Buffalo sperm is sensitive to cryoinjuries, thus improving sperm cryoresistance is a critical approach for wide spreading the assisted reproductive technologies in buffalo. The intention of this work was to assess the effect of propolis-loaded in nanoliposomes (PRNL) supplementation of semen extender on semen quality, antioxidant status and some apoptotic genes of cryopreserved buffalo semen. PRNL were prepared using cholesterol (Chol) as well as soybean lecithin and their physicochemical properties were characterized. Egyptian buffalo bulls (4–6 years) were involved, and the semen samples were collected using the artificial vagina method. Buffalo semen was pooled (n = 25 ejaculates) and cryopreserved in tris extender containing PRNL at 0 (PRNL0), 2 (PRNL2), 4 (PRNL4) and 6 µg/mL (PRNL6), respectively. The PRNL had a size of 113.13 nm and a negative zeta potential (− 56.83 mV). Sperm progressive motility, viability, membrane integrity, abnormalities, chromatin damage, redox status, apoptosis status, and apoptotic genes were investigated after post-thawed buffalo semen. Using 2 or 4 µg/mL PRNL significantly increased sperm progressive motility, viability, and membrane integrity, while sperm abnormalities and the percentage of chromatin damages were the lowest in PRNL2 group. Moreover, the PRNL2 group exhibited the best results for all antioxidative activities (TAC, SOD, GPx and CAT) with significantly higher levels than the other groups (P < 0.05). The levels of ROS and MDA were significantly lower in the PRLN2 compared with other groups. The sperm caspase 3 enzyme activities showed the lowest values in PRNL2 groups followed by PRNL4 and PRNL6 groups with significant differences compared with the control. Adding 2 µg/mL PRNL to freezing media significantly reduced apoptotic genes such as Bax and Caspase 3 in sperm, while significantly increase in Bcl2 expression compared with the control (P < 0.001). The expression of Bcl2, Caspase 3 and Bax genes in sperm were not affected by the 6 µg/mL PRNL addition (P > 0.05). The electron micrography descriptions exemplified that the fortification of 2 or 4 µg/mL PRNL maintained the acrosomal and plasma membrane integrities as well as sustained the ultrastructure integrity of the cryopreserved buffalo spermatozoa when compared with control group, whereas the 6 µg/mL of PRNL demonstrated highest injury to the acrosome and plasma membranes. Results show supplementation of the buffalo freezing extender with 2 or 4 µg/mL of PRNL enhanced post-thawed sperm quality via boosting the antioxidant indices, diminishing the oxidative stress and apoptosis as well as maintained the ultrastructure integrity of frozen-thawed buffalo sperm.

This study aimed to compare the effect of using PMSG at a level of 2500 IU in term of one dose, two doses (1250 IU for each at 12 h interval) or 4 doses (650 IU for each at 12 h interval) on superovulatory response (SOVR), and yield and quality of embryos. A total of 21 Friesian cows (450-550 kg LBW, 3.5-5.5 y old and 1-3 parities) were used as embryo donors. Cows were fed and managed under the same conditions. All cows were i.m. injected with 3 ml PGF2α/cow (Estrumate) for estrous synchronization. Cows in the 1st protocol (P1) were i.m. injected with one dose of PMSG (2500 IU/cow) on day 10 of estrous cycle (control), while those in the 2nd and 3rd protocols were i.m. injected with two doses of PMSG (1250 IU/dose/cow) at 12 h interval on day 10 of estrous cycle (P2) and with four doses of PMSG (625 IU/dose/cow) at 12 h interval on day 10 and 11 of estrous cycle (P3), respectively. After 48 hours of the last PMSG dose in all protocols, all cows were i.m. injected with 2 ml PGF2α/cow. Both AI and GnRH (5 ml Receptal/animal) injections were done on day 14 in P1 and P2 and day 15 in P3. Embryos were un-surgically flushed 7 days post-AI. Number of total follicles (TFN), large follicles (LFN) and CLs (CLN), and diameter of follicles/cow on day of AI and flushing were determined. Ovulation rate (OR), embryo recovery rate (ERR) and quality of non-surgically recovered 7 days post-AI were determined. Results showed that on day of AI, FN, LFN and follicular diameter tended to be the highest in P3, moderate in P2 and the lowest in P1. On day of flushing, total and large follicles were the greatest and the widest in P2, followed by P1, and the least and the narrowest in P3. Ovulatory sites (CLN) was greater in P1 and P3 (2.00 and 2.07/ovary) than in P2 (1.57/ovary). The effect of protocol, ovarian side and their interaction on follicular number and diameter, and CLs number was not significant on AI and flushing days. The response to CLs formation on day of flushing on the right, left or both ovaries was 100% in each protocol. Showing CLs was higher on the right than on the left ovaries in P1 (85.7 vs. 71.4%), being the opposite (71.4 vs. 85.7%) in P2 and similar in P3 (85.7 on each side). OR was higher (P<0.05) in P1 than in P2 (69.9 vs. 52.3%), but did not differ in P3 (59.1%) from that in P1 and P2. Percentage of cows produced embryos was highest in P3 (71.4%), moderate in P1 (57.1%) and the lowest in P2 (14.2%) with average number of 1.71, 1.86 and 0.57 embryos/cow, respectively. ERR was higher in P1 and P3 (46.5 and 41.3%) than in P2 (18.1%). About 25% of cows in P1 produced 5 embryos, and 40% in P3 produced 2 embryos versus one cow in P2 (14.3%) produced 4 embryos. Distribution of embryos at morula stage was higher (50%) in P2 than in P1 (30.8%) and P3 (33.4%). Distribution of embryos at compact morula stage was the highest in P1 and P3 (53.8 and 41.7%) than in P2 (25%) versus 15.4 and 16.6% at blastocyst stage in P1 and P3. Distribution of transferable embryos was 95.3, 50.0 and 83.3% in P1, P2 and P3, respectively. Superovulation of Friesian cows with PMSG (2500 IU/cow) on day 10 of the estrous cycle, in term of 4 doses (625 IU) at 12-h interval, showed the best follicular response (number and diameter) on day of AI and the higher ovulatory response, in term of number of un-ovulated follicles and CLs as well as percentage of cows responded to produce embryos (71.4%) on day of flushing. The same PMSG as a single dose showed the highest response, in term of number of transferable embryos/cow (1.71/cow).

