Bioscience Methods 2025, Vol.16, No.6, 308-316 http://bioscipublisher.com/index.php/bm 310 sperm membrane and acrosome are also more likely to successfully conceive. These biological characteristics of water buffaloes themselves - whether it is the estrous cycle, ovarian structure, or gamete function - all indicate one thing: reproductive management cannot be simply copied from the water buffaloes. Strategies must be adjusted according to the particularity of water buffaloes, and at the same time, various assisted reproductive technologies must truly "adapt" to their physiological rhythms in order to increase the success rate. 3 Overview of Assisted Reproductive Techniques (ARTs) 3.1 Development and limitations of Artificial insemination (AI) Although artificial insemination (AI) has been used in the field of buffalo breeding for many years, the results have never been satisfactory. Compared with cattle, the water buffalo's less "cooperative" estrus behavior has long been an old problem - sometimes the cycle is unstable, sometimes the season is particularly affected, and the estrus is not obvious. These factors make estrus detection quite a headache and it's also difficult to grasp the timing of mating. To bypass this problem, the timed artificial insemination (FTAI) program was later introduced. This approach does not require observing estrus and controls ovulation time through medication, which can slightly increase the success rate of mating and is also convenient for centralized management (Lui, 2025). But then again, even so, the conception rate has not been able to increase steadily, especially during the non-breeding season. Factors such as heat stress, the operator's experience level, semen quality, and even the reproductive state of the cow at that time can all affect the final outcome. Some studies have also pointed out that even with concurrent estrus treatment, the overall success rate of water buffaloes is still lower than that of cattle (Atabay et al., 2020; Neglia et al., 2020; Atabay et al., 2024; Kolachi et al., 2025; Maylem et al., 2025). 3.2 In vitro embryo production (IVF) and embryo transfer (ET) techniques The entire set of IVF and ET technologies developed from cattle is now also being attempted to be applied to water buffaloes. However, the problem is that the number of follicles in the ovaries of water buffaloes is relatively small, and the maturity of oocytes is often insufficient, which makes the entire in vitro embryo production (IVEP) process more complicated than that of water buffaloes. The entire process roughly includes the collection, maturation, fertilization and culture of oocytes, and finally the embryo is transplanted into the recipient cow that is already "ready". Although it sounds like a standardized operation, different donors, breeding bulls, and even environmental conditions can all affect the embryo development rate and yield. Even so, many studies have confirmed that normal pregnancy and even successful calving can be achieved whether using fresh embryos or cryopreserved vitrized embryos. This also indicates that these techniques do have their potential in genetic improvement and protection (Baruselli et al., 2023; Bandeo et al., 2025). 3.3 Oocyte pickup and in vitro maturation (OPU-IVM) Nowadays, more and more breeding projects are beginning to pay attention to OPU, especially the use of ultrasounds guided transvaginal oocyte retrieval (OPU) or laparoscopic methods (LOPU) to repeatedly retrieve eggs from live cows or even pre-estrus heifers. One of the benefits of doing so is that it can accelerate the replacement of generations and shorten the breeding cycle. Although the OPU-IVM technology of water buffaloes developed more slowly than that of cattle, in recent years, by adjusting the hormone stimulation methods, such as using FSH or eCG, there has been an improvement in both the quantity and quality of eggs. However, at present, the number of available embryos that can be retrieved from each OPU is still less than that from cattle, and the differences in ovarian responses among individuals are also quite obvious. Therefore, the subsequent focus may still have to be on how to optimize the cultivation conditions and stimulation schemes. Only in this way can the OPU-IVM technology truly play a stable and long-term role in the buffalo breeding program (Currin et al., 2022; 2023). 4 Mechanisms of ARTs in Enhancing Buffalo Fertility 4.1 Physiological mechanisms for increasing conception rate and shortening estrus cycle In terms of breeding management, the problem with water buffaloes has never been merely "incompatibility", but more often it is "inability to develop passion". The estrus is not obvious and the ovulation time is unstable. Under such circumstances, relying on manual observation of estrus for mating is not very reliable. As a result, methods
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