Adult Leydig cell marker
Insulin-like peptide 3	INSL3	139	Ss03393127_u1	Marker of Leydig cell function
Corticotropin releasing hormone receptor 1	CRHR1	65	Ss03373289_g1	Fetal Leydig cell marker
Platelet-derived growth factor alpha	PDGFRα	59	Ss04955107_g1	Stem Leydig cell marker
Beta-2-microglobulin	B-2-M	60	Ss03391154_m1	Endogenous control
Eukaryotic ribosomal (r) 18 S rRNA	18 S rRNA	69	Hs03003631_g1	Endogenous control

Table 4.

Expression of selected genes associated with testicular function, steroid metabolism, and Leydig cell differentiation/funtional status in the larger vs. smaller testis from immunocastrated male pigs with unilateral testis atrophy

Parameters	Case 1	Case 2	Case 3
ESR1	6.37	5.67	13.12
ESR2	2.14	−2.26	1.12
FSHR	−1.57	−10.67	1.04
LHCGR	−1.45	−4.20	1.08
GnRHRII	4.12	10.59	106.24
INHBA	−1.48	−3.08	−1.22
INHA	−2.46	−4.36	−1.52
AR	−1.91	−9.35	1.13
STAR	2.76	−11.26	1.93
HSD17β7	1.63	−4.71	1.55
BHMT	3.02	11.45	304.78
INSL3	−1.04	−1.17	4.55
CRHR1	−1.02	2.11	9.55
PDGFRα	−3.96	1.28	−1.62

Mean fold changes (FC) in the expression were calculated using the 2^−ΔΔCt method. FC values < 1 were substituted with a negative inverse of the original FC values

ESR1 estrogen receptor 1, ESR2 estrogen receptor 2, FSHR follicle-stimulating hormone receptor, LHCGR luteinizing hormone/choriogonadotropin receptor, GnRHRII gonadotropin-releasing hormone receptor II, INHBA inhibin subunit beta A, INHA inhibin subunit alpha, AR androgen receptor, STAR steroidogenic acute regulatory protein, HSD17β7 hydroxysteroid 17-beta dehydrogenase 7, BHMT Betaine Homocysteine S-Methyltransferase, INSL3 Insulin-like peptide 3, CRHR1 Corticotropin releasing hormone receptor 1, PDGFRα Platelet-derived growth factor receptor alpha

The mRNA expression of genes related to testicular function, steroidogenesis, and Leydig cell differentiation/function had some similarities but also distinct case-related patterns. In all cases, increased expression of estrogen receptor 1 (ESR1; mediating estrogenic effects), gonadotropin-releasing hormone receptor II (GnRHRII; involved in LH-independent control of gonadal function) and betaine homocysteine S-methyltransferase (BHMT; adult Leydig cells marker) was observed in larger testes. In contrast, expression of INHBA and inhibin subunit alpha (INHA) was decreased, implying a disturbance of FSH secretion. Decreased expression of ESR1, GnRHRII and BHMT was most pronounced in the smaller testis of Case 3, suggesting early-onset atrophy, as evidenced by histological observations. However, additional evidence would be required to support this conclusion. Unexpectedly, expression of corticotropin releasing hormone receptor 1 (CRHR1), a marker of fetal Leydig cells in rodents [29], was increased in the larger testis.

In addition, the larger testis in Case 1 had increased expression of estrogen receptor 2 (ESR2; mediates estrogenic effects) and steroidogenic acute regulatory protein (STAR; transports cholesterol into mitochondria) compared to the smaller testis, along with decreased expression of INHA (an inhibitor of pituitary FSH secretion) and platelet-derived growth factor alpha (PDGFRα; stem Leydig cell marker). This gene expression pattern implied active steroidogenesis in the larger testis and was consistent with other indicators, e.g., high plasma testosterone concentration, increased GTI and TI, and lower Leydig cell N:C ratio (see Tables 1 and 2). In contrast, Case 2 (a mature IC boar with a good response to immunocastration) had an opposite gene expression pattern. The larger testis had decreased expression of several key genes, including FSHR, luteinizing hormone/choriogonadotropin receptor (LHCGR), INHBA, INHA, AR, STAR, and hydroxysteroid 17-beta dehydrogenase 7 (HSD17β7), consistent with low plasma testosterone and other features, including low GT and TI and an increased Leydig cell N:C ratio in both testes.

