Optimizing ovulation synchronization in Holstein heifers: implementing a half-dose GnRH analogue (Buserelin Acetate) in a nanofabricated form within the ovsynch protocol to enhance the ovarian activity and reproductive performance outcomes

The objective of this study was to evaluate the efficacy of a nanofabricated formulation of gonadotropin-releasing hormone analogue, buserelin acetate (GnRH-BA), conjugated with chitosan nanoparticles (CH-NPs) as a nanocarrier. The resulting nano-GnRH-BA formulation was incorporated into the Ovsynch (OVS) protocol at a 50% reduced dose to improve ovulation synchronization and reproductive performance in dairy heifers.

CH-NPs and nano-GnRH-BA were synthesized via the ionic gelation method. Key characteristics, including particle shape, size, Polydispersity Index (PdI), and drug-loading efficiency (DLE%), were evaluated. A total of 210 Holstein-Friesian heifers were assigned to either a standard OVS (

n

= 82) or nano-OVS (

n

= 128) protocol. Ovarian dynamics were monitored on days 0 (baseline), 9 (before the second GnRH administration), and 27 post-inseminations (post-AI) during the protocol. Reproductive parameters, including estrus induction rate (EIR), first conception rate (FCR), and pregnancy loss (PL), were also recorded. Additionally, the association between ovarian activity and FCR was analyzed.

Baseline ovarian activity (day 0) was similar between groups. By day 9, heifers subjected to nano-OVS exhibited improved follicular development, with larger preovulatory follicles (18.9 ± 1.4 mm) than those subjected to the standard OVS group (16.8 ± 1.3 mm;

P

= 0.007). On day 27 post-AI, larger corpora lutea were observed in the nano-OVS group compared with the standard OVS group (21.3 ± 1.3 mm vs. 18.3 ± 1.4 mm, respectively;

P

= 0.04). These findings were associated with higher EIR (78.6%,

P

= 0.03) and FCR (47.7%,

P

= 0.32), and lower PL (13.1%,

P

= 0.01) compared to the standard protocol (65.9%, 47.7%, and 21.2%, respectively). Correlation analysis exhibited different physiological patterns between groups.

Nanofabrication of GnRH may enhance hormone stability and enable sustained release, thereby improving ovulation synchronization and luteal development.

Overall, the nano-GnRH-BA formulation within the OVS protocol resulted in a 50% reduction without compromising fertility, while also improving ovarian function and reducing PL in dairy heifers. This approach may represent a promising alternative to conventional synchronization strategies.