A single, episodic event of unilateral/bilateral scrotal swelling in a group of adult boars at an Austrian boar stud.

BACKGROUND: Scrotal swelling is a clinical situation which can be caused by different aetiologies. In this case report, we describe a multi-week episode of unilateral and bilateral scrotal swelling in boars at an Austrian boar stud and its diagnostic work-up. CASE PRESENTATION: In the summer of 2020, the herd veterinarian of an Austrian boar stud reported that over a period of six weeks, five out of 70 boars presented with unilateral severe swelling of the left scrotum and three out of 70 boars with bilateral severe swelling of the left and moderate swelling of the right scrotum, respectively. A complete history was obtained and an on-site evaluation of the facility was done. Five boars were necropsied, and a variety of samples harvested for further diagnostic investigations. Infectious differential diagnoses associated with unilateral swelling of the scrotum or the testis were excluded through serological and tissue testing. In three of the five boars, histopathology revealed complete acute haemorrhagic necrosis of the left testis concurrent with strongly congested blood vessels. Review of the collected information with a group of experts in the field of boar stud management resulted with consensus that, most likely, trauma was the etiologic event causing the clinical signs and pathology. Coincident with discussion of implementing video recording cameras in the boar housing area, no further clinical cases followed. As this case occurred during the first lockdown of the COVID-19 pandemic, we propose that the distress and travelling restrictions may have contributed to frustration among boar stud workers, which was consequently expressed as misbehaviour against boars. CONCLUSIONS: Once all known infectious causes of unilateral swelling of the scrotum were excluded, a critical diagnostic work-up focused on non-infectious causes. Non-infectious causes, such as trauma, need to be carefully evaluated, as it may also include human misbehaviour against boars. Summarizing all findings of this case report, the authors hypothesize that a blunt trauma was the reason for the series of mainly unilateral swelling of the scrota of boars.

Effects of postpartum diseases on antral follicle count and serum concentration of Anti-Müllerian hormone in dairy cows.

The antral follicle count (AFC) and Anti-Müllerian hormone (AMH) concentration are validated markers for ovarian reserve in cattle, but their use as fertility markers is controverse. Here we assessed the effects of postpartum diseases on AFC and AMH concentration, as well as the influence of parity and breed on these parameters. Cows (n = 513, mostly Holstein Friesian and Brown Swiss, parity 3.0 ± 1.8) underwent a single ultrasonography examination 28-56 days after parturition and categorized as having low (n ≤ 15 follicles), intermediate (n = 16-24 follicles), or high (n ≥ 25 follicles) AFC based on objective video analysis of recorded sequences. Blood samples for AMH determination were collected at the time of examination and animals divided into low (< 0.05 ng/ml) and high AMH (≥ 0.05 ng/ml) group, respectively. No effects of postpartum diseases or breed on either AFC or AMH groups could be observed. There was a strong interaction between parity and AFC, primiparous cows having less follicles (13.6 ± 6.2 vs. 17.1 ± 7.0, P < 0.001) than pluriparous cows. The AFC did not affect reproductive parameters or productivity of the cows. In comparison, pluriparous cows with high AMH concentration had shorter calving to first service (86.0 ± 37.6 vs. 97.1 ± 46.7 days, P < 0.05) and calving to conception (123.8 ± 51.9 vs. 135.8 ± 54.4 days, P < 0.05) intervals, but lower milk yield (8440.3 ± 2292.9 vs. 8927.9 ± 2192.5 kg, P < 0.05) compared to cows with low AMH. In conclusion, no effect of postpartum diseases on AFC or AMH concentration of dairy cows could be observed. However, an interaction between parity and AFC, as well as associations of AMH with fertility and productivity in pluriparous cows, were demonstrated.

Relationship between Clinical Uterine Findings, Therapy, and Fertility in the Mare.

