Stallion Testes Research Articles

Optimizing the preparation of paraffin sections from stallion testes.

The preparation of paraffin sections is an important experimental technique in animal histological research, and key factors that determine the quality of a section include the dehydration time, waxing time, and drying temperature of the paraffin section. Paraffin sections obtained from testis tissue of adult horses exhibited higher quality with clear tissue structure and complete cell morphology after they underwent gradient dehydration for 6 hours, were immersed in wax for 60 minutes, and were dried in a 75-degree oven for 15 minutes. The detailed, optimized procedures that are developed in the current study may simplify histological experiments and research on equine testes.

Brain-derived neurotrophic factor and neurotrophic tyrosine receptor kinase-2 in Stallion Testes: Insights into Seasonal Changes and Potential Roles in Spermatogenesis

Brain-derived neurotrophic factor and neurotrophic tyrosine receptor kinase-2 in Stallion Testes: Insights into Seasonal Changes and Potential Roles in Spermatogenesis

Changes in characteristics of spermatogonial stem cells in response to heat stress in stallions

Spermatogonial stem cells (SSCs) are essential for the maintenance of male fertility and survival of species. Environmental conditions, notably heat stress, have been identified as important causes of male infertility and have a negative impact on SSCs. Animals with cryptorchid testes (CT) are optimal models for the study of long-term heat stress-related changes in germ cells. The effect of heat stress on germ cells differs depending on the spermatogenesis stage. Thus, verifying whether the specific phase of spermatogenesis is dependent or independent of heat stress in stallions is important. We evaluated the heat stress-related response of SSCs by comparing the relative abundance of mRNA transcripts and expression patterns of the undifferentiated embryonic cell transcription factor 1 (UTF-1) and deleted in azoospermia-like (DAZL) in the seminiferous tubules of CT and normal testes (NT) of stallions using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunofluorescence, and western blotting. We also analyzed the relative abundance of mRNA of different proliferative markers, including minichromosome maintenance 2 (MCM2), marker of proliferation Ki-67 (MKI-67), and proliferating cell nuclear antigen (PCNA). Testicular tissues from four Thoroughbred unilateral cryptorchid postpubertal stallions were used in this study during the breeding season. The relative abundance of the mRNA transcripts of UTF-1 and MCM2 was significantly upregulated in the CT group than that of those in the NT group. In contrast, the relative abundance of the mRNA transcripts of DAZL was significantly downregulated in the CT group than that of those in the NT group. Western blot quantification showed that the relative intensity of UTF-1 protein bands was significantly higher, while that of DAZL protein bands was significantly lower in the CT group than in the NT group. Immunofluorescence studies showed that the number of germ cells immunostained with UTF-1 was significantly higher while immunostained with DAZL was significantly lower in the CT group than that in the NT group. The higher expression level of UTF-1 in the CT group shows that undifferentiated SSCs are not affected by long-term exposure to heat stress. These results also indicate that germ cells after differentiation phase are directly affected by heat-stress conditions, such as cryptorchidism, in stallions.

Expression pattern of germ cell markers in cryptorchid stallion testes.

Cryptorchidism affects spermatogenesis and testis development, often resulting in stallion subfertility/infertility. This study aims to identify the specific germ cells impacted by cryptorchism in stallions. In a previous study, we found that PGP9.5 and VASA are molecular markers expressed in different germ cells within stallions. Herein, we assessed the heat stress-induced response of spermatogonial stem cells (SSCs) in the seminiferous tubules (ST) of cryptorchid stallion testes (CST) and normal stallion testes (NST). This goal was accomplished by comparing PGP9.5 and VASA expression patterns through reverse transcription quantitative PCR and immunofluorescence assays. We also compared the cross-sectional ST area between groups. Six post-pubertal Thoroughbred unilateral cryptorchid stallions were used. The relative abundance of the mRNA transcripts of PGP9.5 and VASA was significantly upregulated in the NST group than in the CST group. Additionally, the cross-sectional ST area and localization of PGP9.5 and VASA in germ cells were significantly higher in the NST group than in the CST group. Regarding Leydig cells, PGP9.5 staining was observed in both groups. Spermatogonia, primary spermatocytes and secondary spermatocytes were immunostained with VASA in the NST group, while immunostaining was only observed in spermatogonia in the CST group. These results indicate long-term exposure to heat stress conditions, such as cryptorchidism, directly impacts germ cell proliferation and differentiation, leading to impaired spermatogenesis and compromised fertility in stallions.

