Submitted:
29 November 2025
Posted:
01 December 2025
You are already at the latest version
Abstract
Keywords:
1. Introduction
2. Materials and Methods
2.1. Animals and Breeding Soundness Evaluation
2.2. Sample Collection
2.3. Endometrial Histomorphometric Evaluation
2.3.1. Measurement of Luminal Epithelium Height and Glandular Size Parameters
2.3.2. Measurement of Glandular Density Parameters
2.4. Statistical Analysis
3. Results
3.1. Breeding Soundness Evaluation
3.2. Histological Characteristics of the Endometrial Tissue
3.3. Endometrial Histomorphometric Evaluation
3.3.1. Results of Luminal Epithelium and Endometrial Glandular Size Parameters
3.3.2. Results of Glandular Density Parameters
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
| #G | Number of glands |
| AI | Artificial Insemination |
| ARTs | Assisted reproductive technologies |
| GA | Glandular area |
| GE | Glandular epithelium |
| GT | Glandular tissue percentage |
| KDGS | Kenney and Doig biopsy grading system |
| LD | Lumen diameter |
| LE | Luminal epithelium |
| SC | Stratum compactum |
| SS | Stratum spongiosum |
References
- Food and Agriculture Organization (FAO). Available online: Domestic Animal Diversity Information System (DAD-IS) https://www.fao.org/dad-is/en/ (accessed 13th March 2025).
- Camillo, F.; Rota, A.; Biagini, L.; Tesi, M.; Fanelli, D.; Panzani, D. The Current Situation and Trend of Donkey Industry in Europe. J Equine Vet Sci 2018, 65, 44–9. [Google Scholar] [CrossRef]
- Hedrick, P.W.; Garcia-Dorado, A. Understanding Inbreeding Depression, Purging, and Genetic Rescue. Trends Ecol Evol 2016, 31, 940–52. [Google Scholar] [CrossRef]
- Hildebrandt, T.B.; Hermes, R.; Goeritz, F.; Appeltant, R.; Colleoni, S.; de Mori, B.; Diecke, S.; Drukker, M.; Galli, C.; Hayashi, K.; Lazzari, G.; Loi, P.; Payne, J.; Renfree, M.; Seet, S.; Stejskal, J.; Swegen, A.; Williams, S.A.; Zainuddin, Z.Z.; Holtze, S. The ART of bringing extinction to a freeze – History and future of species conservation, exemplified by rhinos. Theriogenology 2021, 169, 76–88. [Google Scholar] [CrossRef]
- De Oliveira, J.V.; Oliveira, P.V.L.F.; e Oña, C.M.M.; Guasti, P.N.; Monteiro, G.A.; da Silva, Y.F.R.S.; de Mello Papa, P.; Alvarenga, M.A.; Dell’Aqua Junior, J.A.; Ozanam Papa, F. Strategies to improve the fertility of fresh and frozen donkey semen. Theriogenology 2016, 85, 1267–1273. [Google Scholar] [CrossRef]
- Panzani, D.; Fanelli, D.; Camillo, F.; Rota, A. Embryo technologies in donkeys (Equus Asinus). Theriogenology 2020, 156, 130–7. [Google Scholar] [CrossRef]
- Fanelli, D.; Moroni, R.; Bocci, C.; Camillo, F.; Rota, A.; Panzani, D. Interspecific and Intraspecific Artificial Insemination in Domestic Equids. Animals 2023, 13:582. [CrossRef]
- Hidalgo, M. Sperm vitrification in horses and donkeys. J Equine Vet Sci 2025, 145, 105340. [Google Scholar] [CrossRef]
- Vidament, M.; Vincent, P.; Martin, F.X.; Magistrini, M.; Blesbois, E. Differences in ability of jennies and mares to conceive with cooled and frozen semen containing glycerol or not. Anim Reprod Sci 2009, 112, 22–35. [Google Scholar] [CrossRef]
- Rota, A.; Panzani, D.; Sabatini, C.; Camillo, F. Donkey jack (Equus asinus) semen cryopreservation: Studies of seminal parameters, post breeding inflammatory response, and fertility in donkey jennies. Theriogenology 2012, 78, 1846–1854. [Google Scholar] [CrossRef]
- Vilés, K.; Rabanal, R.; Rodríguez-Prado, M.; Miró, J. Effect of ketoprofen treatment on the uterine inflammatory response after AI of jennies with frozen semen. Theriogenology 2013, 79, 1019–1026. [Google Scholar] [CrossRef]