Persistent breeding-induced endometritis (PBIE) is a major cause of subfertility in horses and the susceptibility is increased by several factors. The aim of this study was to determine the effects of clinical uterine findings and PBIE therapies, respectively, on pregnancy rate in mares. The analysis included records from 220 mares (390 cycles) inseminated at an artificial insemination (AI) center in Switzerland. Gynecological examinations were performed repeatedly before and after AI to determine cervical tone, uterine edema, and intrauterine fluid accumulation. Pregnancy rate was lower (p < 0.001) in barren mares compared to mares of other reproductive status. A more flaccid cervix (p = 0.009) was observed at the time of ovulation in pregnant cycles, but there was no difference (p > 0.05) regarding uterine edema. Intrauterine fluid accumulation reduced pregnancy rate (p = 0.002). Oxytocin administration had beneficial effects on pregnancy rate (p = 0.015), especially for barren mares, while uterine lavage did not have any effect (p > 0.05). The results show that cervical tone and intrauterine fluid accumulation, but not its degree, are useful parameters for assessment of fertility in mares. Oxytocin treatment improved pregnancy rates in mares with PBIE while uterine lavage had a limited effect.

Impact of negative energy balance and postpartum diseases during the transition period on oocyte quality and embryonic development in dairy cows.

Transition period is a critical time for dairy cows because a large proportion of clinical and subclinical diseases are observed in the first month after parturition. Occurrence of negative energy balance is associated with depressed immunity and these conditions can affect oocyte quality and further embryonic development. The aim of this study was to assess the effects of negative energy balance-associated disorders on in vitro embryo production (IVP) in dairy cattle. We hypothesized that subclinical metabolic and/or inflammatory disorders have a negative effect on oocyte developmental competence and morphokinetic parameters of the resulting embryos. The study was conducted on 30 lactating Holstein-Friesian cows which were assigned into four groups: healthy (HEAL, n = 6), metabolic disease (META, n = 8), inflammatory disease (INFL, n = 8), or combined metabolic and inflammatory disease (COMB, n = 8). Ovum pick-up (OPU) was performed twice weekly on all cows over a period of four weeks (n = 8 OPU sessions/cow) starting on the fifth week postpartum, and the collected oocytes were subjected to routine IVP. Donor's health status did not affect the number of oocytes/OPU or the recovery rate (p > 0.05). The number of quality 1 oocytes collected from INFL and COMB cows was lower compared to HEAL cows (p < 0.05). Also, the percentage of quality 1 embryos was reduced in META and COMB compared to HEAL cows (p < 0.05). Cleavage, blastocyst and hatching rates were similar among groups (p > 0.05). Presence of disease did not affect the time required by zygotes to reach specific developmental stages, as recorded by means of time-lapse monitoring. Nevertheless, there was a higher probability of direct cleavage after IVF in oocytes of COMB cows compared to those of HEAL cows (p < 0.05). In conclusion, oocytes and embryos derived from dairy cows diagnosed with subclinical metabolic and/or inflammatory diseases during the transition period showed reduced quality but similar developmental potential and morphokinetics when compared to healthy cows. These results shed light on the consequences of subclinical disease on embryonic development in dairy cows which might be important for embryo transfer programs.

Sexual Differentiation and Primordial Germ Cell Distribution in the Early Horse Fetus.

It was the aim of this study to characterize the development of the gonads and genital ducts in the equine fetus around the time of sexual differentiation. This included the identification and localization of the primordial germ cell population. Equine fetuses between 45 and 60 days of gestation were evaluated using a combination of micro-computed tomography scanning, immunohistochemistry, and multiplex immunofluorescence. Fetal gonads increased in size 23-fold from 45 to 60 days of gestation, and an even greater increase was observed in the metanephros volume. Signs of mesonephros atrophy were detected during this time. Tubular structures of the fetal testes were present from day 50 onwards, whereas cell clusters dominated in the fetal ovary. The genital ducts were well-differentiated and presented a lumen in all samples. No sign of mesonephric or paramesonephric duct degeneration was detected. Expression of AMH was strong in the fetal testes but absent in ovaries. Irrespective of sex, primordial germ cells selectively expressed LIN28. Migration of primordial germ cells from the mesonephros to the gonad was detected at 45 days, but not at 60 days of development. Their number and distribution within the gonad were influenced (p < 0.05) by fetal sex. Most primordial germ cells (86.8 ± 3.2% in females and 84.6 ± 4.7% in males) were characterized as pluripotent according to co-localization with CD117. However, only a very small percentage of primordial germ cells were proliferating (7.5 ± 1.7% in females and 3.2 ± 1.2% in males) based on co-localization with Ki67. It can be concluded that gonadal sexual differentiation in the horse occurs asynchronously with regard to sex but already before 45 days of gestation.