Molecular Responses of HSP60 and HSP90 to Temperature Variations in Stallion Testes during Breeding and Non-breeding Seasons

Molecular Responses of HSP60 and HSP90 to Temperature Variations in Stallion Testes during Breeding and Non-breeding Seasons

Effects of therapeutic ultrasound and moderate heat on stallion testes

Transplantation of stem cells into dysfunctional testes is currently being investigated as a therapeutic option for men and stallions with advanced testicular degeneration. This series of “proof of concept” studies aimed to identify a safe and efficient method of inducing severe testicular degeneration to create an optimal equine recipient model for intratesticular stem cell transplantation (SCT). Two ex vivo and two in vivo experiments were conducted. At first, forty testes obtained from castrations were used to identify an effective therapeutic ultrasound (TUS) device and the protocol for increasing intratesticular temperature in stallions. Six min of treatment using the Vetrison Clinic Portable TUS machine raised the intratesticular temperature by 8°C-12.5 °C. This protocol was applied to treat three scrotal testes in three Miniature horse stallions, three times, every other day. Contralateral testes served as controls. There were signs of slight tubular degeneration in treated testes two and three weeks after TUS treatment. The number of seminiferous tubules (STs) with exfoliated germ cells (GCs) was increased in one testis only, three weeks after treatment. The degree of apoptosis of GCs was higher in each treated testis in comparison to the contralateral control testis. Next, the ability of various heating devices to increase intratesticular temperatures to at least 43 °C in stallion testes was tested, using twenty testes obtained from castrations. ThermaCare® Lower Back & Hip Pain Therapy Heatwrap (TC heat wrap) reliably increased intratesticular temperatures and kept them continuously between 43 °C and 48 °C for seven to 8 h. In the follow-up in vivo study, the left testes of three Miniature horse stallions were treated with TUS, after which both testes of each stallion were treated with moderate heat provided by the TC heat wrap (three times, every other day, for 5 h each time). There were signs of moderate tubular degeneration in the samples from all treated testes obtained three weeks after treatments (Heat only or Heat/TUS): areas with hypospermatogenesis, spermatogenic arrest, vacuolized Sertoli cells, numerous STs with exfoliated GCs, increased degree of GCs apoptosis, and changes in three histomorphometric numeric attributes of STs. We concluded that TUS or TC wraps increase intratesticular temperature of the isolated stallion testes. Further, treatment with TUS or moderate heat may induce mild to moderate degenerative changes in stallion testes. However, to achieve more robust result – severe testicular degeneration, our treatment protocol has to be modified.

Seasonal changes in the expression of molecular markers of stallion germ cells

The economic impacts of infertility and subfertility of stallions greatly influence the horse breeding industry. Self-renewal and differentiation of spermatogonial stem cells are the initial processes to maintain an adequate sperm population. Thus, understanding these processes may provide useful information to reveal the causes and remedies of subfertile and infertile stallions. Stallions are seasonal breeders. About 50% of the sperm population is reduced during the non-breeding season (NBS) in stallions. The seasonal regulation of spermatogenesis renders stallions as ideal models to understand the process of sperm production. Furthermore, comparing internal and external factors related to spermatogenesis during the breeding season (BS) and NBS may provide a solution for subfertile/infertile stallions. It is especially pertinent to study the expression pattern of different protein markers during undifferentiated, differentiating, and differentiated spermatogonia. Deleted in azoospermia-like (DAZL), undifferentiated cell transcription factor 1 (UTF-1), and protein gene product 9.5 (PGP9.5) are the molecular markers expressed at different stages of spermatogenesis. However, whether the expression pattern of these molecular markers is similar throughout the year in stallion remains undetermined. The objectives of this study were to (1) investigate the expression pattern and localization of DAZL, UTF-1, and PGP9.5 within seminiferous tubules and (2) evaluate the relative mRNA levels of these three germ cell markers in stallion testes during BS and NBS. Immunohistochemistry was performed to check and compare the expression pattern and localization of DAZL, UTF-1, and PGP9.5 antibodies. Reverse transcription-quantitative PCR analysis was performed to calculate the relative mRNA expression levels in the testes. Testicular tissues from thoroughbred stallions were collected during routine castration that was carried out in field conditions. Immunostaining of germ cells with DAZL and UTF-1 in BS and NBS were not significantly different. However, the relative mRNA expression levels of DAZL and UTF-1 were significantly different in both groups. Interestingly, the immunolabeling and the relative mRNA expression of PGP9.5 were significantly different between BS and NBS. From these results, it is hypothesized that the expression level of these putative molecular markers might be gonadotropin-dependent in stallion testes.

