Molecular expression and identification of caprine estrogen receptor gene 1 for fertility status in bucks

Estrogen and its receptors are essential for sexual development and reproduction. Oestrogen receptor alpha (ERα) is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene ESR1 (oestrogen receptor1) responsible for better fertility. ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. The present study was undertaken to investigate the expression and presence of ESR1 gene in fertile and low-fertile male goat breeds. We identified ESR1 gene through various molecular tools. Genotyping was carried out by high resonance melting analysis using Roche Light Cycler 480(LC-480) system and found three different genotypes. Genotypic frequency-AA (blue-0.67), BB(Red-0.2), AB(Green-0.08) with allele frequency A(0.71 and B (0.29). The predominance of this gene in head of epididymis in fertile bucks was confirmed by SDS-PAGE, Western blotting and immunohistochemistry. From the results, we corroborated that the present study provides a useful and effective way to predict male fertility in goat breeds, which in turn increases the percentage of fertility in flock leading to more number of offspring in a kidding season.     

RBP4基因在绵羊性成熟前后附睾中表达的研究

试验旨在研究视黄醇结合蛋白4（retinol binding protein 4,RBP4）基因在绵羊性成熟前和性成熟后附睾中的表达水平,探讨其在绵羊生殖机能调控方面的作用。以绵羊性成熟前和性成熟后附睾为材料,采用实时荧光定量PCR方法检测RBP4基因mRNA在绵羊性成熟前、后附睾中表达量的差异,采用免疫组化技术检测RBP4基因在绵羊性成熟前、后附睾头和附睾尾中蛋白的表达水平。结果显示,RBP4基因在绵羊附睾中性成熟前的表达量为1.3368,性成熟后的表达量为0.6450,虽然二者间的表达量差异不显著（P>0.05）,但性成熟前的表达量高于性成熟后的表达量。RBP4蛋白在性成熟前、后附睾头上皮层、平滑肌层、组织间质中均有阳性表达,主要位于上皮细胞质、平滑肌细胞质;在附睾尾上皮层、组织间质中有阳性表达,主要位于上皮细胞质。推测RBP4基因可能与附睾主细胞分泌功能和附睾头处的收缩功能有关,预示RBP4可能参与附睾微环境的调控,有利于精子成熟、运动和储存。     

Effect of Storage Time and Temperature of Equine Epididymis on the Viability,Motion Parameters,and Freezability of Epididymal Sperm

The recovery of sperm from the epididymal cauda may be the last chance to obtain genetic material when sudden death or serious injuries occur in valuable stallions. However, the lack of technical knowledge regarding the storage and transportation of the epididymis often prevents the preservation of the sperm. Therefore, the aim of this study was to compare sperm parameters of sperm obtained immediately after orchiectomy with sperm recovered from epididymal cauda at different times after storage at 5°C and at room temperature (RT). For that, 48 stallions of different breeds were used. In group 1 (control group), eight stallions were used, and the harvest of the epididymal sperm was performed immediately after orchiectomy. In group 2, 40 stallions were used, which were divided into five groups according to the storage time of the epididymis after orchiectomy (6, 12, 18, 24, or 30 hours), making a total of eight stallions per group. One epididymis of each stallion was stored at 5°C, and the contralateral epididymis was stored at RT, both for the same period. The sperm parameters of total motility, progressive motility, progressive linear velocity, curvilinear velocity, percentage of rapid sperm, and plasma membrane integrity were evaluated in all the groups after sperm recovery, resuspension in a sperm freezing diluent, and thawing. In conclusion, the storage of the testis-epididymis complex at 5°C provided better preservation of epididymal sperm than the storage at RT, and regardless of the temperature, the progressive motility is the sperm parameter that is most sensitive to storage time.     

Genetic Resistance of Barbari and Jamunapari Kids to Natural Infection with Gastrointestinal Nematodes

An investigation was made in 252 Barbari and Jamunapari kids to assess their resistance to natural infection with gastrointestinal (GI) nematodes and to establish indicator traits for such resistance in Indian goats. The indicator traits, faecal egg counts (FEC) and packed cell volume (PCV) were affected by various genetic and non-genetic factors. There were no breed differences in the FEC or PCV at 3, 6 or 9 months of age. Jamunapari male kids had a higher FEC than the female kids at 6 months of age. However, Barbari female kids had a higher FEC than the respective male kids at 9 months of age. At 6 months of age in both breeds, the kids born in the spring (March–April) had a higher FEC than those born in the autumn (October–November). The FEC of kids at 9 months of age was higher than at 3 or 6 months of age. Sire had a significant effect on PCV at 6 and 9 months of age. The kids born in the autumn had a higher PCV than those born in the spring. The PCV of male Barbari kids differed significantly from that of female kids at all the ages. The correlation coefficient of FEC on both body weight and body weight gain was negative, and there was a loss of body weight in the individuals with a high FEC.     