Discussion and conclusions

In this case report, assessing testicular asymmetry in a cohort of young (pubertal) boars and young and mature IC, there was a mean prevalence of PTA of 5.85%, similar to the prevalence reported in healthy adolescent boys at mid-puberty (5.94%; [1]), but roughly half the prevalence in Göttingen minipigs (11.4%; [8]). Given the comparable prevalence of testicular asymmetry in boars and IC, severe cases of unilateral testicular atrophy identified in this study likely represented random findings rather than effects attributable to immunocastration.

The prevalence of PTA > 20%, a threshold commonly used to define a persistent small testis [4], was only 1.55% in the present study. This was markedly lower than the prevalence reported in inbred Duroc strains (22.3%; [3]) and in culled boars (21.4%; [4]), in which the high incidence of testicular asymmetry was attributed to testicular atrophy occurring after sexual maturity. Although the incidence of testicular asymmetry in the general Duroc population is not known, this breed appears to have an unidentified genetic predisposition to orchitis or epididymo-orchitis that negatively affects spermatogenesis and significantly increases the prevalence of testicular asymmetry [3]. According to Creasy et al. [24], epididymo-orchitis may develop as a result of an autoimmune condition. Interestingly, Case 2 was also a Duroc boar that had been eliminated from an AI centre due to low sperm quality ([19]), one of the most common reasons for culling boars (24%; [30]). However, histological signs of inflammation were not observed in Case 2 (mature Duroc boar). In contrast, Case 1 (a Large White × Landrace crossbreed) had lymphocytic infiltration in the interstitium of the smaller testis, suggesting orchitis likely contributed to testicular atrophy, consistent with Noguchi et al. [3].

Despite differences in immunocastration efficiency reflected in plasma testosterone concentrations, urogenital tract indices (GTI, TI) and histomorphometric parameters, qPCR analysis revealed changes in gene expression common to all three cases. In particular, BHMT, GnRHRII and ESR1 were downregulated, whereas INHBA and INHA were moderately upregulated in atrophic testes.

The most pronounced downregulation of the marker for adult Leydig cells BHMT (FC > 300-fold)) was observed in Case 3. In combination with histologic features, which include partially luminized seminiferous tubules and gonocytes, we inferred that in Case 3, testicular atrophy occurred at a prepubertal stage of testicular development before appearance of adult Leydig cells. In Cases 1 and 2, the difference in BHMT expression between larger and smaller testes was moderate (3- and 11-fold in Cases 1 and 2, respectively), suggesting that atrophy occurred in the pubertal/postpubertal stage. Zhang et al. [31] reported that stem, immature and adult Leydig cells were detectable in the testes of black Guanzhong pigs at puberty, whereas only adult Leydig cells were detected in sexually mature animals. However, it was recently reported that up to 20% of fetal Leydig cells persisted into adulthood in mice and rats [32]. In support of the latter, increased expression of CRHR1 was observed in the larger testes of Cases 2 and 3, suggesting the presence of fetal Leydig cells. However, this interpretation should be made with caution, as CRHR1 has only been validated as a fetal Leydig cell marker in rodents [29]. These data suggested that fetal Leydig cells can be maintained in both young (182 days old; Large White × Landrace) and mature (480 days old; Duroc) boars of modern pig breeds, in contrast to black Guanzhong pigs, in which they disappear by puberty (90 days; [31]). This may reflect breed-specific patterns of Leydig cell development, possibly due to selection for reproductive performance and leanness, and warrants further study.

The present results regarding changes in GnRHRII expression also supported its role as an LH‑independent regulator of steroidogenesis in the porcine testis [33] and underlined its involvement in spermatogenesis [34, 35]. Regarding ESR1, data from rodent models suggest that ESR1 expression is decreased in testicular atrophy, consistent with our observation [36].