Epigenetic Changes in Equine Embryos after Short-Term Storage at Different Temperatures.

In embryos subjected to assisted reproductive techniques, epigenetic modifications may occur that can influence embryonic development and the establishment of pregnancy. In horses, the storage temperature during transport of fresh embryos before transfer is a major concern. The aim of this study was, therefore, to determine the effects of two storage temperatures (5 °C and 20 °C) on equine embryos, collected at day seven after ovulation and stored for 24 h, on: (i) morphological development; (ii) expression of candidate genes associated with embryo growth and development, maternal recognition of pregnancy, methylation and apoptosis, and (iii) gene-specific and global DNA methylation. Embryos (n = 80) were collected on day seven or day eight after ovulation and assigned to four groups: day seven control (E7F, fresh); day seven, stored for 24 h at 5 °C (E5C); day seven, stored for 24 h at 20 °C (E20C) and day eight control (E8F, fresh 24h time control). The embryos and the storage medium (EquiHold, holding medium, Minitube, Tiefenbach, Germany) from all treatment groups were analyzed for (i) medium temperature, pH, and lipid peroxidation (malondialdehyde; MDA) and (ii) embryo morphology, mRNA expression and DNA methylation (immunohistochemistry and gene-specific DNA methylation). The size of embryos stored at 5 °C was larger (p < 0.01), whereas embryos stored at 20 °C were smaller (p < 0.05) after 24 h. There were no changes in pH and MDA accumulation irrespective of the group. The mRNA expression of specific genes related to growth and development (POU5F1, SOX2, NANOG), maternal recognition of pregnancy (CYP19A1, PTGES2), DNA methylation (DNMT1, DNMT3A, DNMT3B) and apoptosis (BAX) in the E5C and E20C were either up or downregulated (p < 0.05) when compared to controls (E7F and E8F). The immune expression of 5mC and 5hmC was similar among treatment groups. Percentage of methylation in the CpG islands was lower in the specific genes ESR1, NANOG and DNMT1 (p < 0.001) in E20C embryos when compared to E8F (advanced embryo stage). Therefore, our study demonstrates for the first time the gene-specific and global DNA methylation status of fresh equine embryos collected on days seven and eight after ovulation. Although our results suggest some beneficial effects of storage at 20 °C in comparison to 5 °C, the short-term storage, regardless of temperature, modified gene expression and methylation of genes involved in embryo development and may compromise embryo viability and development after transfer.

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.

Early luteal phase progestin concentration influences endometrial function in pregnant mares.

In the horse, it is still unclear if and to what extent low progestin concentration contributes to early conceptus loss. In the present study, we have investigated if reduced or elevated progestin concentration in the early luteal phase influences endometrial function and conceptus development. We hypothesized that reduced progestin concentration via delayed downregulation of endometrial progesterone receptors (PR) influences endometrial function in healthy fertile mares while progestin substitution does not. Genitally healthy estrous mares (n = 8; age 4-14 years) were inseminated and treated with either altrenogest (0.044 mg/kg once daily orally) on days 5-10 after ovulation (ALT), cloprostenol (125 µg once daily intramuscularly) on days 0-3 after ovulation (CLO) or left untreated (CON). ALT and CLO treatment were chosen to increase and decrease total peripheral progestin concentration, respectively. Each treatment was given to every mare in consecutive cycles. On day 14 after ovulation, endometrial fluid was collected with a cotton roll inserted into the uterus and an endometrial biopsy for immunohistological demonstration of progesterone (PR) receptor distribution was collected. In endometrial fluid, free amino acid concentrations were analyzed by ion exchange liquid chromatography with an amino acid analyzer. Cell nuclei staining positive for the PR were determined in the luminal and glandular epithelium as well as in the stroma. Pregnancy rate tended to differ among treatments. The percentage of luminal epithelial cells staining positive for PR differed among treatments (p < 0.05) and was higher in CLO (84.1 ±â€¯1.9%) than in ALT (70.7 ±â€¯4.7%) and CON cycles (72.8 ±â€¯4.1%). Concentrations of the amino acids isoleucine (CON 0.17 ±â€¯0.03, CLO 0.14 ±â€¯0.02, ALT 0.23 ±â€¯0.04 µmol) and lysine (CON 0.27 ±â€¯0.08, CLO 0.18 ±â€¯0.05, ALT 0.44 ±â€¯0.13 µmol) were influenced by treatment (p < 0.05) and lower in CLO than in ALT and CON cycles. In conclusion, impaired luteal function due to CLO treatment during the early luteal phase of pregnant mares delayed downregulation of progesterone receptors in the endometrial epithelium on day 14. This influenced endometrial function as reflected in lower concentrations of the amino acids lysine and isoleucine in endometrial secretions. Enhanced progestin concentration had less clear effects in healthy fertile mares.