- Miró, J.; Papas, M. Post–Artificial Insemination Endometrial Inflammation and Its Control in Donkeys. J Equine Vet Sci 2018, 65, 38–43. [Google Scholar] [CrossRef]
- Canisso, I.F.; Panzani, D.; Miró, J.; Ellerbrock, R.E. Key Aspects of Donkey and Mule Reproduction. Veterinary Clinics of North America - Equine Practice 2019, 35, 607–642. [Google Scholar] [CrossRef]
- Abdelnaeim, M. Morphological Characteristics of the Donkey (Equus asinus) Uterus during Estrus: Light, Scanning and Transmission Electron Microscopic Study. Journal of Agricultural and Veterinary Sciences, Qassim University 2008, 2, 45–57. [Google Scholar]
- Alfradique, V.A.P.; Netto, D.L.S.; Alves, S.V.P.; Machado, A.F.; Novae,s C.M.; Penitente-Filho, J.M.; Machado-Neves, M.; Lopes, M.S.; Guimaraes, S.E.F The impact of FSH stimulation and age on the ovarian and uterine traits and histomorphometry of prepubertal gilts. Domest Anim Endocrinol 2023, 83, 106786. [CrossRef]
- Wang, C.K.; Robinson, R.S.; Flint, A.P.F.; Mann, G.E. Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels. Reproduction 2007, 134, 365–371. [Google Scholar] [CrossRef]
- Lefranc, A.C.; Allen, W.R. ; Influence of breed and oestrous cycle on endometrial gland surface density in the mare. Equine Vet J 2007, 39, 506–510. [Google Scholar] [CrossRef]
- Abdoon, A.S.S.; Soliman, S.S.; Nagy, A.M. Uterotubal junction of the bovine (Bos taurus) versus the dromedary camel (Camelus dromedarius): Histology and histomorphometry. Reproduction in Domestic Animals 2024, 59, e14665. [Google Scholar] [CrossRef]
- Mansour, G.D.; Ferreira, A.M.R.; Fernandes, F.T.; Henry, M. Histomorphometry of epithelial structures of the mare’s endometrium. Revista Brasileira de Ciência Veterinária 2004, 11, 44–48. [Google Scholar] [CrossRef]
- Dubrovskaya, A.B.; Lebedeva, L.F.; Schukis, K.A. Comparative histomorphological characteristics of the endometrium of young and aged mares in estrus and diestrus. IOP Conf Ser Earth Environ Sci 2019, 341, 012067. [Google Scholar] [CrossRef]
- Herrera, M.; Herrera, J.M.; Cantatore, S.; Aguilar, J.; Felipe, A.; Fumuso, E. Comparative histomorphological study of endometrium in mares. Journal of Veterinary Medicine Series C: Anatomia Histologia Embryologia 2018, 47, 153–158. [Google Scholar] [CrossRef]
- Rasch, K.; Schoon, H.A.; Sieme, H.; Klug, E. Histomorphological endometrial status and influence of oxytocin on the uterine drainage and pregnancy rate in mares. Equine Vet J 1996, 28, 455–460. [Google Scholar] [CrossRef]
- Herrera, M.F.; Otermin, M.; Herrera, J.M.; Simoy, M.V.; Bianchi, C.P.; Aguilar, J.J.; Fumuso, E.A. Effect of Mycobacterium cell wall fraction on endometrial histomorphometry of mares resistant and susceptible to persistent breeding-induced endometritis. Theriogenology 2020, 156, 2–10. [Google Scholar] [CrossRef]
- Love, C.C. Evaluation of reproductive efficiency. In Equine breeding management and artificial insemination, 2nd ed.; Samper J.C. Publisher: Saunders: Philadelphia, USA, 2009; pp. 289–94. [Google Scholar] [CrossRef]
- Card, C. Post-breeding inflammation and endometrial cytology in mares. Theriogenology 2005, 64, 580–588. [Google Scholar] [CrossRef]
- Hinrichs, K.; Cummings, M.R.; Sertich, P.L.; Kenney, R.M. Clinical significance of aerobic bacterial flora of the uterus, vagina, vestibule, and clitoral fossa of clinically normal mares. J Am Vet Med Assoc 1988, 193, 72–75. [Google Scholar] [CrossRef]
