Reproductive efficiency is crucial for dairy farm productivity, but achieving consistent fertility remains challenging. TAI improves pregnancy rates by synchronizing ovulation and enabling precision breeding. Despite the advancements in hormone synchronization protocols, outcomes vary due to genetic, physiological, and metabolic differences among cows. This review examines current TAI protocols, including gonadotropin-releasing hormone (GnRH)-based and estradiol/progesterone (P4)-based synchronization methods, emphasizing their effectiveness and practical limitations. We also examined how to integrate emerging omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, into TAI protocols, marking a significant shift in reproductive management. These tools unveil molecular biomarkers that enable the precise tailoring of TAI protocols to the genetic, metabolic, and physiological profiles of individual animals, addressing challenges in variable fertility responses. Key factors influencing TAI success, such as animal health, environment, and insemination timing, are explored. This review identifies gaps in the existing literature, such as the integrated omics methodologies and data integration across platforms, and proposes a framework for future research to refine TAI protocols to address genetic variability and apply omics technologies to identify validated biomarkers for early pregnancy detection, which will significantly enhance the practical impact of TAI. Future directions highlight the need for interdisciplinary approaches combining molecular insights with robust on-farm applications to improve fertility outcomes and reduce reliance on blanket synchronization methods. By combining traditional synchronization methods with cutting-edge molecular tools, TAI offers promising opportunities for improving reproductive efficiency and sustainability in dairy farming.

The success of assisted reproductive technology is contingent upon the growth potential of embryos post-vitrification process. When compared to in vivo embryos, it has been found that the high intracellular lipid accumulation inside the in vitro-derived embryos results in poor survival during vitrification. Based on this finding, the present study assessed the impact of incorporating forskolin and linoleic acid (FL) entering in vitro culture (IVC) on the embryos’ cryo-survival, lipid content, and viability throughout vitrification. Lipid metabolomics and single-cell RNA sequencing (scRNA-seq) techniques were used to determine the underlying mechanism that the therapies were mimicking. It was observed that out of 726 identified lipids, 26 were expressed differentially between the control and FL groups, with 12 lipids upregulated and 14 lipids downregulated. These lipids were classified as Triacylglycerol (TG), Diacylglycerol (DG), Phosphatidylcholine (PC), and so on. A total of 1079 DEGs were detected between the FL and control groups, consisting of 644 upregulated genes and 435 downregulated genes. These DEGs were significantly enhanced in the arachidonic acid metabolism, lipolysis, fatty acid metabolism, cAMP signaling pathway, and other critical developmental pathways. Based on the observation, it was concluded that forskolin and linoleic acid decreased the droplet content of embryos by modulating lipid metabolism, thus enhancing the vitrified bovine embryos’ cryo-survival.

The astringent selection criteria for milk-oriented traits in dairy cattle have rendered these animals prone to various metabolic disorders. Postpartum lactational peak and reduced feed intake lead to negative energy balance in cattle. As a compensatory mechanism, cattle start mobilizing fat reserves to meet the energy demand for vital body functions. Consequently, diminished glucose concentrations and elevated ketone body levels lead to poor ovarian function. The impaired follicular development and subpar oocyte quality diminish the conception rates, which poses significant economic repercussions. Follicular fluid is integral to the processes of follicular growth and oocyte development. Hence, the present study was performed to identify potential alterations in metabolites in the follicular fluid under in vitro culture conditions mimicking negative energy balance. Our results revealed nine distinct metabolites exhibiting differential expression in follicular fluid under negative energy balance. The differentially expressed metabolites were predominantly associated with pathways related to amino acid metabolism, lipid metabolism, signal transduction mechanisms, and membrane transport, alongside other biological processes. The identified signature metabolites may be further validated to determine oocyte fitness subjected to in vitro fertilization or embryo production from slaughterhouse source ovaries.

Porcine embryo cryopreservation remains challenging due to high lipid content, oxidative stress, and ice recrystallization that compromise post-thaw survival and developmental competence. We evaluated an integrated vitrification approach combining antioxidants (berberine, melatonin), iron oxide (Fe₃O₄) nanoparticles, and antifreeze protein I (AFP I) with post-thaw interventions (glutathione and zona pellucida digestion) to synergistically improve cryosurvival and developmental competence of porcine parthenogenetic embryos. In vitro-matured parthenogenetic embryos were vitrified on Cryotop using a protocol that included berberine and melatonin in embryo culture, Fe₃O₄ nanoparticles and AFP I in cryoprotectant solutions, and post-warming treatment with glutathione and a brief zona pellucida digestion. Survival, hatching, adenosine triphosphate (ATP) content, reactive oxygen species (ROS) levels, cytoskeletal integrity, and the expression of BAX, BCL2, OCT4, and SOX2 genes were measured. Both the dual antioxidant (berberine + melatonin) and nanoparticle + AFP interventions produced greater improvements than individual additives. Fully integrating all components yielded the highest post-thaw viability, with ~94% survival and ~90% hatching, values statistically equivalent to those of fresh embryos. Treated embryos also showed significantly higher ATP levels, lower ROS accumulation (approaching levels in fresh embryos), and preserved microtubule structure (~91% normal). Vitrification alone upregulated BAX and downregulated BCL2, OCT4, and SOX2, whereas the integrated protocol restored their expression levels to near control levels. This multi-component antioxidant, nanoparticle, antifreeze strategy synergistically enhances the cryotolerance and developmental competence of vitrified porcine embryos by mitigating oxidative stress and cryoinjury. Post-thaw viability and molecular markers were restored to near-fresh conditions, demonstrating a promising approach to improve embryo cryopreservation outcomes in swine and potentially other species.

The objective of this study was to determine if the use of human chorionic gonadotrophin (hCG) and ketoprofen would affect the pregnancy percentages of dairy cows following timed artificial insemination (TAI). This experiment was conducted on a commercial dairy farm in China involving 799 healthy Holstein cows (2–4 parities) between October and December 2024. Cows were randomly assigned to three groups: hCG-3 = treated with (0 d: GnRH, 100 μg; 7–8 d: PGF_2α, 0.4 mg; 52 h: GnRH, 100 μg; 14–16 h: TAI) + hCG 3 vials (300 µg)/cow on day 7 of TAI; hCG-2 group = TAI + 2 vials (200 µg) hCG/cow on day 7; and hCG+ketoprofen = TAI + 300 µg hCG/cow + ketoprofen 10 mL/cow on days 15 and 16 of TAI. Metabolomic profiling (untargeted and targeted) of 22 pregnant cows was conducted on serum collected on days 17 and 21 post-TAI. Results indicated greater pregnancy percentages in the cows of the hCG+ketoprofen-treated group compared to those in the other two groups (60.1% compared with 49.6% and 41.9%). The cows treated with hCG+ketoprofen had less oxidative stress markers, downregulation of arachidonic acid metabolism and upregulation of glycerophospholipid metabolism on day 17 after TAI, indicating that there was upregulation of tryptophan and serotonergic pathways, increased amino acid metabolism and continued anti-inflammatory effects on day 21 after TAI. These findings were confirmed by evaluation data collected by conducting the targeted metabolomic procedures, as indicated by the greater progesterone and melatonin and lesser 17-estradiol and 21-deoxycortisol concentrations. These findings indicate that combined hCG+ketoprofen administrations following the TAI treatment regimen improve pregnancy percentages in dairy cattle as a result of metabolic and endocrine milieu modulations.