Anti-Müllerian hormone profiling in prepubertal horses and its relationship with gonadal function.

Anti-Müllerian hormone (AMH) has gained increasing interest as a biomarker for assessment of gonadal activity. The ability to predict the ovarian follicular reserve of prepubertal female horses (fillies) or to identify stallions with testicular pathologies already during their prepubertal life has not been analyzed so far. Both would help to select fertile horses and reduce costs associated with keeping animals. The objectives of the present study were to (1) assess AMH, LH, FSH, progesterone (females) and testosterone (males) dynamics in prepubertal horses from birth onwards and (2) determine whether AMH concentrations detected in plasma of prepubertal female and male horses are correlated with postpubertal gonadal development. Warmblood foals (n = 30, 14 females, 10 normal males and 6 males with abnormal testicular development) born between February and May of two consecutive years (n = 28 in the first year and n = 2 the next year), were included in the study. Information on gestational length, parity of the dam and placental weight was collected for all foals. Blood samples for hormone analysis were collected from birth onwards every four weeks up to the age of one year. At two years, blood samples were collected on the day when antral follicle count (AFC) and total testicular volume (TTV) were assessed. AMH was detectable in the plasma of all animals from birth onwards and its concentration was significantly higher (P < .001) in males than in females, regardless of testicular development. In males, AMH and testosterone concentration were similar for all animals during the first year of life, regardless of testicular development. At two years, AMH concentration was higher (P < .05) in males with abnormal testicular development than in those with normal testes. In females, AMH concentration at two years was correlated with AMH concentration at birth (P < .05) and with AFC (P < .001). At birth, LH concentration was lower (P < .05) in stallions with abnormal testes (0.3 ±â€¯0.2 ng/ml) than in controls (0.6 ±â€¯0.2 ng/ml). A high negative correlation between AMH concentration and gestation length was observed in males during the first eight weeks of life (P < .01, r = -0.64 to -0.71). Elevated progesterone concentrations over 1 ng/ml were observed in several females starting with 20 weeks of age. This was paralleled by an increase in AMH concentration and was preceded by FSH and LH increases. In conclusion, AMH determination can be reliably used from two years onwards to identify stallions with abnormal testicular development, but it is inconclusive before puberty. In female horses, determination of AMH concentration at a prepubertal age allows for prediction of AMH and AFC after puberty. We suggest that premature luteinisation occurs before the onset of puberty in female horses and that LH secretion in the perinatal period is involved in testicular development and descent in the horse.

Cooled-storage of equine semen does not induce major changes in sperm DNA methylation.

A decrease in fertility of equine semen during cooled-storage so far has mainly been attributed to changes in sperm membrane function. In the present study we hypothesized that cooled-storage also changes the sperm DNA methylation level. For this purpose, semen was collected from 10 fertile stallions and processed for cooled-storage at 5 °C. Two final concentrations, 50 × 106 and 100 × 106 cells/mL, were used. Semen was analyzed for total motility, progressive motility, membrane integrity, phosphatidylserine translocation (PST), mitochondrial membrane potential and chromatin condensation, immediately after processing and at 24 h-intervals until 72 h of storage. DNA cytosine methylation was assessed by ELISA after DNA extraction and denaturation. DNA methylation did neither change over time nor was affected by semen concentration. Total motility, progressive motility, membrane integrity, PST, mitochondrial membrane potential and chromatin condensation changed over storage time, but no differences between semen concentrations could be demonstrated. The results demonstrate that cooled-storage of equine semen does not induce changes in sperm DNA cytosine methylation. In cooled-semen of good quality, a concentration of 100 × 106 sperm/mL does not affect semen longevity. It can be concluded that a better fertility of cooled-stored than cryopreserved stallion semen is at least in part a result of only minor influences of cooled-storage on DNA integrity and methylation.

