Production, Evaluation, Cultivation and In vitro Fertilization of Cattle Oocytes: Recent Trends in Reproductive Biotechnologies in Animal Breeding : A Review

The article presents the results of assessing reproductive biotechnology for cattle breeding. The issues of obtaining genetic material from bulls-producers and oocytes from donor cows, for their further cultivation and obtaining embryos, in order to replicate highly productive offspring from valuable animals, are considered. Oocyte production was analyzed in three different ways: puncture, section, and aspiration. A total number of 156 oocyte-cumulus complexes (OCCs) were collected out of 40 ovaries by puncture – 50 pcs, section – 47 pcs, and aspiration – 59 pcs. The results showed that puncture and section gave significantly higher total OCCs per an ovary (4.16 and 4.0, respectively) than aspiration (3.68), but a higher number of normal (grade A and B) OCCs per an ovary was observed with aspiration (2.5) than with puncture (1.82) and section (2.00). During aspiration, oocyte-cumulus complexes were collected from the surface of follicles with a diameter of 3 to 8 mm using a needle. During puncture, all surfaces were pierced with a hypodermic needle; during section, incisions were made along the entire ovarian surface with a scalpel, that is, all sizes of superficial follicles were collected. OCCs were divided into 4 classes based on cumulus and nucleus cells: grade A – oocytes completely surrounded by cumulus cells; Grade B – oocytes partially surrounded by cumulus cells; Grade C – oocytes not surrounded by cumulus cells; and grade D – degeneration observed in both oocyte and cumulus cells. Grades A and B were considered normal, while grades C and D were considered to be damaged. The obtained oocytes were cultured and fertilized in vitro, which means that their fertilization with sperm occurred under artificially maintained optimal conditions outside the body. Normal fertilization results are zygote formation with male and female pronuclei (PN). As a result of the conducted fertilization of 251 mature oocytes, 142 fertilizations were successful, which amounted to 56.57%.

INTERACTION OF FOLIAR NUTRIENTS AND HERBICIDES INTO PRODUCTION

A field trial was carried out at the fields of Baghdad University - Jadriya for the first location during the summer season of 2018, while the second location was in Mishkab Rice Research Station, Al-Najaf al-ashraf governorate during the summer season of 2019. This study was aimed investigate rice (Oryza sativa cv. Anber) response to foliar application of nutrition with the use of different herbicides. The design was RCBD wihin using split plot arrangement four replicates. The nutrition treatments; (Humic acide, Seaweed, Nano fertilizer, Normal fertilization and Without fertilizer) were in main plots. Herbicides treatments (Ronstar, Oscar, Rainbow, Super flag, weed free and weedy) occupied the sub plots.The results indicated that Ronstar herbicide was superior in giving the lowest average weeds density value 6.55 and 9.60 plant m-2 respectively, and highest average of grain yield was 2.53 and 3.42 t.h-1, respectively. The normal fertilization treatment was superior gave the highest average grain yield value 3.07 and 4.13 t h-1. As for the effects of interaction, the Ronstar combination with normal fertilization gave highest grain yield value 3.55 and 4.59 t h-1, respectively. Concluded from this trial that the Ronstar herbicide is the best among the herbicides used with different fertilization treatments by achieving the highest averages with most of the studied charactristics.

Oocytes with smooth endoplasmic reticulum aggregates do not impact blastocyst euploidy rate

Objective: To investigate whether the euploidy rate of blastocysts derived from smooth endoplasmic reticulum (SERa) positive cycles and oocytes are impacted.Design: Retrospective cohort study.Setting: A tertiary hospital-based reproductive medicine center.Patient(s): A total of 601 preimplantation genetic testing (PGT) cycles with obtained oocytes in our center between April 2017 and May 2021 were included in the study. Intervention(s): Women＞35 years and PGT cycles with chromosomal structural rearrangements (PGT-SR) were excluded. Embryological and blastocyst ploidy outcomes were compared between SERa+ oocyte, sibling SERa- oocytes and oocytes in SERa- cycles.Main Outcome Measure(s): Embryological outcomes and blastocyst euploidy rate.Results: No significant difference was observed in the normal fertilization rate (82.1 % vs. 77.8 % vs. 83.1 %, respectively, P=0.061), blastocyst formation rate (71.0 % vs. 72.5 % vs. 68.4 %, respectively, P=0.393), good quality blastocyst formation rate (46.4 % vs. 48.3 % vs. 42.6 %, respectively, P=0.198) between the SERa+ oocyte group, sibling SERa- oocyte group and SERa- oocyte group. No significant difference was observed in the euploidy rate (50.0 % vs. 62.5 % vs. 63.3 %, respectively, P=0.324), mosaic rate (12.5 % vs. 9.7 % vs. 13.4 %, respectively, P=0.506) and aneuploidy rate (37.5 % vs. 27.8% vs. 23.2 %, respectively, P=0.137) between the three groups.Conclusion: Our results suggest that the euploidy rate of blastocysts derived from SERa+ cycles and oocytes are not impacted.