The use of ultrasonography in the reproductive evaluation of boars

The objective was to study the use of ultrasound as a complementary test in the breeding soundness evaluation in male pigs and study the pattern of echogenicity of the testicular parenchyma in boars of different racial groups. Twenty‐six adult boars from four different racial groups were used, 10 from the Piau breed (group 1), four from the commercial and finishing group (group 2), six Pietrain breed (group 3) and six from the Duroc breed (group 4). All animals were evaluated for breeding soundness evaluation and the ultrasound examination of the testicles. The groups of animals that were evaluated showed no difference in the main semen parameters that were evaluated, except for the sperm volume, concentration of the ejaculated sperm and the supravital staining; the lowest figures were for the animals from the Piau breed (group 1). In relation to the testicular biometrics, Duroc animals (group 4) had a greater scrotal width compared to the other groups. But when we assessed the intensity of pixels of the testicles, there was a difference between groups. The groups 2 (finishing animals), 3 (Pietrain) and 4 had no difference between themselves. Group 3 had greater pixel intensity in relation to group 1. Of the 26 animals studied, five showed an abnormality during ultrasound evaluation, like hydrocele, hyperechoic mass in the testicular parenchyma, cyst in the head of the epididymis and the presence of fluid in the head and tail of the epididymis. The various animal groups studied did not differ in the principal reproductive parameters evaluated, showing that despite the great variability of reproductive traits between breeds and within the same breed, the breeding soundness evaluation, the more complete it is, is essential for the selection of breeders and the ultrasonography of the reproductive system becomes an important addition in this examination.     

Abnormalities of the Testes and Epididymis in Bucks and Rams Slaughtered at Debre Zeit Abattoir,Ethiopia

A study was conducted at Debre Zeit export abattoir on 404 bucks belonging to four breeds and 167 rams of two breeds. The animals were selected from the slaughter flock using stratified sampling based on breed. Ante-mortem and post-mortem examinations were carried out after each animal was carefully identified. Various genital abnormalities were observed, among which testicular atrophy and epididymitis were the predominant genital problems in both bucks and rams. The prevalence of cryptorchidism was higher (p<0.05) in bucks than in rams, while epididymitis (p<0.05), sperm granuloma (p<0.01) and epididymal cysts (p<0.05) were more frequent in rams. In bucks, none of these abnormalities was significantly associated with age (p>0.05). However, testicular atrophy (p<0.01), epididymitis (p<0.01) and haemorrhagic lesions (p<0.05) were associated with breed, the prevalence being higher in Woito-Guji and Boran bucks than in the Arsi-Bale and Afar breeds. In rams, age affected (p<0.05) the incidence of testicular atrophy, epididymitis and sperm granulomas. Epididymitis, sperm granuloma and epididymal cysts were more frequent (p<0.01) in Adal rams than in Black Head Somalis.     

Gene and protein expression in the reproductive tract of Brazilian Somalis rams

Brazilian Somalis is a locally‐adapted breed of rams raised in tropical climate and native pastures. The present study was conducted to evaluate gene expression and proteome of the reproductive tract of such rams. Samples were collected from testes, epididymides, seminal vesicles and bulbourethral glands of four rams. Expression of clusterin (CLU), osteopontin (OPN) and prostaglandin D2 synthase (PGDS) genes were evaluated in all samples by real‐time PCR. Shotgun proteomic analysis was performed using samples from the head, corpus and cauda epididymides and from all other structures as well. Gene ontology terms and protein interactions were obtained from UniProtKB databases and MetaCore v.6.8 platform. CLU trasncripts were detected in the testes, epididymides, seminal vesicles and bulbourethral glands of the Somalis rams. The initial region and body of the epididymis had the greatest CLU expression. OPN mRNA was localized in all tissues of the ram reproductive tract. PGDS mRNA was detected in the testes and epididymides. Lable‐free mass spectrometry allowed the identification of 137 proteins in all samples. Proteins of the epididymis head mainly participate in cellular processes and response to stimulus, participating in catalityc activity and binding. Proteins of epididymis body acted as regulatory proteins and in cellular processes, with binding and catalytic activity. Cauda epididymis molecules were associated with cellular processes and regulation, with binding function and catalytic activity as well. Testis proteins were mainly linked to cell processes and response to stimuli, and had catalytic function. Seminal vesicle proteins were involved in regulation and mainly with binding functions. Most bulbourethral gland proteins participated in cellular processes. The present study is the first to evaluate the proteome and gene expressions in the reproductive tract of Brazilian Somalis rams. Such pieces of information bring significant cointribution for the understanding of the reproductive physiology of locally‐adapted livestock.     

