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.

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.

The aim of this study was to determine the relationship between the oocyte yield by the ovum pick-up (OPU) technique with the Anti-mullerian Hormone (AMH) concentrations of the donors. Ten healthy Holstein heifers aged 12 to 15 months were included in the study. AMH measurements were performed with Bovine VIDAS® Anti-Mülleian Hormone kits (Biomeriux, Marcy l’Etoile, France) using the Mini Vidas device. A total of 67 OPU sessions were performed on a random day of the cycle. Oocytes were classified according to their quality, and viability evaluation of oocytes was made according to the cell layer number and cumulus integrity in the cumulus-oocyte complex (COC), the homogeneity of their cytoplasm. The average oocyte yield in OPU sessions per animal was range from 4–8. There was an significant negative correlation between the collected oocyte numbers and plasma AMH levels. In conclusion, it was observed that AMH concentration did not affect the number of viable and the quality oocytes collected in weekly OPU administration in animals. It was thought that OPU applications performed without knowing the day of the cycle did not provide the expected correlation with AMH data.

Background: This study aimed to determine the effects of environmental temperature on the number and quality of oocytes and embryo production rates obtained by performing ovum pick up (OPU). Heat stress leads to long-term, short-term, visible, and invisible effects in dairy cows. Its effects on reproduction are evident in all stages, from oocyte development to birth. Disturbance in ovarian follicle development, follicular dominance deficiency, anoestrus, polyspermia, embryoniclosses, decreased fetal growth, and abortion are some examples of responses to these effects. The aim of the present study was aimed to determine the effects of ambient temperature on oocyte quality and number and embryo production rates.Materials, Methods & Results: The animal material used in this study comprised 10 Holstein heifers. At the beginning of the study, the heifers were 13-15 months old. OPU was performed at different times of the year, and weather conditions were recorded. Grouping according to ambient temperature was done as < 10°C (group 1), 10-25°C (group 2), and > 25°C (group 3). The veterinary ultrasonography device and a set of compatible intravaginal OPU probe, catheter, and aspiration device were used for OPU application. All antral follicles with diameters of 2-8 mm in the ovaries were aspirated. The aspirated follicle fluids were examined under a stereo microscope, and the cumulus-oocyte complexes (COC) were collected and classified according to their structure. A, B, and C-quality oocytes were included in the in vitro embryo production process. After performing 69 OPUs on random days of the cycle, the number of oocytes per OPU was found to be 8.72, 6.32, and 6.85 in groups 1, 2, and 3, respectively (P < 0.05). The number of viable oocytes per OPU was 6.83, 4.64, and 4.65 in groups 1, 2, and 3, respectively (P < 0.05). The statistical difference between the first group and the other groups was significant for cleavage and blastocyst counts (P < 0.05).Discussion: All the negative effects of heat stress on animals resulted from the increased body temperature. Reproductive performance is adversely affected by high temperatures and humidity during periods of high ambient temperatures. Metabolic heat is released, and the heat load increases due to the metabolism of nutrients in cattle. Internal body temperature is regulated via the dissipation of metabolic heat to the environment. The amount of heat dissipated via conduction andconvection depends on the unit body weight, surface area, skin and coat color, difference in temperature gradient of the animal and ambient temperature, and humidity. In the present study, it was determined that the blastocyst development rates of the oocytes obtained in the warm season (>25°C [group 3]) were lower than those of the other groups. It was concluded that this may be because the oocytes developed under chronic heat stress in the animals, and several cycles were required to enhance oocyte quality and developmental potential. Additional studies are needed to investigate the response of oocytes obtained with OPU to heat stress during embryonic developmental stages and to determine the sensitivity and effects of embryonic tissue damage according to developmental stages. Based on the results of the present study, it was concludedthat performing OPU and in vitro embryo production (IVEP) when the ambient temperature is close to the thermoneutral limits may increase the blastocyst development rates. Keywords: blastocyst, heat stress, heifer, in vitro embryo production, oocyte quality, ovum pick-up.

The aim of this study is to determine the effects of different treatments used to synchronize follicular waves prior to superstimulation of responses and in vitro embryo production (IVEP). The study used 12 healthy Holstein heifers. In GnRH + P4, an intravaginal progesterone (P4) device was inserted into the vagina on a random day of the estrous cycle, and 10 µg of the gonadotropin-releasing hormone (GnRH) was administered intramuscularly at the same time. After these applications, 25 mg of PGF2α was administered intramuscularly on the third and fourth days of treatment. The intravaginal progesterone device was removed on day 5. At the same time, 125 µg of follicle-stimulating hormone (FSH) was administered intramuscularly as a single dose. Two days after the FSH injection, oocytes were retrieved using transvaginal ultrasound. In contrast to GnRH + P4, hCG + P4 was performed with 1500 IU hCG (human chorionic gonadotropin) injection instead of GnRH. In DFR + P4, dominant follicles were removed. In the control group, the oocytes were collected on a random day of the estrous cycle. The ovarian follicles were measured by ultrasound prior to oocyte collection. The number of medium-diameter follicles (3–8 mm) was found to be greater in hCG + P4 and DFR + P4 than in the control group (p<0.05). It was determined that the numbers of viable oocytes, Grade A oocytes, cleaved oocytes, and blastocysts obtained per ovum pickup (OPU) were greater in hCG + P4 and DFR + P4 than in the control group (p<0.05). In addition, the numbers of Grade A oocytes and blastocysts obtained per OPU were greater in the DFR + P4 group than in the GnRH + P4 group (p<0.05). As a result, it was thought that synchronization of follicular wave emergence with DFR and hCG in the short synchronization protocols used before superstimulation could increase the success rate of OPU/IVEP programs.

Prostaglandins may be one of the important factors affecting the development and quality of the embryo in in vitro production (IVP) and may have an inhibitory effect on the development of the embryo. The supplementation of non-steroidal anti-inflammatory drugs (NSAIDs) in the in vitro culture medium may facilitate embryo development. The objective of this study was to assess the effect of non-steroidal anti-inflammatory drugs on embryo development and quality during bovine in vitro embryo culture. The study involved the performance of a series of procedures, including in vitro embryo production processes, the supplementation of different NSAIDs in the in vitro culture, and the evaluation and staining of embryos. In the initial 3 d of the in vitro culture stage, three different NSAIDs were administered to the groups: flunixin meglumine (FM), meloxicam (Mel), and carprofen (Car). No treatment was applied to the control group at this stage. All groups were incubated in the same culture medium, with the solutions being changed starting from the fourth day of the in vitro culture. On the seventh day of culture, the embryos were evaluated according to their developmental stages and quality. Subsequently, the developing embryos were subjected to differential staining and the TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) method for the assessment of their developmental competence, quality, and apoptotic index. Following the supplementation of NSAIDs in the culture medium, the blastocyst formation rates were found to be higher in the FM, Car, and Mel groups compared to in the control group (P<0.05). Furthermore, the inner cell mass, trophectoderm, and total cell counts were observed to be higher in the FM, Car, and Mel groups in comparison to the control group. In addition, the number of apoptotic cells and the apoptotic index were found to be lower in the FM, Car, and Mel groups compared to the control group. Consequently, it was established that the supplementation of non-steroidal anti-inflammatory drugs in the culture medium during in vitro embryo production resulted in an enhanced rate of blastocyst development and an improved quality of blastocysts.