Objectives: 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. Methods: 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. Results: The ¼ OVS protocol achieved a significantly greater follicular response (p < 0.05) than other protocols. On day 4, following the first GnRH administration, the OVS group exhibited a higher number of subordinate follicles (p < 0.05) and a greater diameter of the dominant follicles (DFs), whereas the ¼ OVS group showed a greater subordinate follicle diameter (p < 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 (p < 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). Conclusions: 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).

Aim of this study was to determine the effect of double-Ovsynch (DO) and pre-synchronization (PS) protocols on ovarian follicular dynamics, progesterone (P4) concentration and pregnancy rate at first postpartum AI in Friesian cows. Total of 44 lactating cows on 39-45 day post-partum (12 primiparous and 32 multiparous cows) were used in this study. Cows were divided into two similar groups (22 cows in each). Cows in G1 were pre-synchronized by two PGF2α (Estrumate) injections at 14 d interval (on days 42 and 56±3 post-partum), then initiate the Ovsynch-timed AI protocol after 12 days. Cows in G2 were injected on day 0 with 1st dose of GnRH (Receptal) on day 51±3 post-partum, with PGF2α on day 7 and 2nd dose of GnRH after 72 h, then initiate the Ovsynch-timed AI protocol after 7 days. Cows in both groups were treated with the same Ovsynch protocol: 1st GnRH on days 68±3 post-partum, PGF2α after 7 days, 2nd GnRH 56 h after PGF2α, and timed AI after 16 h. Cows were subjected to ultrasonography device to examine follicular number (FN) and diameter of dominant follicles (DF) as well as counting the number of corpora lutea (NCLs). Blood samples were collected to determine P4 in blood serum. Results showed that FN or NCLs/ovary on day 0 or during pre-synchronization treatment was not affected significantly by treatment. FN/ovary at 2nd GnRH of Ovsynch-breeding protocol was greater in G1 than in G2 (1.6 vs. 2.0 foll./ovary). However, NCLs and DF diameter during Ovsynch-breeding in G1 and G2 were not affected significantly by treatment. Concentration of P4 in blood serum of cows on days 0, pre-synch, 1st GnRH and PGF2 injections was not affected significantly by treatment. On day of 2nd GnRH injection, P4 concentration was higher (P0.05) in pregnant than in non-pregnant cows during pre-synch protocol. Progesterone concentration on day 75 showed an opposite trend, being lower (P3 ng/ml) on day 0 and at 1st GnRH of Ovsynch breeding protocol and lower P4 concentration (<1 ng/ml) at 2nd GnRH. Thus, pre-synchronization with Presynch-Ovsynch increased cyclicity and fertility of Frisian cows at first service postpartum in Frisian cows. However, Double-Ovsynch gave higher ovulation rate than presynch-Ovsynch.