Characterization of Cytokines in Donkey Follicular Fluid and Seminal Plasma: A Pilot Study

ABSTRACT

Post‐breeding endometrial inflammation is a well‐known physiological response in jennies, especially after artificial insemination with frozen–thawed sperm. Bioactive molecules present in reproductive fluids, including cytokines, may contribute to regulating these immune processes. The aim of this study was to characterize the cytokine profile of preovulatory follicular fluid (FF) and seminal plasma (SP) in donkeys and to explore their potential relevance for reproductive physiology. To this end, 10 FF samples were collected from five fertile Catalan jennies, and 10 samples were isolated from 10 different Catalan jackasses' ejaculates. Cytokine concentrations were measured using a Luminex MagPix system with an equine‐specific Milliplex MAP panel. Three cytokines were detected in the FF, with IP‐10 being the most abundant, followed by IL‐10 and eotaxin. The SP, in contrast, showed a broader cytokine profile, with 10 detectable mediators. Fractalkine was the most abundant cytokine in this biofluid, followed by FGF‐2 and G‐CSF. GRO and IL‐1β were present at intermediate concentrations, and IL‐8, IL‐13 and IL‐12 were observed at low concentrations. From the 10 cytokines detected, IP‐10 and eotaxin were found at the lowest concentrations. These results provide the first description of cytokines in donkey FF and SP. The presence of chemotactic and immunomodulatory cytokines in these reproductive fluids suggests they may contribute to the regulation of follicular processes and the post‐breeding inflammatory response in jennies.

1. Introduction

In jennies, natural mating or artificial insemination (AI) induces an acute physiological endometrial inflammatory response characterized by a marked influx of polymorphonuclear (PMN) cells. The use of frozen–thawed semen further exacerbates this response and is associated with reduced pregnancy rates (0%–28%), limiting the effectiveness of AI with frozen semen in conservation and recovery programs for endangered breeds (Company et al. 2025).

Several bioactive molecules present in the reproductive tract may modulate this inflammatory response. During ovulation, the follicular fluid (FF) is released together with the oocyte, and a fraction of this fluid reaches the oviduct (Hansen et al. 1991). In parallel, seminal plasma (SP) contains numerous immunomodulatory factors that can influence the interaction between semen and the female reproductive tract (Bromfield 2014). Among them, cytokines are key mediators of intercellular communication and play important roles in regulating inflammation and in tissue remodelling and repair (Dinarello 2007). Data on cytokine composition in the FF and SP from donkeys are, however, scarce.

In this context, the aim of the present study was to characterize the cytokine profiles of preovulatory FF and SP in donkeys, as this may provide some insights into their potential role in post‐breeding reproductive physiology in jennies.

2. Materials and Methods

2.1. Animals and Samples

All animals used in this study were clinically healthy, had proven fertility, and were housed at the Equine Reproduction Service, Autonomous University of Barcelona (UAB, Bellaterra, Spain), under standard husbandry and welfare conditions. All procedures were approved by the Ethics Committee of the UAB (Code: CEEAH 1424) and conducted in accordance with the ARRIVE guidelines and EU Directive 2010/63.

2.1.1. Follicular Fluid Collection

Ten samples of preovulatory FF were collected from five Catalonian jennies (two follicles per animal collected in different cycles), aged 3–12 years, during the breeding season (May–July). Follicular development was monitored by ultrasonography, and follicles were considered preovulatory when they reached ≥ 40 mm in diameter, were associated with uterine edema, and no corpus luteum was present. Follicular aspiration was performed by ultrasound‐guided flank puncture. The ipsilateral flank was clipped and aseptically prepared, and jennies were sedated with detomidine hydrochloride (Domidine, 0.02 mg/kg IV; Dechra Veterinary Products, Northwich, UK) and locally anaesthetised with lidocaine (Lidor, Richter Pharma, Wels, Austria). The ovary was positioned against the abdominal wall by transrectal palpation, and the preovulatory follicle was aspirated using a 20‐gauge needle attached to a 20 mL syringe under ultrasonographic guidance. Samples were centrifuged (2000 × g, 10 min, 4°C) to remove cellular debris and any blood contamination, and the supernatant was aliquoted and stored at −80°C until analysis.