Evaluation of testicular echotexture with Ecotext as a diagnostic method of testicular dysfunction in stallions

This study aimed to assess if Ecotext, a new software for evaluation of testicular echotexture, is a good method for diagnosis of stallions with testicular dysfunction (TD). Relationships between Ecotext parameters and sperm motility and production, testicular volume, and testicular blood flow were also studied. Ecotext provides a total of six echotexture parameters: Ecotext 1 (black pixels), 2 (white pixels) and 3 (grey pixels), and another 3 parameters related to hypoechogenic areas: Ecotext tubular density (ETD), Ecotext tubular diameter (ETd), and Ecotext tubular area (ETA). Stallions (n = 33) were assessed using proven diagnostic techniques (spermiogram, B-mode and Pulse Doppler ultrasound), and subsequent analysis with Ecotext. Animals were classified as “control stallions” (n:21, acceptable semen quality), and “stallions with TD” (n:12, poor semen quality (TM < 60%, PM < 45% and total nº of sperm with PM < 2000 × 106 spz), that were subdivided into “induced TD group” (immunized, anti-GnRH vaccine) and “acquired TD group”. The acquired TD group showed differences in all Ecotext parameters in relation to controls (Ecotext 1:0.11 ± 0.17 vs 2.82 ± 2.52, Ecotext 2:1584.0 ± 575.8 vs 388 ± 368.2, Ecotext 3:134.2 ± 9.26; ETA: 2.14 ± 0.59 vs 5.40 ± 1.90; ETd: 65.66 ± 6.27 vs 86.93 ± 10.65 and ETD: 92.35 ± 11.24 vs 132.10 ± 16.35, p ≤ 0.001). Results suggest acquired TD stallions were suffering testicular degeneration with loss of architecture and function as all Ecotext parameters were altered in relation to controls. Induced TD horses only showed a reduction in ETD (116.2 ± 8.59 vs 132.10 ± 16.35, p ≤ 0.001), despite all sperm parameters being worse. These findings suggested immunized stallions probably only experience an acute loss of testicular functionality and parenchyma architecture is likely not affected since differences in Ecotext parameters with control stallions were not detected. ETD was the best parameter to identify animals with TD (AUC: 0.84, optimal cut-off value of 124.3 seminiferous tubules/cm2). Correlations were found between ETD and Doppler indices (PI: 0.60; RI: 0.47 p ≤ 0.001), total testicular volume (r: 0.48; p ≤ 0.05) and sperm motility (TM:0.51; and PM:0.54; p ≤ 0.001) and production (r:0.51; p ≤ 0.001). In summary, Ecotext could identify changes in testicular echotexture of stallions with TD. Results open the possibility for new research focused on establishing the relationship between Ecotext parameters and histomorphometry features in stallion testes.

Olfactory receptor (OR7D4 and OR1I1) expression in stallion testes

Olfactory receptor (OR7D4 and OR1I1) expression in stallion testes

Effects of intravenous multiple busulfan injection on suppression of endogenous spermatogenesis in recipient stallion testes.

