Bioscience Methods 2025, Vol.16, No.6, 308-316 http://bioscipublisher.com/index.php/bm 309 insemination (FTAI), in vitro embryo transfer (IVEP), and embryo transfer, among others. They do not simply increase the number of mating times. Instead, they bypass the natural reproductive problems of water buffaloes through means such as synchronous ovulation, concentrated estrus, and shortened intervals - especially during periods when estrus is not obvious or the season is incorrect, they can also improve reproductive efficiency (Srirattana et al., 2022; Maylem et al., 2025). However, these technologies are not simply "put on". They must be optimized around the physiological characteristics of water buffaloes themselves and managed more meticulously in order to truly reduce costs and improve effects (Baruselli et al., 2018). This study aims to emphasize the economic and genetic value of water buffaloes in global animal husbandry, analyze the main reasons and impacts of the species' low reproductive capacity, showcase the application and case studies of assisted reproductive technology in enhancing reproductive capacity, and is expected to provide information for breeding strategies, increase productivity, and support sustainable water buffalo farming. 2 Biological Basis of Reproduction in Water Buffalo 2.1 Characteristics of the reproductive cycle and estrus behavior in buffalo Although the average estrus cycle of water buffaloes is 21 days, it is actually not stable and often fluctuates. The most troublesome thing is that their estrus is not very obvious, unlike that of cattle which is easy to notice. Sometimes it only lasts for five or six hours, and sometimes it can extend to a whole day (5 to 27 hours), which makes it very difficult for breeders to accurately grasp the timing for mating. Moreover, unlike cows, water buffaloes do not have obvious homosexual climbing behavior. Without even this "hint", it is even more difficult to detect estrus. Ovulation usually occurs between 24 and 48 hours after the onset of estrus. However, in poor conditions, water buffaloes are prone to delayed sexual maturity, slow postpartum recovery, and prolonged estrus periods (Nava-Trujillo et al., 2020; Kolachi et al., 2025), which is also quite a headache. 2.2 Constraints on reproductive performance There are actually many reasons that affect the reproductive efficiency of water buffaloes, and they are often intertwined. The first is seasonality. Water buffaloes prefer to breed under short-day conditions, so their breeding performance is even better in autumn and winter. However, as the duration of sunlight increases in spring and summer, their estrus actually decreases and their ovulation also drops. This is because light exposure affects the secretion of melatonin, which in turn regulates the entire hypothalamic-pituitary-ovarian axis (Ramadan, 2017; Currin et al., 2022). Another problem is the corpus luteum. Sometimes it is embedded in the ovary and is not easily observed. Coupled with weak luteal function and low progesterone secretion, the estrous cycle and pregnancy rate will be affected (Devkota et al., 2022). Nutrition should not be overlooked either. Malnutrition, extremely hot weather or parasitic infections can all cause water buffaloes to enter a "low desire" mode and not cooperate with breeding arrangements, especially during the non-breeding season or when feed is tight (Zicarelli, 2019). There is another phenomenon called "silent estrus" or "ovaries without a cycle". Many water buffaloes just quietly "miss" the estrus period, and the breeders are completely unaware of it. Naturally, they cannot be mated and the conception rate is also low. 2.3 Physiological differences in sperm and oocyte traits Buffaloes also have some special features in terms of gametes. Let's start with the female. The ovaries of water buffaloes are inherently smaller than those of cows, and they contain fewer primordial follicles, which means that the "raw materials" available for the development of oocytes are limited. Moreover, their follicular development usually follows only one main path, with one dominant follicle, and the others are prone to premature atresia. As follicles grow, the gene expression of oocytes and granulosa cells within them also changes. This difference directly affects the continued development of follicles and the quality of oocytes (Xu et al., 2024). Let's take a look at male water buffaloes. The performance of their sperm is actually related to the season. The quality of sperm is generally better in winter - including indicators such as motility, membrane integrity, and fallopian tube binding ability. In contrast, it is poorer in summer. Some studies have also pointed out that some genes related to gonadotropin release, such as SNP polymorphisms in GnRH, can affect sperm motility, concentration and morphology (Saraf et al., 2019; Wang et al., 2020). Generally, the offspring of bulls with good integrity of the
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