Extracellular vesicles (EVs) have been identified in the uterine fluid in different species and have been pointed as key players in the embryo-maternal dialogue, maternal recognition of pregnancy and establishment of pregnancy. However, little is known about the uterine EVs in the mare. Therefore, the present study aimed at characterizing EVs from uterine lavage of cyclic mares by comparing five EVs isolation methods and the combination of them: (1) ultracentrifugation (UC); (2) concentration of lavage volume by Centricon ultrafiltration (CE); (3) the use of CE with different washing steps (phosphate-buffered saline with or without trehalose); (4) size-exclusion chromatography with iZON-qEV columns, and (5) a combination of the methods with best results based on EVs yield, purity, and protein cargo profiles. Transmission electron microscopy and Western blotting confirmed the isolation of EVs by all methods but with quantitative and qualitative differences. Mass spectrometry provided differences in protein profiles between methods, number of identified proteins, and protein classes. Our results indicate that the combination of CE/trehalose/iZON/UC is an optimal method to isolate equine uterine EVs with good yield and purity that can be applied in future studies to determine the role of equine uterine EVs in embryo-maternal interactions.

Background and Aim: The present study aimed to evaluate and compare the overall and breed‐specific seasonal breeding patterns, fertility rates, cyclicity, and follicular dynamics of Arab, Thoroughbred, and Percheron mares under the subtropical conditions of Pakistan. Materials and Methods: A retrospective analysis of climatic data and breeding records of eleven breeding studs spanning four years (2020–2023) was made to find out the overall seasonality in the breeding pattern of mares. Fifty mares of each breed ( n = 150 in total) were scanned by ultrasonography for a calendar year to find the cyclicity pattern and follicular dynamics (follicular growth rate, size of ovulatory follicle, and days from estrus till ovulation). Results and Discussion: The statistical analysis of breeding records demonstrated a clear pattern of seasonal breeding ( p < 0.05). The highest monthly foalings were noted in March (247 ± 45.37), and overall breeding activities peaked in Spring season ( p < 0.05). Breed‐specific results of Arab, Thoroughbred, and Percheron mares revealed that Arab mares maintained stable breeding activity throughout the year, with the highest activity in spring and peak conception rate in winter (56.25% ± 32.78; p  > 0.05). Thoroughbred mares experienced significant seasonal declines from spring to winter with a peak conception rate in winter (63.89% ± 27.37, p  > 0.05). Percherons showed the most pronounced seasonal effects, especially with a high fall conception rate (73.04% ± 19.61) and a sharp decrease in winter breeding metrics ( p < 0.05). Furthermore, Thoroughbred and Percheron mares displayed the most pronounced seasonal effects on the percentage of cyclic mares 77.3% and 56% in winters ( p < 0.05). Moreover, the follicular dynamics of the three breeds also exhibited significant differences ( p < 0.05). Conclusion: The current study concludes that seasonal and breed‐specific variability exists among the reproductive parameters of Arab, Thoroughbred, and Percheron mares in subtropics, necessitating breed‐specific reproductive management measures to maximize mare breeding efficiency.

The objective of current study was to assess the trend in various luteal characteristics viz luteal size (LS), plasma progesterone (P⁴) concentration, and luteal blood flow (LBF) using color Doppler imaging (CDI) and power Doppler imaging (PDI) modes in pregnant and nonpregnant Nili‐Ravi buffaloes. Lactating, cyclic, and healthy Nili‐Ravi buffaloes (n = 09) without any reproductive abnormality were selected in present study. Buffaloes were synchronized using Ov‐Synch, and fixed‐time artificially insemination was performed (day = 0). Pregnancy was diagnosed on 30‐day post‐AI using B‐mode ultrasonography based on presence or absence of embryonic heartbeat. Ovaries of all animals were scanned from day 5 till 21 post‐AI using both B‐mode and Doppler ultrasonography to measure LS and LBF. After each ovarian ultrasound examination, blood samples were collected via jugular venipuncture to determine plasma P⁴ concentration. According to results, LBF using CDI and PDI was significantly higher (P ≤ 0.05) in pregnant buffaloes on days 13 and 15 post‐AI, respectively. The mean LS and plasma P⁴ concentration did not differ (P ≥ 0.05) between pregnant and nonpregnant animals until day 15 post‐AI. However, a significant difference (P ≤ 0.05) was noticed for both on day 17 and onwards. It is concluded that LBF is a more sensitive luteal character as compared to LS and P⁴ for earlier pregnancy diagnosis in Nili‐Ravi buffaloes when ascertained through CDI.

Mubbashar Hassan, Sanan Raza, Ahmad Yar Qamar, Muhammad Ilyas Naveed, Abdul Mateen, Muhammad Noman, Sayed Aun Muhammad and Abid Hussain Shahzad. Journal of Animal Reproduction and Biotechnology 2024;39:62-5. https://doi.org/10.12750/JARB.39.1.62

Mubbashar Hassan, Ayat Fatima, Sanan Raza, Ahmad Yar Qamar, Muhammad Ilyas Naveed, Abdul Mateen, Sayyed Aun Muhammad and Abid Hussain Shahzad. J Anim Reprod Biotechnol 2026;41:238-41. https://doi.org/10.12750/JARB.41.3.238