Preparation of recipient stallions is critical step to produce donor spermatogonial stem cell (SSC) derived sperm using transplantation technique. This study was conducted to evaluate the effects of intravenous busulfan infusion on germ cell depletion, semen production, and libido in stallions. Six Thoroughbred stallions were separated into two treatment groups: 1) a multiple low-dose (2.5 mg/kg bw for the first 4 weeks and 5 mg/kg bw for the 5th week); and 2) control group treated with PBS. Testicular samples were obtained at 11 weeks and classified into three different patterns of spermatogenesis, such as normal, Sertoli cell only, and destroyed. Semen collection and libido experiments were performed 1 week before treatment, and 4 and 8 weeks after treatment. For the sperm analysis, total spermatozoa and motility were measured using a light microscope with a motility analyzing system. In the multiple low-dose group, the numbers of tubules categorized as Sertoli cell only were significantly higher than those in the control as well as the total population and total/progressive motility of sperm were significantly decreased 8 weeks after the start of the treatment. The sperm production and motility in the multiple low-dose group appears to be reduced, while libido was maintained. In conclusion, multiple administration of 2.5 mg/kg bw busulfan depletes endogenous germ cells in the stallion recipients for SSC transplantation.

Oxytocin receptor expression in stallion testes and epididymides

Endocrine, paracrine, and autocrine factors orchestrate the development and physiology of the stallion reproductive system. Oxytocin (OXT) is one of the critical endocrine, paracrine, and autocrine factors for the male reproductive system. Previous studies have investigated OXT receptor (OXTR) expression in testes and epididymides, including humans, marmosets, macaques, swine, and sheep. This study aimed to explore (1) OXTR localization in the testes and epididymides and (2) the seasonal modification of OXTR expression in the testes. Adult stallion testis and epididymis samples were prepared using routine castration procedures. Reverse-transcription PCR was performed to detect the presence of OXTR messenger RNA (mRNA) in the testes. Western blot procedure was performed to confirm the cross-reactivity of OXTR antibody to horse OXTR. Immunohistochemistry was performed to detect OXTR protein expression in the testes and epididymides. Oxytocin receptor mRNA was detected in the stallion testes. The OXTR protein band was observed at 55 kDa. Interestingly, the relative intensity of the OXTR protein band varied between nonbreeding and breeding season. The OXTR protein level in the testes collected during the breeding season was higher than that during the nonbreeding season. Oxytocin receptor localization was observed in the cytoplasm of Type A spermatogonia and spermatid. Oxytocin receptor protein expression was also observed in the cytoplasmic area of Leydig cells and the membrane of the seminiferous tubules. The cytoplasm of principal and basal cells in the caput, corpus, and cauda was also immunolabeled with OXTR antibody. In conclusion, based on the expression of OXTR in tissues of testes and epididymides, OXT–OXTR system may be a critical factor for stallion testicular and epididymal function. In addition, according to the seasonal alteration of intensity, the OXT–OXTR system may be associated with seasonal changes in the reproductive system in stallions.

Comparative Transcriptomics Analysis of Testicular miRNA from Cryptorchid and Normal Horses.

Simple SummaryThe testis is an important organ for mammals, and testicular microRNA expression is associated with male fertility to a certain extent. Cryptorchidism is the failure of one or both testes to descend into the scrotal sac. It is a common congenital malformation in horses. The major clinical consequence of this abnormality is impaired fertility. The expression of testicular microRNAs is influenced by many factors, including high temperature and disease, in cryptorchid horses. Here, we investigated the microRNA expression levels of normal and retained testes. Their expression patterns showed significant differences. In addition, we obtained comprehensive expression data for equine testicular microRNA, which is fundamental information for further analysis.In the biological process of testicular spermatogenesis, the expression and interaction of many genes are regulated by microRNAs (miRNAs). However, comparisons of miRNA expression between descended testes (DTs) and undescended testes (UDTs) are rarely done in horses. In this study, we selected two UDTs (CKY2b and GU4b) from Chakouyi (CKY) and Guanzhong (GU) horses and eight DTs (GU1–3, CKY1, CKY3, CKY2a, GU4a, and GU5). Three groups were compared to evaluate expression patterns of testicular miRNA in stallion testes. Group 1 compared normal CKY horses and GU horses (CKY1 and CKY3 vs. GU1–3). Group 2 (CKY2a and GU4a (DTs) vs. CKY2b and GU4b (UDTs)) and group 3 (GU1–3, CKY1, CKY3 (DTs) vs. CKY2b and GU4b (UDTs)) compared the expression levels in unilateral retained testes to normal testes. The results show that 42 miRNAs (7 upregulated and 35 downregulated) had significantly different expression levels in both comparisons. The expression levels of eca-miR-545, eca-miR-9084, eca-miR-449a, eca-miR-9024, eca-miR-9121, eca-miR-8908e, eca-miR-136, eca-miR-329b, eca-miR-370, and eca-miR-181b were further confirmed by quantitative real-time PCR assay. The target genes of differentially expressed miRNAs in three comparisons were predicted, and the functions were annotated. The putative target genes of the 42 co-differentially expressed miRNAs were annotated to 15 functional terms, including metal ion binding, GTPase activator activity, zinc ion binding, intracellular, cytoplasm, and cancer pathways, and osteoclast differentiation. Our data indicate that the differentially expressed miRNAs in undescended testis suggests a potential role in male fertility and a relationship with cryptorchidism in horses. The discovery of miRNAs in stallion testes might contribute to a new direction in the search for biomarkers of stallion fertility.

