Buffalo is economically an important dairy animal in Europe, South Asia and South America owing to its low maintenance requirements, adoptability to harsh environment and high feed conversion efficiency [1], [2]. Its importance is strengthened by the fact that it accounts for over 12% of the global milk demand and contains higher fat and protein contents compared to cow milk [3]. But, at the same time, it demonstrates poor reproductive efficiency as evident by its poor ovarian reserve as compared to cow, long calving interval and seasonality in breeding [4], [5], [6]. Reduced attention by the researchers has further predisposed this specie to the declining levels [7].

The growing global population is driving an increased demand for animal-derived proteins, raising significant concerns about sustainability and food security. A well-managed buffalo production system offers a potential solution to these challenges, as dairy products and beef provide high-quality protein sources [8], [9]. Genetic selection aimed at enhancing beef or milk yield, improving adaptability to elevated environmental temperatures, boosting fertility, and reducing metabolic diseases and reproductive disorders are crucial objectives for achieving low-input farming and economically sustainable production systems [10], [11].

IVEP is an effective strategy for genetic selection, enabling more offspring per year compared to natural or artificial insemination methods. Since the introduction of embryo transfer (ET), buffalo genetics have continued to improve [12]. Advancements in embryo retrieval, culture systems, and vitrification techniques have significantly enhanced pregnancy success rates [13]. However, pregnancy success and live birth rate following ET remains low in both cattle and buffalo [14], [15], with buffalo exhibiting lower oocytes quality, reduced cleavage rates, and decreased blastocysts production as compared to cattle [16].

One factor contributing to this limitation is the challenge of identifying and selecting the most competent blastocysts for transfer [17]. Embryo competence is often assessed through subjective methods, with the International Embryo Transfer Society (IETS) coding system being the standard for classifying blastocyst stages and quality[18]. While this system relies on evaluator expertise, it still results in suboptimal pregnancy outcomes [15], [19], [20]. Pre-implantation genetic testing for aneuploidy (PGT-A) and karyotype testing are also utilized for embryo selection, with digital karyotype results assisting in this process [21]. However, even the transfer of euploid blastocysts results in pregnancy rates of only 30-50% in humans [22], [23], [24], [59].6% in bovines [25]. It is widely acknowledged that achieving a successful pregnancy requires the identification of the most competent blastocyst for transfer. Unfortunately, no current methods accurately predict implantation or developmental competence.

Conflicting evidence exists in the literature regarding pregnancy outcomes with blastocysts at various stages of development. In mice, rapidly developing blastocysts are considered healthier, while slow-developing blastocysts more closely mimic the typical in vivo timeline [26]. Early-cleaving bovine embryos reach the blastocyst stage faster than late-developing embryos, but pregnancy rates are unrelated to development timing [27]. In humans, transfers of day 5 blastocysts result in higher clinical pregnancy and live birth rates compared to day 6 transfers [28], [29], [30]. However, slower-developing bovine embryos tend to activate genes associated with cell survival and apoptosis [31]. Mouse embryos with slower development rates are more similar to in vivo-derived embryos in terms of cell number, embryo volume, and expression of metabolic markers [26].

The molecular factors driving the development of competent buffalo blastocysts are progressively being identified. RNA-Seq analysis of key gene expression levels provides predictive insights into the cellular status of embryos [32], [33], [34]. In pigs, transcriptional changes at various developmental stages (2-cell and 4-cell) in in vitro-produced embryos have been compared with in vivo-developed embryos to understand the causes of reduced developmental potential. These studies identified altered transcript levels in apoptosis-related factors, spindle components, and cell cycle regulators, which contribute to the diminished developmental potential of in vitro embryos [35]. In swine, embryos that cleave more rapidly show higher expression of genes involved in the meiotic cell cycle [36], while slow-cleaving bovine embryos exhibit increased caspase activity compared to fast-cleaving ones [37]. Furthermore, differences in gene expression profiles have been noted in somatic cell nuclear transfer embryos versus in vivo-derived pig embryos [38]. However, the specific gene expression patterns that can reliably predict the most competent buffalo blastocysts remain unclear.

Keeping above discussion in view, we hypothesized that the developmental timing of buffalo blastocyst expansion reliable predictor of embryo competence. So, this study aims to predict the most competent buffalo blastocysts by comparing developmental potential and pregnancy outcomes following the transfer of blastocysts expanded on day 5 (fast-growing), day 6 (moderately growing), or day 7 (slow-growing). Using the SMART-Seq2 technology, RNA-Seq analysis was further conducted to identify differentially expressed genes (DEGs) between day 5 and day 7 embryos. The insights derived from RNA-Seq will help bridge the gap in understanding the molecular events occurring in blastocysts at various developmental stages, providing a more robust system for grading blastocysts for transfer. This research aims to support more informed decision-making in embryo selection during assisted reproductive techniques.