- Dascanio, J.J. Uterine Lavage. In Equine Reproductive Procedures, 2nd ed.; Dascanio, J.J., McCue, P., Eds.; Publisher: John Wiley & Sons: Hoboken, USA, 2021; pp. 127–130. [Google Scholar] [CrossRef]
- Aguilar, J.; Hanks, M.; Shaw, D.J.; Else, R.; Watson, E. Importance of using guarded techniques for the preparation of endometrial cytology smears in mares. Theriogenology 2006, 66, 423–430. [Google Scholar] [CrossRef]
- Rasmussen, C.D.; Petersen, M.R.; Bojesen, A.M.; Pedersen, H.G.; Lehn-Jensen, H.; Christoffersen, M. Equine Infectious Endometritis—Clinical and Subclinical Cases. J Equine Vet Sci 2015, 35, 95–104. [Google Scholar] [CrossRef]
- Álvarez-Delgado, C.; Ruedas-Torres, I.; Sánchez-Carvajal, J.M.; Priego-Capote, F.; Castillo-Peinado, L.; Galán-Relaño, Á.; Moreno, P.J.; Díaz-Bueno, E.; Lozano-Buenestado, B.; Rodríguez-Gómez, I.M.; Carrasco, L.; Pallarés, F.J.; Gómez-Laguna, J. Impact of supplementation with dihydroxylated vitamin D3 on performance parameters and gut health in weaned Iberian piglets under indoor/outdoor conditions. Porcine Health Manag 2023, 9, 15. [Google Scholar] [CrossRef]
- Leishman, D.; Miller, R.B.; Doig, P.A. A Quantitative Study of the Histological Morphology of the Endometrium of Normal and Barren Mares. Can J Comp Med 1982, 46, 17–20. [Google Scholar]
- Kenney, R.M. Cyclic and pathologic changes of the mare endometrium as detected by biopsy, with a note on early embryonic death. J Am Vet Med Assoc 1978, 172, 242–262. [Google Scholar] [CrossRef]
- Kenney, R.M.; Doig, P.A. Equine endometrial biopsy. Current Therapy in Theriogenology 1986, 2, 723–729. [Google Scholar]
- Vilés, K.; Rabanal, R.; Rodríguez-Prado, M.; Miró, J. Influence of seminal plasma on leucocyte migration and amount of COX-2 protein in the jenny endometrium after insemination with frozen-thawed semen. Anim Reprod Sci 2013, 143, 57–63. [Google Scholar] [CrossRef]
- Van Camp, S.D. Endometrial biopsy of the mare. A review and update. Vet Clin North Am Equine Pract 1988, 4, 229–245. [Google Scholar] [CrossRef]
- Causey, R.C. Mucus and the mare: How little we know. Theriogenology 2007, 68, 386–394. [Google Scholar] [CrossRef] [PubMed]
- Troedsson, M.H.T.; Liu, I.K.M.; Thurmond, M. Function of uterine and blood-derived polymorphonuclear neutrophils in mares susceptible and resistant to chronic uterine infection: phagocytosis and chemotaxis. Biol Reprod 1993, 49, 507–514. [Google Scholar] [CrossRef] [PubMed]
- Petersen, M.R.; Skive, B.; Christoffersen, M.; Lu, K.; Nielsen, J.M.; Troedsson, M.H.T.; Bojesen, A.M. Activation of persistent Streptococcus equi subspecies zooepidemicus in mares with subclinical endometritis. Vet Microbiol 2015, 179, 119–125. [Google Scholar] [CrossRef] [PubMed]
- Ferris, R.A.; Mccue, P.M.; Borlee, G.I.; Glapa, K.E.; Martin, K.H.; Mangalea, M.R.; Hennet, M.L.; Wolfe, M.L.; Broeckling, C.D.; Borlee, B.R. Model of Chronic Equine Endometritis Involving a Pseudomonas aeruginosa Biofilm. Infection and Inmunity 2017, 85, e00332–17. [Google Scholar] [CrossRef]
- Skive, B.; Rohde, M.; Molinari, G.; Braunstein, T.H.; Bojesen, A.M. Streptococcus equi subsp. zooepidemicus invades and survives in epithelial cells. Front Cell Infect Microbiol 2017, 7, 465. [Google Scholar] [CrossRef]
- Köhne, M.; Hüsch, R.; Tönissen, A.; Schmidt, M.; Müsken, M.; Böttcher, D.; Hirnet, J.; Plötz, M.; Kittler, S.; Sieme, H. Isolation and characterization of bacteriophages specific to Streptococcus equi subspecies zooepidemicus and evaluation of efficacy ex vivo. Front Microbiol 2024, 15, 1448958. [Google Scholar] [CrossRef]