Anti-Muellerian hormone, inhibin A, gonadotropins, and gonadotropin receptors in bull calves after partial scrotal resection, orchidectomy, and Burdizzo castration.

Eight-week-old calves were either castrated by partial scrotal resection (SR) without removing the testes (n = 10), Burdizzo (BZ) clamp (n = 10), orchidectomy (OR; n = 10), or were left gonad intact as controls (CO; n = 10). Concentrations of anti-Muellerian hormone (AMH), inhibin A, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in plasma were determined from 16 to 48 weeks of age. At 18 months, testes of SR, BZ, and CO bulls were obtained and the immunolocalization of LH and FSH receptors and AMH analyzed. Concentration of AMH in plasma of CO and SR bulls decreased with increasing age (P < 0.001). A similar AMH profile in CO and SR indicates that SR did not induce a true cryptorchid state. In groups OR and BZ, AMH was undetectable. Plasma inhibin concentration was higher in groups CO and SR than BZ and OR (P < 0.001). Plasma LH and FSH concentrations decreased over time (P < 0.001) and were higher in groups BZ and OR than SR and CO (P < 0.001). In the testes, immunolabeling for AMH existed in Sertoli cells of CO and SR but not BZ bulls. FSH receptors were localized in Sertoli cells, Leydig cells, spermatocytes, and the epididymis of CO and SR animals, whereas LH receptors were restricted to Leydig cells. In BZ animals, FSH and LH receptors and AMH were absent, indicating complete testicular degeneration. In conclusion, AMH is a more reliable marker for the presence of testicular tissue in bulls than inhibin. Scrotal resection did not induce a true inguinal cryptorchid state but affected testicular responsiveness to gonadotropic stimulation.

Cytosine methylation of sperm DNA in horse semen after cryopreservation.

Semen processing may contribute to epigenetic changes in spermatozoa. We have therefore addressed changes in sperm DNA cytosine methylation induced by cryopreservation of stallion semen. The relative amount of 5-methylcytosine relative to the genomic cytosine content of sperm DNA was analyzed by ELISA. In experiment 1, raw semen (n = 6 stallions, one ejaculate each) was shock-frozen. Postthaw semen motility and membrane integrity were completely absent, whereas DNA methylation was similar in raw (0.4 ± 0.2%) and shock-frozen (0.3 ± 0.1%) semen (not significant). In experiment 2, three ejaculates per stallion (n = 6) were included. Semen quality and DNA methylation was assessed before addition of the freezing extender and after freezing-thawing with either Ghent (G) or BotuCrio (BC) extender. Semen motility, morphology, and membrane integrity were significantly reduced by cryopreservation but not influenced by the extender (e.g., total motility: G 69.5 ± 2.0, BC 68.4 ± 2.2%; P < 0.001 vs. centrifugation). Cryopreservation significantly (P < 0.01) increased the level of DNA methylation (before freezing 0.6 ± 0.1%, postthaw G 6.4 ± 3.7, BC 4.4 ± 1.5%; P < 0.01), but no differences between the freezing extenders were seen. The level of DNA methylation was not correlated to semen motility, morphology, or membrane integrity. The results demonstrate that semen processing for cryopreservation increases the DNA methylation level in stallion semen. We conclude that assessment of sperm DNA methylation allows for evaluation of an additional parameter characterizing semen quality. The lower fertility rates of mares after insemination with frozen-thawed semen may at least in part be explained by cytosine methylation of sperm-DNA induced by the cryopreservation procedure.

Effect of stallion age on the expression of LH and FSH receptors and aromatase P450 in equine male reproductive tissues.