Artificial insemination of frozen epididymal sperm in beagle dogs

Freeze-storage of epididymal sperm is an important technique for the preservation of gametes in animals, including those becoming extinct. We froze canine sperm recovered from the cauda epididymis and investigated the fertility. The qualities of sperm from the cauda epididymis before freezing were: mean sperm motility, 89.4 +/- 1.6 (SE) %; sperm viability, 89.1 +/- 1.1%; and these were significantly higher than those of sperm from the caput-corpus epididymis (P<0.01, P<0.05). The number of sperm recovered from both cauda epididymides varied among animals: 6.3-122.3 x 10(7), mean 61.5 +/- 10.0 x 10(7). Freezing was used only for sperm recovered from the cauda epididymis. The sperm motility and viability after thawing were 19.5 +/- 2.5% and 53.1 +/- 3.3%, respectively. These were slightly lower than those of frozen-thawed ejaculated sperm, but the differences were not significant. When 2 x 10(8), 3 x 10(8), or 4 x 10 (8) sperm were inseminated in the unilateral uterus, only one animal inseminated with 3 x 10(8) sperm was fertilized (1/16, 6.3%). When 1 x 10(8) sperm were inseminated in the bilateral uterine tubes, one of six animals (16.7%) was fertilized. Therefore, although the qualities of epididymal sperm after thawing were similar to those of ejaculated sperm, the conception rate obtained with frozen-thawed epididymal sperm was low in beagle dogs. It is necessary to investigate the differences in damage between epididymal sperm after thawing and ejaculated sperm and to develop a method for improving the conception rate.     

Clinical,Ultrasonographic and Pathological Findings in a Bull with Segmental Aplasia of the Mesonephric Duct

On assessment for use in an AI stud, a 12‐month‐old bull was found to produce low volume ejaculates with 41% of the sperm having morphological abnormalities. No left epididymal tail was palpable and the head of the epididymis on the left was twice the size compared with the right. Ultrasound examination showed the left testis to contain a large central area of decreased echogenicity, which could be followed proximally to a 15‐mm echolucent lesion at the site of the epididymal head. Postmortem examination revealed a 15‐mm diameter cyst in the region of the left epididymal head, and absence of the body and tail of the epididymis. The mediastinum testis of the left testis was dilated, corresponding to the area of decreased echogenicity observed on ultrasonography. No left seminal vesicle was present and the ampulla was significantly smaller than the same structure on the right. Histological examination revealed incomplete or absent spermatogenesis involving the majority of seminiferous tubules in the left testis, and a small proportion of those of the right testis. The cystic structure at the site of the left epididymal head was lined by irregular, sometimes attenuated, epithelium and contained sparse spermatozoa. This case demonstrates the adverse impact, which segmental aplasia of the mesonephric duct had on the testicular and epididymal function of a bull, and highlights the importance of careful clinical assessment in its diagnosis.     

Localization of Oestrogen Receptors in the Epididymis During Sexual Maturation of the Domestic Cat

Oestrogens are involved in regulation of spermatogenesis and sperm maturation and are essential for male fertility. To study the role of oestrogens on epididymal function in the domestic cat, we analyzed the localization patterns of oestrogen receptors (ERs) within the epididymis of juvenile, pubertal and adults using immunohistochemistry. Cat epididymal tissues obtained during routine castrations were fixed in chilled Bouin's solution and processed for immunohistochemistry with ER-specific antibodies. For a certain receptor type, ER localization was influenced by donor age. In the juvenile epididymis, ERα was localized in the nuclei of epithelial cells of efferent ducts and undifferentiated epithelium of the ductus epididymis. During puberty, ERα localization in the undifferentiated epithelium of the epididymis shifted from the nuclei to the cytoplasm and plasma membrane. Oestrogen receptor-α level was highest in the pubertal and adult epididymis, especially within the cytoplasm and in plasma membranes of caput epithelial cells. This finding was suggestive of a role in fluid reabsorption within the efferent ducts and the epididymis. In corpus and cauda regions, ERα was less abundant, suggesting a minor role for oestrogens in sperm storage areas. Interestingly, localization of ERβ was neither influenced by age nor location within the epididymis and was ubiquitous throughout. Results demonstrate that oestrogen actions within the epididymis may be predominantly mediated through ERα during sexual maturation in the domestic cat.     

