Evaluating the Impact of Minimized GnRH and PGF2α Analogues-Loaded Chitosan Nanoparticles on Ovarian Activity and Fertility of Heat-Stressed Dairy Cows

<b>Objectives</b>: This study aimed to evaluate the effectiveness of gonadotropin-releasing hormone-loaded chitosan-TPP nanoparticles (GnRH-CNPs) and prostaglandin F_2α-loaded chitosan-TPP nanoparticles (PGF_2α-CNPs) within the Ovsynch protocol for enhancing reproductive performance in heat-stressed dairy cows. <b>Methods</b>: Thirty-six cyclic purebred Friesian cows not detected in standing heat for more than 90 days postpartum were randomly allocated to three treatment groups. The control group (OVS, n = 12) followed the standard Ovsynch protocol with conventional doses. The ½ OVS group (n = 12) received 5 µg GnRH-CNPs on days 0 and 9, along with 250 µg PGF_2α-CNPs on day 7. While the ¼ OVS group (n = 12) was administered 2.5 µg GnRH-CNPs on days 0 and 9, with 125 µg PGF_2α-CNPs on day 7. Ovarian follicular dynamics and corpus luteum (CL) development were monitored on days 0, 4, 7, and 9 of the protocol. Serum progesterone (P₄) concentrations were measured throughout the synchronization period and on days 15 and 30 post-AI. Pregnancy was diagnosed on day 30 post-AI. <b>Results</b>: The ¼ OVS protocol achieved a significantly greater follicular response (<i>p</i> < 0.05) than other protocols. On day 4, following the first GnRH administration, the OVS group exhibited a higher number of subordinate follicles (<i>p</i> < 0.05) and a greater diameter of the dominant follicles (DFs), whereas the ¼ OVS group showed a greater subordinate follicle diameter (<i>p</i> < 0.05) and a higher number of DFs. On day 9, after PGF_2α administration, the ¼ OVS group maintained an elevated number of subordinate follicles, while larger subordinate follicle diameters were observed in the ½ OVS and OVS groups. No significant differences in DF numbers and diameters were observed among groups. P₄ concentrations remained similar across groups during treatments. Compared to control, a significantly higher value of P₄ concentration (<i>p</i> < 0.05) was recorded on day 15 post-AI in the ½ OVS group and on day 30 post-AI in the ¼ OVS group. These findings correlated with a higher pregnancy rate in the ¼ OVS group (65%) compared to the ½ OVS and OVS groups (40% in each). <b>Conclusions</b>: Nanofabrication reduced GnRH and PGF_2α dosage by 50% and 75% without impairing ovarian response and pregnancy rates. The ¼ OVS protocol notably enhanced the ovarian activity and fertility, highlighting the use of GnRH-CNPs and PGF_2α-CNPs as promising and practical approaches to enhance the fertility in dairy cattle under heat stress (HS).