Expression of enzymes involved in polyunsaturated fatty acid synthesis in the stallion testis and epididymis.

The aim of the present study was to characterise key enzymes involved in polyunsaturated fatty acid (PUFA) synthesis in the testis and epididymis collected from 2-year-old healthy warmblood stallions (n=10). The mRNA expression of fatty acid synthase, the Δ9-, Δ6-, Δ5- and Δ4-desaturases and elongases 6, 5 and 2 (encoded by the fatty acid synthase (FASN), the stearoyl-CoA desaturase (SCD), the fatty acid desaturase 2 (FADS2), the fatty acid desaturase 1 (FADS1), the delta 4-desaturase, sphingolipid 1 (DEGS1), ELOVL fatty acid elongase 6(ELOVL6), ELOVL fatty acid elongase 5 (ELOVL5), ELOVL fatty acid elongase 2 (ELOVL2) genes respectively) was determined in equine testis and epididymis. All enzymes were present in testicular tissue and along the epididymis, but mRNA expression differed among localisations. The protein localisation of FADS1, FADS2 and ELOVL5 was determined by immunohistochemistry. In the testes, FADS1 was expressed in the germinal cells and ELOVL5 was expressed in germinal and Leydig cells; FADS2 was not detected. In the epididymis, FADS1 and FADS2 were expressed in the principal and basal cells, whereas ELOVL5 was found only in the principal cells of the caput. All three enzymes were present in epididymal vesicles secreted by an apocrine mechanism. These results suggest active PUFA metabolism during spermatogenesis and epididymal sperm maturation in stallions.

Dexamethasone downregulates expression of several genes encoding orphan nuclear receptors that are important to steroidogenesis in stallion testes

Glucocorticoids impair testosterone synthesis by an unknown mechanism. Stallions treated with the synthetic glucocorticoid dexamethasone had testes collected at 6 or 12 hours postinjection. The testicular expression of selected genes encoding nuclear receptors and steroidogenic enzymes was measured. At 6 hours, dexamethasone treatment decreased levels of NR0B2, NR4A1, NR5A1, and NR5A2 messenger RNAs (mRNAs) and NR5A2 mRNA levels remained depressed at 12 hours. In contrast, dexamethasone increased levels of NFKBIA mRNA at both time points. At 6 hours, dexamethasone did not alter levels of NR0B1, NR2F1, NR2F2, NR3C1, CYP11A1, CYP17A1, CYP19A1, DHCR24, GSTA3, HSD3B2, HSD17B3, LHCGR, or STAR mRNAs. In primary cultures of Leydig cells, 10 -9 and 10 -7 M dexamethasone decreased levels of NR4A1 and NR5A1 mRNAs and increased those of NFKBIA mRNA. Our discovery that dexamethasone downregulates NR4A1, NR5A1, and NR5A2 genes, known to be important for testicular functions, may be part of the mechanism by which glucocorticoids acutely decreases testosterone.