Tris–Egg Yolk–Glycerol (TEY) Extender Developed for Freezing Dog Semen is a Good Option to Cryopreserve Bovine Epididymal Sperm Cells

Cryopreservation of epididymal spermatozoa is often performed after shipping the excised testis–epididymis complexes, under refrigeration, to a specialized laboratory. However, epididymal spermatozoa can be collected immediately after excision of the epididymis and sent extended and refrigerated to a laboratory for cryopreservation. In this experiment, we evaluated the effect of both methods of cold storage bovine epididymal spermatozoa as well as of two different extenders on spermatozoa characteristics after freeze–thawing. For that, spermatozoa collected from the caudae epididymis of 19 bulls were extended and cryopreserved in either AndroMed^® or a Tris–egg yolk (TEY)‐based extender. Cryopreservation of sperm cells was performed immediately after castration (Group A, n = 9) or after cold storage for 24 h diluted in the two extenders and (Group B, n = 9) and also after cold storage for 24 h within the whole epididymis (Group C, n = 10). Sperm subjective progressive motility (light microscopy), plasma membrane integrity (hypoosmotic swelling test) and sperm viability (eosin–nigrosin) were evaluated. In vitro fertilization and culture (IVF) was performed to assess the blastocyst rate. No differences (p > 0.05) were observed on post‐thaw sperm parameters between samples from Group A, B and C. TEY extended samples presented a higher (p < 0.01) percentage of progressive motile and live sperm, than those extended in AndroMed^®. Blastocyst rate after IVF differed only (p < 0.05) between the reference group (IVF performed with frozen semen with known in vitro fertility) and Group A extended in AndroMed^®. We conclude that when cryopreservation facilities are distant from the collection site, bovine epididymal sperm can be shipped chilled overnight either within the epididymal tail or after dilution without deleterious effect on post‐thaw sperm quality. TEY extender was more suitable for cold storage and freezing bovine epididymal sperm, than the commercial extender AndroMed®.     

Optimization of the in vitro fertilization protocol for frozen epididymal sperm with low fertilization ability in Ban—A native Vietnamese pigs

The aim of the present study was to improve the penetration during in vitro fertilization (IVF) of a frozen lot of epididymal sperm with a notoriously low fertilization ability of a Ban boar which is a native Vietnamese breed by optimizing different parameters of the IVF system. In Experiment 1, we determined that Pig‐fertilization medium was superior medium to Tyrode's albumin lactate pyruvate‐polyvinyl alcohol medium for IVF and defined the optimum the sperm concentration (1 × 10⁶ sperm/ml). In Experiment 2, we clarified that partial removal of cumulus cells from cumulus‐oocyte complexes by hyaluronidase treatment before IVF enhances sperm penetration, whereas complete cumulus removal reduces penetration. Finally, in Experiment 3 the elevation of concentration of caffeine in Pig‐fertilization medium from 2 to 5 mmol/L and the prolongation of the co‐culture of gametes from 3 to 5 hr significantly increased the total penetration rate from 15.2% to over 50%. In conclusion, the combination of partial oocyte denudation, an elevated caffeine concentration in Pig‐fertilization medium and an extended interval of IVF with using an optimized sperm concentration was a potent way to improve the fertilization results for a frozen epididymal Ban sperm lot with low fertility.     