2.1.2. Seminal Plasma Isolation

Ten ejaculates were collected from different Catalonian donkeys aged 4–11 years. Semen was collected using a Hannover artificial vagina (Minitube GmbH, Tiefenbach, Germany) and filtered through a nylon mesh to remove the gel fraction. The SP was isolated by centrifugation following the protocol described by Catalán et al. (2025), aliquoted, and stored (−80°C) until analysis.

2.2. Cytokine Analysis

Cytokine concentrations in the FF and SP were measured using a Luminex MagPix system (Luminex, Austin, TX, USA) with an equine‐specific Milliplex MAP magnetic bead panel (EMD Millipore, Billerica, MA, USA), according to the manufacturer's instructions. The panel simultaneously detects 23 cytokines, including interleukins (IL‐1α, IL‐1β, IL‐2, IL‐4, IL‐5, IL‐6, IL‐8, IL‐10, IL‐12p70, IL‐13, IL‐17A, IL‐18), tumour necrosis factor‐α (TNF‐α), interferon‐γ (IFN‐γ), chemokines (IP‐10/CXCL10, GRO/CXCL1, MCP‐1/CCL2, eotaxin/CCL11, fractalkine/CX3CL1, RANTES/CCL5), and growth factors (FGF‐2, G‐CSF, GM‐CSF). Data were analysed using Milliplex Analyst software (EMD Millipore), and concentrations were calculated from standard curves using a five‐parameter logistic regression model, as previously described by Martin et al. (2017). All samples were examined in duplicate.

2.3. Descriptive Statistics

Given the exploratory nature of the study, only descriptive statistics were performed using R (version 4.0.3, R Core Team, Vienna, Austria). Cytokine concentrations are presented as mean ± standard error of the mean (SEM), and the coefficient of variation (CV, %) was calculated to assess inter‐individual variability.

3. Results

Detectable concentrations of three cytokines were identified in the FF (Table 1). IP‐10 was the most abundant cytokine in the FF, followed by IL‐10, whereas eotaxin was detected at the lowest concentrations.

TABLE 1.

Concentrations of cytokines detected in the follicular fluid of jennies.

Cytokine	pg/mL	CV (%)
IP‐10	90.51 ± 4.40	15.36
IL‐10	81.25 ± 13.10	51.00
Eotaxin	6.72 ± 0.03	1.61

Note: Data are presented as mean ± SEM (n = 10), together with the coefficient of variation (CV, %).

Abbreviations: IL‐10, interleukin‐10; Eotaxin, (CCL11); IP‐10, interferon gamma‐induced protein 10 (CXCL10).

Detectable concentrations of 10 cytokines were found in SP (Table 2). Fractalkine was the most abundant cytokine, followed by FGF‐2 and G‐CSF. GRO and IL‐1β were present at intermediate concentrations, and IL‐8, IL‐13 and IL‐12 were detected at lower levels. Eotaxin and IP‐10 showed the lowest levels.

TABLE 2.

Concentrations of cytokines detected in donkey seminal plasma.

Cytokine	pg/mL	CV (%)
Eotaxin	7.23 ± 0.19	8.16
FGF‐2	488.06 ± 52.72	34.16
Fractalkine	656.05 ± 5.80	2.80
G‐CSF	241.53 ± 39.07	51.15
GRO	193.97 ± 6.79	11.07
IL‐1b	181.11 ± 45.43	79.32
IL‐8	71.11 ± 12.07	53.66
IL‐12	16.06 ± 1.01	19.85
IL‐13	39.76 ± 3.65	29.02
IP‐10	9.85 ± 1.43	45.92

Note: Data are presented as mean ± SEM (n = 10), together with the coefficient of variation (CV, %).

Abbreviations: Eotaxin (CCL11); FGF‐2, fibroblast growth factor‐2; fractalkine (CX3CL1); G‐CSF, granulocyte colony‐stimulating factor; GRO, growth‐regulated oncogene (CXCL1); IL‐1β, interleukin‐1 beta; IL‐8, interleukin‐8; IL‐12, interleukin‐12; IL‐13, interleukin‐13; IP‐10, interferon gamma‐induced protein 10 (CXCL10).

4. Discussion

This study provides a preliminary characterization of the cytokine profile present in preovulatory FF and SP in donkeys. Marked inter‐individual variability was observed in cytokine concentrations in both biological fluids, with coefficients of variation varying across cytokines.