Relationship of sperm motility with clinical outcome of percutaneous epididymal sperm aspiration–intracytoplasmic sperm injection in infertile males with congenital domestic absence of vas deferens: a retrospective study

Summary Congenital domestic absence of vas deferens (CBAVD) is a common factor in male infertility, and percutaneous epididymal sperm aspiration (PESA) combined with intracytoplasmic sperm injection (ICSI) is a primary clinical treatment, but the effect of the sperm obtained on pregnancy outcome remains to be explored. This study aimed to investigate the relationship between sperm motility with clinical outcome of PESA–ICSI in infertile males with CBAVD. A cohort of 110 couples was enrolled. In total, 76 infertile males were included in the high motility group, while the remaining 34 males were placed in the low motility group. Clinical pregnancy, embryo implantation rate and live birth rate were included as the primary outcome. After all follow-ups, we found that the high motility group achieved higher normal fertilization rates, cleavage rates, transplantable embryo rates and high-quality embryo rates than those in low motility group (normal fertilization rate, 78.2 ± 11.7% vs. 70.5 ± 10.2%, P = 0.003; cleavage rate, 97.1 ± 2.9% vs. 92.3 ± 3.0%, P = 0.000; transplantable embryo rate, 66.8 ± 14.9% vs. 58.6 ± 12.6%, P = 0.009 and high-quality embryo rate, 49.9 ± 10.5% vs. 40.5 ± 11.2%, P = 0.000). Additionally, compared with the low motility group, the clinical pregnancy rates, embryo implantation rates, and live birth rates in the high motility group were significantly increased (pregnancy rate, 61.8% vs. 26.5%, P = 0.009; embryo implantation rate, 36.5% vs. 18.0%, P = 0.044; live birth rate, 55.3% vs. 17.6%, P = 0.000). We concluded that the motility of sperm obtained by PESA affected the clinical outcome of ICSI in infertile males with CBAVD.

P–155 Oocyte recovery 39 hours (from 39h to 41h) after administration of follicular maturation trigger does not affect clinical results

Study question What is the clinical outcome of oocytes recovered after 39 hours from ovulation inducing drug administration? Summary answer Oocytes obtained after 39 hours from follicular maturation triggering are equally viable to those obtained at the standard time of 36 hrs. What is known already In the clinical setting of ART, ovum pick-up (OPU) is generally performed around 36 hours after the administration of ovulation inducing drugs (OID). However, there are cases where OPU cannot be performed at this time often due to long operating lists. As the time elapsed between the administration of ovulation inducing drugs and OPU becomes longer, there is a concern about time-related oocyte aging. Nevertheless, there are few reports of clinical results of OPU after 36 hours from OID. Study design, size, duration We conducted a review of 1187 cycles and 1951 patients in which OPU and embryo transfer was performed in 2017–2018. All cycles underwent a ‘freeze-all’ of embryos and the transfer cycle was in the thawed embryo transfer cycle for all cases. Participants/materials, setting, methods The time from the administration of OID to the end of OPU was divided into 36h group and over 39h group and the MII and normal fertilization rate of oocytes obtained from OPU after ovarian stimulation were compared. After confirmation of fertilization, the D3 good-quality embryo and the D5 and 6 good-quality blastocyst rates of embryos that continued to be cultured and the pregnancy and miscarriage rates of cleavage-stage embryos and blastocyst transfers were compared. Main results and the role of chance The MII rate in the 36h and >39h groups was 78.1% vs. 80.0%, and the normal fertilization rate was 77.9% vs. 78.1% (ICSI) and 65.4% vs. 67.6% (Conventional-IVF). The D3 good-quality embryo rate (good-quality embryos are embryos with less than 5% fragmentation in 7–9 cells and compaction with more than 50% adhesion between split spheres) was 21.8% vs. 25.3%, the D5 good-quality blastocyst rate (at least 3BB according to Gardner classification) was 33.6% vs. 40.1%, and the D6 good-quality blastocyst rate was 31.1% vs. 37.5%, all of which were not significantly different. The pregnancy rate for cleavage-stage embryo transfer was 26.6% vs. 6.7%, and the miscarriage rate was 25.3% vs. 42.9%, both of which were not significantly different. The pregnancy rate for blastocyst transfer was 45.4% vs. 50.0%, and the miscarriage rate was 22.2% vs. 20.0%, both of which were not significantly different. (The significance difference test was a χ-square test) Limitations, reasons for caution The study was a retrospective study. Wider implications of the findings: Even if OPU is conducted after 36h of the administration of OID, to the extreme range of 39h–41h, oocyte aging does not seem apparent and pregnancy outcomes are similar to the standard time interval of 36 hours. Trial registration number ‘not applicable’