Solexa Profiling Identifies Differentially Expressed MiRNAs Between Sexually Immature and Mature Equine Testis

ABSTRACT MicroRNAs (miRNAs) are a class of short non-coding RNAs identified as potent regulators of gene expression. Previous studies have suggested that miRNAs are involved in mammalian spermatogenesis. Stallion fertility is an important trait for the horse breeding industry, but stallion fertility traits are largely ignored in the industry. In this study, we generated expression profiles of miRNAs in foal (immature) and stallion (mature) testes using Solexa sequencing. We identified 438 known and homologous equine miRNAs and 199 novel miRNAs which were distributed among all the chromosomes. The two developmental stages showed significant differences in miRNA expression patterns. Our result expands the horse miRNA database and provided additional information on the stallion fertility and possible spermatogenesis regulation through specific miRNAs.

Technical aspects of mesenchymal stem cells transplantation into the stallion testes

Changes in anatomical, behavioural and seminal characteristics were measured throughout the year in seven Beni Arouss bucks maintained at a latitude of 35 °N. Testicular size varied significantly. There were the greatest values during the summer and autumn for scrotal circumference and least in the winter for testicular length. Values for most variables related to sexual behavior were not affected by season except the number of mounts before first ejaculation which was least in the summer. No differences were detected between seasons in terms of semen volume. The sperm concentration, viability and percentage of normal sperm, however, were less during the winter period. The size of sperm heads was larger in the autumn and motility of spermatozoa was greater in the summer and autumn. Results from the correlation analysis indicated that the majority of conventional seminal characteristics were correlated with testis measurements (P < 0.05; r < 0.5). Values for conventional and CASA motility variables indicated there were positive correlations between viability and progressive motility (r = 0.4; P < 0.05) and between the percentage of normal sperm and straight line and average path velocity (r = 0.4; P < 0.05). A global reproduction performance score was established for each buck, which allowed for assessment of magnitude of seasonal changes for each individual. These global score values indicated there was a greater reproductive performance for all bucks during summer and autumn. This study described for the first time seasonal variations of reproductive characteristics of Beni Arouss bucks and results indicate that even though there is a maximal capacity for reproductive performance during the summer and autumn, breeding should be possible throughout the year.

Anti-Müllerian hormone as a biomarker for acute testicular degeneration caused by toxic insults to stallion testes

Recently, anti-Müllerian hormone (AMH) was validated as a reliable marker of testicular damage caused by various chemotherapy drugs in humans and in mice. In horses, the reference values of AMH concentrations in normal stallions, during different seasons of a year, have been recently reported. However, this hormone was not evaluated in subfertile or infertile stallions with testicular damage. Therefore, the objective of this study was to investigate the effects of experimentally induced testicular degeneration on the concentration of AMH in stallions. Severe but transient testicular degeneration was induced in six Miniature horse stallions, in two, separate experiments (three stallions in each experiment), by the administration of a single dose of the contraceptive compound RTI-4587-073(l). Six different stallions served as controls (three stallions in each experiment). Treated and control stallions were switched between the experiments. Concentrations of AMH were determined in 78 samples of blood plasma collected during the first experiment and in 24 samples collected during the second experiment. Furthermore, the expression of AMH in 30 samples of testicular parenchyma, collected from these stallions during the second experiment, was also evaluated, using immunohistochemistry (IHC) and objectively analyzed using computerized methods. During the first experiment, the concentrations of AMH in blood increased significantly in treated stallions (P < 0.05), reaching a 62–151% change from the baseline by day 10 after treatment, before gradually decreasing to the pretreatment levels. There was no change in blood AMH concentration in control stallions. Only a trend to increase AMH concentration was observed in treated stallions during the second experiment (P = 0.055). The labeling for immunoreactive AMH in the Sertoli cells gradually increased after treatment, which was confirmed by the significantly increased IHC optic density score value (P < 0.05) and significantly decreased percentage contribution of negative pixels at fourth week after treatment (P < 0.05). We concluded that AMH is a promising candidate as a biomarker of testicular damage in stallions caused by toxic insults that lead to testicular degeneration.