Cauda epididymides associated with smaller (atrophic) testes had substantial histomorphometric changes, including reduced luminal content, epithelial hyperplasia (cribriform changes), and increased thickness of the peritubular musculature. The latter two changes were only observed in Cases 1 and 2. Cribriform changes result from epithelial folding and bridging caused by contractions of epididymal ducts [37], which reduce duct diameter and occur in response to decreased sperm and seminal fluid production of atrophic testes [24]. Increased thickness of the peritubular smooth muscle layer likely reflected an adaptive response of the epididymis to increased duct contractions associated with testicular atrophy.

The very small sample size (n = 3) limited generalizability of findings and the retrospective design may have introduced biases, restricting causal interpretations. In addition, the absence of contemporaneous controls for environmental factors complicated analysis, as these factors could have influenced the observed outcomes. Finally, the use of a limited gene panel through targeted qPCR restricted the scope of molecular insights, underscoring the need for more comprehensive, transcriptome-wide analyses to better understand underlying mechanisms.

To conclude, unilateral testis atrophy was not reflected by plasma testosterone concentrations nor the efficacy of immunocastration. Instead, it was associated with impaired spermatogenesis, Leydig cell atrophy and decreased expression of genes such as GnRHRII, ESR1 and BHMT, plus histoarchitectural changes in the epididymis. These findings highlighted the importance of assessing histological and molecular changes and not simply measuring plasma testosterone concentrations in cases of unilateral testicular atrophy.

Abbreviations

AR

Androgen receptor

B-2-M

Beta-2-microglobulin

BHMT

Betaine homocysteine S-methyltransferase

CRHR1

Corticotropin releasing hormone receptor 1

Ct

Threshold cycle

EM

Entire male

ESR1

Estrogen receptor 1

ESR2

Estrogen receptor 2

FC

Fold change

FSHR

Follicle-stimulating hormone receptor

GnRH

Gonadotropin-releasing hormone

GnRHRII

Gonadotropin-releasing hormone receptor II

GTI

Genital tract index

HE

Hematoxylin and eosin

HSD17β7

Hydroxysteroid 17-beta dehydrogenase 7

IC

Immunocastrated pigs

INHA

Inhibin subunit alpha

INHBA

Inhibin subunit beta

INSL3

Insulin- like peptide 3

LHCGR

Luteinizing hormone/choriogonadotropin receptor

N

C ratio: Nucleus to cytoplasm ratio

PDGFRα

Platelet-derived growth factor alpha

PTA

Percentage of testicular asymmetry

qPCR

Quantitative polymerase chain reaction

STAR

Steroidogenic acute regulatory protein

TI

Testes index

ΔCt

Delta Ct

18 s rRNA

Eukaryotic ribosomal (r) 18s RNA

Authors’ contributions

PF performed histomorphometric examinations and data presentation. GF conducted genital tract measurements and sample collection at the processing line and critically revised the manuscript. MŠ helped interpret the data and critically revised the manuscript. MV designed the study and was a major contributor in preparing the manuscript. All authors read and approved the final version of the manuscript.

Funding

The authors received financial support from the Slovenian Research and Innovation Agency programs P4-0053 and P4-0133 and ERA NET SusAn SuSi (631 − 10/2015/7; Sustainability in pork production with immunocastration).

Data availability

All data generated or analyzed during this study are included in this published article.

Declarations

Ethics approval and consent to participate

The authors confirm that the study was conducted in accordance with all relevant institutional and EU regulations. Immunocastration with Improvac^® (Zoetis), a licensed veterinary medicinal product approved for use in pigs, is considered a standard husbandry practice rather than a scientific procedure and therefore does not require ethical approval. All tissue samples were collected post-mortem after slaughter, ensuring that no animals were euthanized or subjected to additional procedures for research purposes. Thus, according to Directive 2010/63/EU [38] and the decision of the Ethical Committee of the Veterinary Faculty, University of Ljubljana (decision No. 5-4-2018/5), the study was not subject to ethical protocols. The Institute of Preclinical Sciences, Veterinary Faculty of the University of Ljubljana, is registered with the Veterinary Administration of the Republic of Slovenia (official registration no. SI-B-07-22-07) and the use of animal by-products for research purposes is authorized (permit no. U34453-1/2024/5).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.