P–096 Real-time ranking of single spermatozoa using artificial vision analysis of complex motility patterns during ICSI aimed at improving fertilization and blastocyst development

Study question Can real-time artificial vision identify beneficial movement patterns of single spermatozoa in a cohort visualized in PVP during ICSI possibly enhancing fertilization and embryo development? Summary answer Artificial vision seems able to identify advantageous movement patterns of individual spermatozoa having a significant impact on both normal fertilization and blastocyst formation. What is known already Spermatozoa isolated from poor semen may reduce the quality of embryo development and blastocyst formation. Normal motility is dependent on general sperm morphology and characteristic movement of the flagellum enabling forward mobility. Spermatozoa roll as they swim. It is known that this rotational motion around their longitudinal axis promotes rheotaxis, which is a mechanism that allows the sperm to navigate to the site of fertilization. Therefore, it is possible that the characteristics of the rotational movement are related to sperm quality. Study design, size, duration Non-intervention study based on a cohort of 132 videos of in-vitro fertilization treatments with ICSI during which the sperm selection process was recorded up to sperm injection. The study was performed at one IVF center within a 6-month period. Injected spermatozoa and their corresponding oocytes were individually assessed from fertilization to blastocyst formation. Videos, where spermatozoa selected for injection could not be identified, were excluded. Relevant outcomes included normal fertilization (2PN), and blastocyst formation. Participants/materials, setting, methods Using a digitizer attached to an optical microscope (640 x 480 pixels), videos were recorded to include the sperm selection process, immobilization, and subsequent injection following standard ICSI protocols. Individual spermatozoa motility features were extracted using a proprietary computer-vision algorithm (SID, IVF 2.0 LTD). The rotational movements of spermatozoa were inferred by computing the variations of the mean intensity of the sperm in the video-sequence across time (MI). Main results and the role of chance Based on SID’s analysis, we found statistically significant differences between the median prominences of the MI of those injected spermatozoa that resulted in successful fertilization in comparison to those with failed fertilization (p-value=0.029, 28 negative fertilization, and 71 positive fertilization) using a one-tailed t-Student test with a significance level of 5%. We also found statistically significant differences between the median prominences of the MI of those spermatozoa that resulted in blastocysts in comparison with the spermatozoa-oocyte cohorts which didn’t reach the blastocyst stage (p-value 0.004, 51 with negative blastocyst formation and 48 with blastocyst formation). Limitations, reasons for caution The size of this database is modest, therefore a larger study with multiple clinics will be necessary to confirm the findings. Large prominence does not necessarily assurance successful fertilization or blastocyst formation since there may be other factors such as oocyte quality or the ICSI technique. Wider implications of the findings: Objective assessment of sperm rotational movement is difficult to quantify and to be objectively assessed during standard sperm selection. Real-time artificial vision tools such as SID could assist embryologists during the sperm selection process for ICSI. Trial registration number NA

The Influence of Male and Female Overweight/obesity on IVF Outcome: A Cohort Study Based on Registration in Western China