Recovery of fertility in quinestrol‐treated or diethylstilbestrol‐treated mice: Implications for rodent management

Fertility control is an alternative strategy to traditional culling for the management of rodent pests. Previous studies have demonstrated that quinestrol is a potential contraceptive for male rodents, but the recovery of fertility in quinestrol‐treated rodents has not been evaluated. This study used C57BL/6J mice to evaluate the recovery rate of male fertility after the administration of quinestrol. Diethylstilbestrol (DES), a non‐steroid estrogenic compound, was used for comparison. Different groups of mice were treated with 1 mg/kg quinestrol, 1 mg/kg DES, or castor oil separately for 7 days. These mice were then killed on days 8, 22 and 50 respectively. Our results indicated that the weight of epididymides and seminal vesicles decreased significantly on days 8 and 22 in quinestrol/DES‐treated mice, with extensive histological changes in the seminiferous tubules. Sperm concentrations in the cauda epididymal fluid were significantly reduced on days 8 and 22 in both quinestrol and DES treatment groups and on day 50 for the DES, but not the quinestrol group. Further analysis revealed that DES‐treated mice exhibited a higher proportion of abnormal sperm accumulation in the epididymis, indicating that the normal sperm transportation to the cauda epididymis was blocked. Our results indicate that the anti‐fertility effects on male mice given quinestrol were of shorter duration than for those receiving DES at the dose of 1 mg/kg body weight.     

Expression of Matrix Metalloproteinases (MMP‐2, MMP‐9) and their Inhibitors (TIMP‐1, TIMP‐2) in Canine Testis,Epididymis and Semen

This study aims to investigate the role of matrix metalloproteinases (MMPs) in determining semen quality and to evaluate the expression and cellular localization of MMP‐2, MMP‐9, tissue inhibitor of metalloproteinase‐1 (TIMP‐1) and TIMP‐2 in the testes, epididymis and ejaculated spermatozoa. Gelatinase activities between normal (n = 21) and abnormal (n = 25) semen samples showed a significant, sixfold increase in proMMP‐2 and MMP‐2 activity in high than low sperm concentration samples (p < 0.001). ProMMP‐9 and MMP‐9 levels were significantly elevated in samples with low sperm counts compared to those with high sperm density (p < 0.001). High levels of proMMP‐2 and MMP‐2 were associated with high sperm motility (≥70%, p < 0.001). Sperm‐rich fraction showed significantly (eight‐fold) higher proMMP‐9 enzymatic activity compared with prostatic fraction. The mRNA expressions of MMP‐2, MMP‐9, TIMP‐1 and TIMP‐2 were confirmed in testicular and epididymal tissues. Immunohistochemical staining illustrated the MMP‐2‐specific strong immunoreactivity in the head of mature spermatids during spermatogenesis, whereas MMP‐9, TIMP‐1 and TIMP‐2 were absent in these cells. Matrix metalloproteinase‐9 immunoreactivity was observed in the spermatocyte and round spermatid, whereas TIMP‐1 was only exhibited in the residual bodies. Immunolabeling of epididymal and ejaculated sperm demonstrated MMP‐2 localization along acrosomal region of sperm, while MMP‐9, TIMP‐1 and TIMP‐2 localization was merely limited to the flagella. In conclusion, spermatozoa initially acquire MMP‐2 during their formation at testicular level, and the presence of this protein persists through the epididymal transit and up to ejaculate. The enzymatic activity of MMP‐2 and MMP‐9 may serve as an alternative biomarker in determining semen quality.     

Estrogen and androgen receptor expression in relation to steroid concentrations in the adult boar epididymis

The steroid hormone regulation of the epididymis in a high estrogen producing animal like the boar is not currently understood. To test the hypothesis that the boar epididymis is an estrogen and androgen responsive tissue, the presence of estrogen and androgen receptors, in conjunction with steroid hormone concentrations were investigated in the boar epididymis. Epididymal (caput, corpus, cauda) and testicular samples of boars (1–2.5 years; n = 5) were collected for immunolocalization of estrogen receptor alpha (ER), estrogen receptor beta (ERβ) and androgen receptor (AR). Concentrations of testosterone, estradiol and estrogen conjugates (EC) in the tissue were also determined. AR and ERβ were localized in the principal and basal cells of all three epididymal regions. ER was localized in the principal cells of the caput, some cells of the corpus and was not present in the cauda. Testosterone (p < 0.0001), estradiol (p < 0.0001) and EC (p < 0.005) were significantly lower in the epididymis compared with the testis. The epididymal regions were not significantly different from each other for testosterone (p > 0.15) or estradiol (p > 0.09). EC were significantly higher in the corpus than either the caput (p = 0.003) or cauda (p = 0.002). These results suggest that the boar epididymis is responsive to both estrogens and androgens and that both steroid hormones are important for proper epididymal function. Since testosterone and estradiol concentrations are similar throughout the epididymis, regional differences in steroid hormone regulation are likely due to differences in receptor expression.     

