The effect of different FSH administration before ovum pick up on superstimulation response and oocyte yield.

This study aims to evaluate the effect of synchronization and different superstimulation protocols on oocyte yield before ovum-pick up (OPU) to provide a homogeneous follicle population. Animals in all study groups except the control group applied a synchronization protocol including modified ovsynch+progesterone and dominant follicle ablation (DFA, on the 6th day following synchronization). In group 1, oocytes were retrieved by ultrasonography only on the 4th day after DFA. In group 2, 250 µg pFSH (100 µg IM, 150 µg SC) was administered as a single dose on the 2nd day following DFA, and oocytes were retrieved on the 2nd day following this injection. In group 3, 250 µg pFSH was administered IM in four equal doses 12 h apart on the 1st and 2nd days following DFA, and oocytes were retrieved 2 days after the last FSH injection. In group 4, 250 µg pFSH (IM) dissolved in Montanide ISA 206 adjuvant was administered as a single dose on the 2nd day following DFA, and oocytes were retrieved 2 days after this treatment. In the control group (group 5), oocytes were retrieved from animals without any hormonal treatment on a random day of the oestrus cycle. In order to assess the follicle population in the ovary on the day of OPU, the number of follicles according to their diameter was determined by ultrasonography in all groups. The ratio of medium-sized follicles (3-8 mm) was higher in the synchronized groups (Groups 1, 2, 3 and 4) than in the control group (Group 5) (p < .05). It was determined that the total number of oocytes obtained after OPU and the number of oocytes of suitable quality (Grade A and B) in in vitro embryo production were higher in the superstimulated groups (Groups 2, 3 and 4) compared to the control group. It was found that the number of Grade-A quality oocytes was higher in the superstimulated groups (Groups 2, 3 and 4) than the other groups. As a result, it was found that the synchronization and superstimulation treatments prior to the OPU increased the ratio of medium-sized follicles and the total number of oocytes obtained. In addition to the synchronization protocol, it was determined that superstimulation treatments increased the oocyte quality obtained with OPU. Furthermore, it was observed that a single dose of FSH dissolved in Montanide ISA 206 adjuvant produced a superstimulation response similar to that produced by repeated doses of FSH.

Does using microfluidic sperm sorting chips in bovine IVEP affect blastocyst development?

This study aimed to determine the effect of microfluidic sperm sorting chip on embryo development and quality in the sperm treatment step in in vitro embryo production in cattle. Only A-quality oocytes obtained from the ovaries of Holstein cattle were included in the study. These oocytes were first placed in in vitro maturation medium, and matured oocytes were randomly divided into two groups at the 24th hour of maturation. Oocytes in the first group with the Microfluidic Sperm Sorting Chip (MFSC, n = 154) were put into a fertilization medium with spermatozoa prepared with microfluidic sperm sorting device. Oocytes in the second group (Con, n = 169) were fertilized with spermatozoa prepared by the routine sperm treatment step of the commercial company. The rate of cleavage (85.71% vs. 76.33%, respectively) and of reaching the blastocyst (44.15% vs. 32.54%, respectively) in the MFSC group were higher than the control group. In addition, it was determined that the numbers of ICM (45.8 ± 2.04 vs. 39.2 ± 1.85, respectively), TE (122.13 ± 2.19 vs. 115.0 ± 2.61, respectively), TC (167.93 ± 2.89 vs. 154.2 ± 2.62, respectively) increased in the MFSC group compared to the control group. A statistically significant difference was found in the number of cells with apoptosis per embryo (5.14 ± 0.77 vs. 11.91 ± 0.79) and apoptotic index rates (3.06 ± 0.47 vs. 7.72 ± 0.55%) of the MFSC and Con groups. As a result, we concluded that using microfluidic sperm sorting chips during sperm treatment in bovine IVEP increases the rate of reaching the blastocyst, embryo development, and quality and reduces the possibility of apoptosis in developing blastocysts. For this reason, it is also thought that the use of microfluidic sperm sorting devices during sperm treatment in bovine IVEP may be a new alternative in this field.

Putrescine supplementation improves the developmental competence of in vitro produced bovine embryos.

The aim of this study was to investigate the effect of putrescine, anti-apoptotic, antioxidant, and a cell proliferation stimulant, on embryo development and quality by supplementing it to in vitro culture medium. In this study, oocytes were obtained from the ovaries of Holstein cattle. Following maturation and fertilization, the presumptive zygotes were randomly assigned to two groups. The first group (Putrescine, n = 435) was supplemented with putrescine at a concentration of 0.5 mM to in vitro culture. The second group (n = 407) was maintained under standard culture conditions without any supplementations to the medium. Following the determination of the developmental stages of the embryos, only those in the blastocyst stage were subjected to differential staining and the cell numbers of the embryos were determined. Moreover, the TUNEL assay was employed to ascertain the extent of cell death and the apoptotic index in the embryos. Additionally, the levels of ROS were determined in the embryos. Furthermore, gene expression analyses were conducted on blastocyst-stage embryos to ascertain the potential of putrescine supplementation in embryo development along specific pathways. Following in vitro culture, the blastocyst formation rate was 44.37 % in the putrescine group and 32.97 % in the control group (P < 0.05). The counts of ICM (60.60 ± 15.79 vs 50.73 ± 16.74), TE (117.70 ± 23.67 vs 94.0 ± 22.46), and TCC (178.30 ± 26.15 vs 144.73 ± 26.86) were found to be statistically higher in blastocysts developing after putrescine supplementation compared to the control group. Furthermore, the number of apoptotic cells (7.69 ± 2.17 vs 9.96 ± 3.99) and the apoptotic index (5.07 % vs 8.01 %) were found to be lower in the putrescine group in comparison to the control group. Nevertheless, it was established that the ROS level in the control group was approximately two-fold higher than in the putrescine group (P < 0.05). The findings also revealed that putrescine up-regulated the gene expression of SOD, GPX4, CAT, BCL2, NANOG and GATA3 while simultaneously down-regulating the BAX expression level. In conclusion, the supplementation of putrescine to the culture medium during in vitro bovine embryo production was found to contribute to the improvement of embryo quality and early embryonic development.