Background: Overweight/obese couples are more likely to present with a lower fertility, but it is still unclear whether the Overweight/obese can reduce the successful rate of in vitro fertilization-embryo transform (IVF-ET).This systematic review uses strict Chinese BMI criterias to evaluate whether male and female overweight/obesity will affect embryo quality and IVF outcomes, and whether there are gender differences.Methods: We reviewed the IVF-ET data of Northwest Women and Children's Hospital from 2015.01 to 2019.06, and divided the included population into four groups: Group 1 (n = 3413, male and female BMI: 18.5≤BMI＜24kg/m2, the control group);Group 2 (n = 1437, female BMI≥24 kg/m2 and male BMI18.5≤BMI＜24 kg/m2, the experimental group 1); Group 3 (n=4283, female BMI18.5≤BMI＜24 kg /m2 and male BMI≥24 kg/m2, the experimental group 2) and Group 4 (n=2176, female BMI≥24 kg/m2 and male BMI≥24 kg/m2, the experimental group 3). .Primary outcomes were clinical pregnancy rate,（CPR), live birth rate(LBR)，available embryo rate and normal fertilization rate.Results: The study found that the overweight/obesity of the independent female group or the combined male and female group manifested with a much less number of available embryos and a much less number of high-quality embryos (p <0.05). Meanwhile, the independent male group displayed a significant reduction of IVF-ET cleavage rate (p=0.019) and the independent female group displayed a significant reduction of the fertilization rate (p=0.029) and the normal fertilization rate (p=0.004). The combined male and female group manifested with a significant reduction of the available embryo rate (p=0.026), the fertilization rate (p=0.010) and the normal fertilization rate (p=0.003)，but overweight/obesity has no significant effect on CPR、LBRand abortion rates. Conclusion: overweight/obesity affects the embryo quality, the fertilization rate, and the cleavage rate, and there exists gender differences. Therefore, in order to improve the embryo quality, the fertilization rate and the cleavage rate of IVF of overweight/obese couples, it is still recommended that one partner or both couples should manage to reduce their weight to a normal value before IVF treatment.

Effects of in vitro maturation media on in vitro fertility of porcine oocytes and early development of embryos

In pigs, embryo productivity is still lower than that in other livestocks. One of the reasons is incomplete maturation of porcine oocytes in in vitro conditions. Therefore in vitro maturation (IVM) plays a crucial role in in vitro production of porcine embryos. It provides prerequisite condition to in fertilization and subsequent development of porcine embryos. In a previous study, effects of NCSU-37-based medium and TCM-199-based media supplemented with porcine follicular fluid (pFF) or Fetal Bovine Serum (FBS) on in vitro maturation of Landrace oocytes collected in Vietnam have been compared, suggesting that NCSU-37 medium supplemented with 10% of porcine follicular fluid (pFF) had the highest rate of oocytes reach to metaphase II stage in comparison to those of the other two TCM-199-based media. In the present study, further experiments were carried out to evaluate the contribution of IVM media on fertilization capability and developmental competence. Porcine oocytes matured in vitro in 3 media: NCSU-37 supplemented with 10% pFF, TCM-199 supplemented with either 10% pFF or 10% FBS were subjected to in vitro fertilization and subsequent in vitro culture to monitor fertility and embryo development. The results showed that penetration and normal fertilization rates in both TCM-199 groups are both higher than that of NCSU-37 group. Moreover, the cleavage and blastocyst rates, and cell numbers of blastocysts which is a criterion for embryo quality were all higher in TCM-199 groups, especially in the group supplemented with pFF. It might be concluded that TCM-199 media supplemented with either pFF or FBS are suitable for effective in vitro maturation of Landrace porcine oocytes collected in Vietnam.

‘Two countries-two labs’: the transnational gamete donation (TGD) programme to support egg donation

Purpose To evaluate the effectiveness, efficiency, and safety of a transnational gamete donation (TGD) programme based on the shipping of vitrified donor oocytes. Methods A retro-prospective observational study was conducted in the Assisted Reproductive Technology Center of the University Hospital of Florence, Italy. The study population included 622 consecutive donor oocyte cycles. A mean number of 6 vitrified oocytes per couple were shipped from two Spanish biobanks. In the receiving centre, gametes were warmed and inseminated and the subsequent embryo transfer (ET) was performed. The main outcome measurement was LBR. Secondary outcomes included oocyte survival rate, ICSI damage rate, normal fertilization, cleavage, and implantation rate (IR) in both ‘fresh’ and cryotransfer cycles. Results A total number of 3536 mature oocytes were warmed with 81.4% surviving oocytes. 2PN oocytes were 1941 with an ICSI normal fertilization rate of 70.4% and a cleavage rate of 93.4%; 857 day-3 embryos were transferred in 498 women, 63 blastocysts in 44. Couples with at least one vitrified embryo were 181 (32.3%). IR was 25.1% and 33.1% for day-3 ET and blastocyst stage respectively. Crude pregnancy rate and LBR after the first ET were 35.5% and 27% correspondingly with a conservative cumulative LBR of 34% and an optimal LBR of 51.4%. Conclusion Imported vitrified donor oocytes retain their competence and are capable of resulting in ongoing pregnancies and healthy babies in a proportion comparable to other existing systems as egg donation with vitrification/warming in the same laboratory and transnational fresh oocyte donation.