Examination of the relationship between the estrogen receptor gene and reproductive traits in swine

The relationship between estrogen receptor (ESR) genotype and reproductive traits in a population of Yorkshire, Large White, and crossbred animals was studied. Reproductive tract and litter data were analyzed for associations with ESR genotype, parity, and breed. Forty-six Yorkshire, 31 Large White, and 70 crossbred females from the above population were mated to Hampshire boars and slaughtered at 75 d of gestation. Data collected included ovulation rate, uterine horn length, number of fetuses, fetal weight, uterine weight, number of mummies, fetal sex, fetal placement, fetal survival, and fetal space. Data were analyzed using a model that included the fixed effects of ESR genotype, breed, parity, and all significant two-way interactions. Litter data representing 212 litter records were analyzed in a model that included the fixed effects of ESR genotype of dam, parity, farrowing month, dam breed, sire breed, and all significant two-way interactions. The ESR genotype was significantly associated with the total litter weight of piglets born and total litter weight of piglets born alive. Dams with the AA genotype had significantly (P = 0.04) heavier litters at birth (14.44 +/- 0.36 kg) than dams with the BB genotype (13.43 +/- 0.47 kg). Ovulation rate was significantly (P < 0.05) different between animals of parity 1 (17.22 +/- 0.41) and parity > or = 3 (19.92 +/- 0.85). Significant breed effects were observed for fetal weight, with purebred Large White animals having a greater fetal weight per horn (3,909 +/- 114 g) than purebred Yorkshire animals (3,553 +/- 92 g). Notable, but nonsignificant, trends with respect to ESR genotype were also observed for number of piglets alive at weaning and total litter weight at weaning. The ESR gene is positively associated with several previously uninvestigated reproductive traits.     

Expression of estrogen receptor 1 and progesterone receptor in primary goat mammary epithelial cells

The exact role and sensitivity of cells to estrogen and progesterone mediated through the steroid receptors during lactation is not known. Expression of estrogen receptor 1 (ESR1) and progesterone receptor (PGR) was quantified in mammary tissue‐derived primary goat mammary epithelial cells (pgMECs) to determine the influence of donor tissue physiology (lactating and juvenile) and cell culture growth conditions (basal and lactogenic) on ESR1 and PGR expression in the derived cells. Relative messenger RNA (mRNA) levels for both receptors were the highest in cell lines derived from mammary tissue of juvenile goats. Maintaining pgMECs in lactogenic conditions resulted in up‐regulation of ESR1 (1.36‐ to 12.35‐fold) and in down‐regulation of PGR (‐2.53‐ to ‐3.62‐fold), compared to basal conditions. Based on Western blotting analysis we suggest that the differences in mRNA expression are translated to the protein level. We suggest that differential expression in lactating conditions is correlated with terminal differentiation of the pgMECs. Double immunostainings showed that estrogen receptor alpha (ER‐α) positive cells do not exclusively belong to the luminal lineage and that ER‐α and PGR can be expressed individually or co‐expressed in the pgMECs. The derived primary cultures/lines in early passages are hormone‐responsive and represent a useful surrogate for mammary tissue in research experiments.     

Mitochondrial DNA Copy Number in Spermatozoa of Fertile Stallions

Predicting male fertility on non‐invasive sperm traits is of big importance to human and animal reproduction strategies. Combining the wide range of parameters monitored by computer‐assisted sperm analysis (CASA) with some molecular traits (e.g. mtDNA content) may help to identify markers of the male fertility. The aim of this study was to characterize variation in the mtDNA copy number in equine sperm and to investigate whether mtDNA content is correlated with quality traits of stallion spermatozoa and the age of the male. Ejaculates collected from 53 fertile stallions were divided into four age groups (3–5, 6–10, 11–14 and >15 years) and were subjected to a complex investigation including conventional analysis, CASA, flow cytometry and mtDNA content (real‐time PCR). The mean (±SD) number of mtDNA copies equalled 14 ± 9 and varied from 3 to 64. Considering the great number of sperm parameters monitored in this study, only few of them were correlated with the mtDNA content: ejaculate volume (a positive correlation), the amplitude of lateral head displacement (ALH; a negative correlation) and the high mitochondrial activity index (a negative correlation). The stallion age was not correlated with the mtDNA copy number. This study provides the first set of data on mtDNA content in equine sperm and confirms phenomena previously described for humans and dog on associations between sperm mtDNA content and selected motility parameters monitored by the CASA. Basing our study on spermatozoa from fertile stallions could however limit the extent of detected associations.