- Veiga, G.A.L.; Miziara, R.H.; Angrimani, D.S.R.; Papa, P.C.; Cogliati, B.; Vannucchi, C.I. Cystic endometrial hyperplasia-pyometra syndrome in bitches: Identification of hemodynamic, inflammatory, and cell proliferation changes. Biol Reprod 2017, 96, 58–69. [Google Scholar] [CrossRef]
- Radar-Chafirovitch, A.; Catarino, J.; Lourenço, L.; Payan-Carreira, R.; Ferreira-Dias, G.; Miró, J.; Quaresma, M.; Pires, M.A. Evaluation of Inflammatory Infiltrated in the Endometrium of the Jenny (Equus asinus). J Comp Pathol 2022, 191, 46. [Google Scholar] [CrossRef]
- Wu, R.; Yu, F.; Holyoak, G.R.; Gao, Y.; Zhu, Y.; Li, J. Use of the endometrial histopathology to improve diagnosis of donkeys with endometritis. Equine Vet Educ 2023, 36, 43–50. [Google Scholar] [CrossRef]
- Lopes Costa, L.M.; Sancler-Silva, Y.F.R.; Albino, M.V.; Fontes, C.S.; Texeira, C.S.; Freitas, M.S.; Kladt, L.V.; Schultz, E.B. Does the uterine inflammatory response differ between mares and donkey jennies inseminated with frozen donkey semen? J Equine Vet Sci 2023, 125, 104703. [Google Scholar] [CrossRef]
- Diaz-Jimenez, M.; Rota, A.; Dorado, J.; Consuegra, C.; Pereira, B.; Camillo, F.; Panzani, D.; Fanelli, D.; Tesi, M.; Monaco, D.; Hidalgo, M. First pregnancies in jennies with vitrified donkey semen using a new warming method. Animal 2021, 15, 100097. [Google Scholar] [CrossRef]
- Nielsen, J.M. Endometritis in the mare: A diagnostic study comparing cultures from swab and biopsy. Theriogenology 2005, 64, 510–518. [Google Scholar] [CrossRef]
- Rudolph, N.; Schoon, H.A.; Schöniger, S. Immunohistochemical characterisation of immune cells in fixed equine endometrial tissue: A diagnostic relevant method. Pferdeheilkunde 2017, 33, 524–537. [Google Scholar] [CrossRef]




| Parameters | Jennies | Mares | |||||||||||
| J1 | J2 | J3 | J4 | J5 | J6 | M1 | M2 | M3 | M4 | M5 | M6 | ||
| Parity status1 | Maiden | Maiden | Foaling | Foaling | Foaling | Foaling | Maiden | Maiden | Foaling | Foaling | Foaling | Foaling | |
| Age (years)2 | 3 | 5 | 10 | 10 | 11 | 12 | 3 | 3 | 12 | 13 | 14 | 16 | |
| PMNs (%)3 | 1.62 | 0.50 | 0.17 | 2.61 | 3.71 | 0.66 | 0.66 | 0.33 | 0.50 | 0.00 | 0.33 | 0.00 | |
| KDGS4 | I | IIa | I | IIa | I | IIa | I | IIa | IIa | IIa | IIa | I | |
| Parameters | Jennies | Mares | |||
| Maiden (n = 2) | Foaling (n = 4) | Maiden (n = 2) | Foaling (n = 4) | ||
| LE (µm) | 14.19 ± 0.47b | 17.88 ± 0.33a | 10.93 ± 0.24c | 18.01 ± 0.37a | |
| GE-SC | 11.23 ± 0.30b** | 12.21 ± 0.26ab | 9.75 ± 0.21c | 12.43 ± 0.25a | |
| GE-SS | 10.10 ± 0.25b | 11.59 ± 0.22a | 9.39 ± 0.23b | 12.43 ± 0.25a | |
| LD-SC | 15.25 ± 1.39a | 15.62 ± 1.27a** | 10.78 ± 0.75b | 9.37 ± 0.55b | |
| LD-SS | 12.80 ± 1.45 | 11.47 ± 0.71 | 12.78 ± 1.47 | 13.71 ± 1.38** | |
| GA-SC | 1322.03 ± 105.85bc | 1908.62 ± 148.64a | 1018.72 ± 74.21c | 1588.38 ± 103.27ab | |
| GA-SS | 1501.21 ± 110.18 | 1599.21 ± 104.16 | 1485.8 ± 127.31** | 1835.28 ± 137.12 | |
| Parameters | Jennies | Mares | ||
| Maiden (n = 2) | Foaling (n = 4) | Maiden (n = 2) | Foaling (n = 4) | |
| #G-SC | 11.68 ± 1.51 | 10.86 ± 1.20 | 8.70 ± 1.66 | 8.83 ± 1.22 |
| #G-SS | 26.25 ± 3.05*** | 35.53 ± 2.31*** | 31.90 ± 4.24*** | 25.91 ± 2.31*** |
| GT-SC | 15.67 ± 1.20ab | 18.31 ± 1.33a | 8.70 ± 1.52c | 12.67 ± 1.27bc |
| GT-SS | 14.37 ± 1.22 | 16.12 ± 0.91 | 12.87 ± 0.81* | 15.41 ± 0.99 |
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