Cytokines within the ovarian follicle act as key regulators of the local microenvironment, contributing to follicular development and oocyte competence (Baskind et al. 2014). The presence of proinflammatory mediators such as IP‐10, the most abundant cytokine detected in the FF, is consistent with the involvement of immune‐related pathways in ovulatory processes, including immune cell recruitment, protease activation, and follicular wall remodelling, as described in other species (Duffy et al. 2019). The immunomodulatory cytokine IL‐10, detected in this study at slightly lower levels, is implicated in regulating inflammatory responses and tissue homeostasis (Saraiva et al. 2020). Eotaxin, which was present in FF at much lower concentrations, has been associated with eosinophil recruitment and angiogenesis (Salcedo et al. 2001) and may also contribute to local immune and vascular processes around ovulation. Collectively, the presence of these cytokines in FF suggests that immune‐related processes may contribute to regulation and tissue remodelling within the FF environment.

In contrast, SP exhibited a broader cytokine profile. Detected chemotactic mediators such as fractalkine, GRO, and IL‐8 are capable of recruiting neutrophils and monocytes (Bromfield 2014), whereas eotaxin attracts eosinophils. Recently, SP has been shown to trigger extracellular traps of neutrophils and eosinophils in the uterus of jennies, highlighting its potential role in modulating early immune responses (Company et al. 2025; Mateo‐Otero et al. 2022). Additionally, the SP contained both proinflammatory and immunomodulatory cytokines, such as IP‐10, IL‐1β, IL‐12 and IL‐13, which are involved in the activation and regulation of immune responses (Ledee et al. 2008; Granot et al. 2012). For its part, FGF‐2 is associated with angiogenesis and tissue remodelling, and G‐CSF is related to immune regulation and endometrial receptivity (Ledee et al. 2008; Zhou and Zhou 2025).

In equids, SP has been shown to contain immunomodulatory mediators capable of influencing the post‐breeding uterine response and the interaction between semen and the female reproductive tract (Troedsson et al. 2024; Sancler‐Silva et al. 2026). Consistently, Podico et al. (2024) reported that the inflammatory response induced by donkey SP is lower than that induced by horse SP, and suggested that this could be associated with differences in cytokine concentrations and other immunomodulatory mediators. Together, the cytokine profile identified in this study supports a potential involvement of SP in the modulation of post‐breeding immune responses in jennies. Nevertheless, the specific biological functions of these cytokines in donkey reproduction remain unknown. On the other hand, it is worth mentioning that the SP is largely removed during semen processing for cryopreservation. Thus, the loss of these immunomodulatory factors may contribute to the exacerbated uterine inflammatory response and reduced fertility frequently reported after AI with frozen–thawed sperm in jennies. In parallel, the FF released during ovulation can reach the oviduct, suggesting that cytokines present in both fluids may act in a complementary manner to regulate early immunological events.

Limitations of the present study include the use of a horse‐specific Milliplex MAP kit, which may have restricted cytokine detection, and the possibility that some cytokines may have been present at concentrations below the assay detection limit. Furthermore, the relatively small sample size of this pilot study should also be considered when interpreting these findings, which constitute a preliminary characterization of cytokine profiles in donkey reproductive fluids.

5. Conclusion

In conclusion, this study provides the first description of the cytokine profile of donkey FF and SP. While these mediators may help regulate the follicular microenvironment and the post‐breeding inflammatory response, their specific roles in donkey reproductive physiology warrant further investigation.

Author Contributions

J.C., J.M. and M.Y. conceived and designed the experiments. S.C., J.C., L.P. and C.M. performed the experiments and data analysis. J.M. and M.Y. provided the resources. S.C. and J.C. drafted the manuscript. J.M. and M.Y. critically revised the manuscript and approved the final version.

Funding

This work was supported by Ministerio de Ciencia e Innovación (PID2020‐113320RB‐I00, PID2023‐149853OB‐I00).

Conflicts of Interest

The authors declare no conflicts of interest.

Company, S. , Catalán J., Padilla L., Maside C., Miró J., and Yeste M.. 2026. “Characterization of Cytokines in Donkey Follicular Fluid and Seminal Plasma: A Pilot Study.” Reproduction in Domestic Animals 61, no. 7: e70271. 10.1111/rda.70271.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.