The aim of the present study was to characterise receptors for LH and FSH (LHR and FSHR, respectively) and aromatase in epididymal and testicular tissue from stallions of different ages (prepubertal, young, mature and old). Gene and protein expression were assessed by real-time quantitative polymerase chain reaction (real-time qPCR), immunohistochemistry and multiple immunofluorescence labelling. There were no differences in LHR mRNA expression in epididymal and testicular parenchyma in stallions of different age. In contrast, expression of FSHR and CYP19A1 in caput, corpus and cauda epididymis and in testicular parenchyma increased with age (P<0.001). Immunolabelling for LHR, FSHR and aromatase was influenced by puberty. In postpubertal stallions, positive staining for LHR and aromatase was detected in Leydig cells, whereas protein expression of FSHR was present in Sertoli cells and primary spermatocytes. In prepubertal colts, staining for LHR, FSHR and aromatase was detected in seminiferous tubules. In epididymal tissue, aromatase was present in the cauda epididymis only, regardless of age. In conclusion, the results highlight the significance of gonadotropin action and oestrogen production for the maturation of male reproductive tissue in the horse. The presence of FSHR in the seminiferous tubules suggests effects of FSH on spermatogenesis in this species. The importance of oestrogen production for maintenance of testicular function in stallions was confirmed.

The Orthology Clause in the Next Generation Sequencing Era: Novel Reference Genes Identified by RNA-seq in Humans Improve Normalization of Neonatal Equine Ovary RT-qPCR Data.

BACKGROUND: Vertebrate evolution is accompanied by a substantial conservation of transcriptional programs with more than a third of unique orthologous genes showing constrained levels of expression. Moreover, there are genes and exons exhibiting excellent expression stability according to RNA-seq data across a panel of eighteen tissues including the ovary (Human Body Map 2.0). RESULTS: We hypothesized that orthologs of these exons would also be highly uniformly expressed across neonatal ovaries of the horse, which would render them appropriate reference genes (RGs) for normalization of reverse transcription quantitative PCR (RT-qPCR) data in this context. The expression stability of eleven novel RGs (C1orf43, CHMP2A, EMC7, GPI, PSMB2, PSMB4, RAB7A, REEP5, SNRPD3, VCP and VPS29) was assessed by RT-qPCR in ovaries of seven neonatal fillies and compared to that of the expressed repetitive element ERE-B, two universal (OAZ1 and RPS29) and four traditional RGs (ACTB, GAPDH, UBB and B2M). Expression stability analyzed with the software tool RefFinder top ranked the normalization factor constituted of the genes SNRPD3 and VCP, a gene pair that is not co-expressed according to COEXPRESdb and GeneMANIA. The traditional RGs GAPDH, B2M, ACTB and UBB were only ranked 3rd and 12th to 14th, respectively. CONCLUSIONS: The functional diversity of the novel RGs likely facilitates expression studies over a wide range of physiological and pathological contexts related to the neonatal equine ovary. In addition, this study augments the potential for RT-qPCR-based profiling of human samples by introducing seven new human RG assays (C1orf43, CHMP2A, EMC7, GPI, RAB7A, VPS29 and UBB).

Expression and immunolocalisation of follicle-stimulating hormone receptors in gonads of newborn and adult female horses.

In mares, FSH and its receptor (FSHR) are essential for ovarian function. The objective of the present study was to analyse FSHR gene expression at the mRNA and protein levels in ovarian tissue from newborn and adult horses. Expression of mRNA was analysed by reverse transcription polymerase chain reaction, whereas FSHR protein was visualised by immunohistochemistry (IHC), immunofluorescence labelling (IF) and western blot. FSHR mRNA was detected in ovarian follicles and luteal tissue from adult mares, as well as in the ovaries of neonates. Follicular growth up to 4mm in diameter was already present in neonates. Using IHC and IF, FSHR protein was detected in granulosa cells, cumulus cells and inconsistently in oocytes, independent of the animal's age or the stage of folliculogenesis. A lower FSHR expression was observed in theca cells in comparison to granulosa cells. FSHR was abundant in the ovarian stroma cells of neonates but not of adults. Luteal cells stained positive for FSHR independent of the stage of corpus luteum development. The presence of FSHR protein in various cell populations of the ovary was confirmed by western blot. In conclusion, FSHR is present in horse ovaries consistently from birth onwards and expression remains constant during the oestrous cycle.