Bioscience Methods 2025, Vol.16, No.6, 308-316 http://bioscipublisher.com/index.php/bm 311 like timed artificial insemination (FTAI) have gradually gained attention. Its core lies in using hormone intervention to bring ovulation time within a "controllable range". Drugs such as GnRH, hCG, prostaglandins and progesterone can all be used. The key is to combine them reasonably and smooth out the rhythm of the follicular phase and the luteal phase. Whether estrus can be detected or not is not that important. Compared with natural estrus or fertilization arranged by experience, the pregnancy rate of this method is significantly more stable (Presicce et al., 2022; Bhat & Dhaliwal, 2023). Sometimes, adding an injection of GnRH or hCG during artificial insemination can further increase the success rate. Moreover, this approach is not expensive in field practice and is easy to promote in operation (Atabay et al., 2020; Selvaraju et al., 2023). 4.2 Herd management through estrus synchronization and superovulation If you only want one or two cows to get pregnant smoothly, using FTAI alone might be sufficient. But when it comes to an entire group of water buffaloes, especially considering management costs and labor constraints, simultaneous estrus becomes even more cost-effective. Synchronization solutions like Ovsynch and CIDR have actually been used quite proficiently by many farms. They can unify the estrous cycles of multiple water buffaloes, eliminating the need to observe each one individually during mating and saving a significant amount of time. As for superovulation, it is more like a tailor-made tool for IVF or embryo transfer. By injecting hormones such as FSH and eCG, cows can release multiple mature follicles at once, thereby increasing the efficiency of egg retrieval. If it is a donor with high genetic value, this approach is obviously more cost-effective and can amplify the "leverage effect" of genetic output more effectively, which is quite practical both in the breeding season and the non-breeding season (Baruselli et al., 2018; 2020). 4.3 Sperm quality control and accelerated genetic progress Ultimately, whether it is artificial insemination or in vitro fertilization, sperm quality is the foundation. Although many semen samples may remain intact when frozen, once the sperm motility and membrane integrity do not meet the standards, mating will be in vain. Nowadays, some technologies can be of assistance, such as proteomic analysis and genetic screening - markers like GnRHR gene polymorphisms have been used to assess sperm expressiveness (Figure 1). This way, bulls with potential infertility or low breeding value can be screened out before mating, saving wasted time (Wang et al., 2017; Karanwal et al., 2023; Kolachi et al., 2025). From a broad perspective, these technologies not only increase the conception rate, but more importantly, they promote the rapid spread of superior genes within the population, accelerate the speed of genetic improvement of water buffaloes, and also provide a practical handle for enhancing breeding efficiency. Figure 1 Sequential stages of the estrous cycle (Adopted from Kolachi et al., 2025)
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