Relationship between echotextural and histomorphometric characteristics of stallion testes

The goal of this study was to investigate correlations between objective measures of testicular echotexture and histomorphometric attributes related to the histological composition of stallion testes. Fifty-four scrotal testes were obtained from three groups of stallions during routine castrations: colts <1 yr old (n = 18), young stallions 1–5 yrs old (n = 27), mature stallions > 5 yrs old (n = 9). In addition, two scrotal testes with degeneration, 16 retained inguinal and 10 retained abdominal testes were surgically obtained. Cross-sectional and longitudinal ultrasonograms were obtained for each testis. Mean numerical pixel values (NPVs) as well as pixel standard deviations (PSDs) were determined for each image (ImageJ-1.5 software). Histomorphometric attributes of the seminiferous tubules (STs) were derived (three tissue samples per each testis) using image analysis software [relative STs area: RSTA = ST area/total cross-sectional area (TA) x 100%; relative STs lumen: RSTL = ST lumen area/TA x 100%; individual ST area; ISTA; individual ST lumen: ISTL; seminiferous epithelium height: SHE]. Degree of fibrosis was graded semi-quantitatively (0–3) in samples from 17 testes. All measures of testicular echotexture as well as all histomorphometric attributes of STs had highest values when obtained from the scrotal testes of young and mature stallions (P < 0.05). The NPVs and PSDs from the ultrasonographic images of the scrotal testes were significantly correlated with all histomorphometric attributes of STs (P < 0.001). However, there was no correlation between the majority of these measures and attributes if each group of the scrotal testes was analyzed separately. The NPVs from the ultrasonographic images of the retained inguinal testes were correlated with RSTA, RSTL, ISTA, and ISTL, while the NPVs from the retained abdominal testes were not correlated with any of the histomorphometric attributes of the STs. Testes with high degree of fibrosis had variable values of pixel intensity and pixel heterogeneity. Based on the results of this study, we concluded that the pixel intensity and pixel heterogeneity of stallion testes increase during the first year of life and remain stable in young and mature stallions. These changes occur in parallel to the development of the seminiferous tubules. Testicular echogenicity in stallions does not seem to reflect degree of testicular fibrosis. Retained abdominal testes have lower and less heterogeneous echogenicity than scrotal testes from stallions that are more than one year old. While pixel analysis cannot replace biopsy in assessing testicular histomorphology in young and mature stallions, testicular echogenicity is a good indicator of peri-pubertal growth and expansion of the seminiferous tubules.

The Lin28 Expression in Stallion Testes.

The molecular markers for specific germ cell stages can be utilized for identifying, monitoring, and separating a particular stage of germ cells. The RNA-binding protein Lin28 is expressed in gonocytes of human fetal testes. The Lin28 expression is restricted to a very small population of spermatogonial cells in human, mice, and monkey. The main objective of this study was to investigate the expression pattern of Lin28 in stallion testes at different reproductive stages. Based on the presence or absence of full spermatogenesis and lumina in seminiferous tubules, the testicular samples were categorized into two reproductive stages pre-pubertal and post-pubertal. We performed a reverse transcription polymerase chain reaction to confirm the presence of Lin28 mRNA in the testicular tissues and a western blot analysis to verify the cross-reactivity of rabbit Lin28 antibody with horse testicular tissue. For immunohistochemistry, Lin28 (rabbit anti-human), GATA4 (goat anti-human) or DAZL (goat anti-human) antibodies were used. The results of RT-PCR confirmed the expression of Lin28 mRNA in the stallion testes. The western blot analysis showed that the expression of 28 kDa Lin28 protein was localized in the cytoplasm of spermatogonia at both reproductive stages. The numbers of Lin28-positive germ cells per 1000 Sertoli cells in pre- and post-pubertal stages were 253 ± 8.66 and 29.67 ± 2.18, respectively. At both reproductive stages, all Lin28 positive cells showed no co-stained with GATA4 antibody, whereas only some of the Lin28-positive germ cells showed co-staining with DAZL antibody. The results from whole-mount staining showed that the Lin28 expression was limited to Asingle (As) and Apaired (Apr) spermatogonia. In conclusion, Lin28 might be utilized as a molecular marker for undifferentiated spermatogonial stem cells when used with DAZL antibody.

Use of thermography for functional evaluation of stallion scrotum and testes

Use of thermography for functional evaluation of stallion scrotum and testes