Somatic Embryo Yield and Quality From Norway Spruce Embryogenic Tissue Proliferated in Suspension Culture

Somatic embryogenesis is being piloted for the commercial production of genetically improved Norway spruce (Picea abies L. Karst) forest regeneration material in Finland. The main challenge to making the process commercially relevant is the dependence on time-consuming and highly skilled manual labor. Automation and scaling up are needed to improve cost-effectiveness. Moving from the proliferation of embryogenic tissue on semisolid media to suspension cultures could improve process scalability. In a series of four experiments (overall, with 20 cell lines, 4–9 per experiment), the suitability of proliferation in suspension culture for Norway spruce somatic embryogenesis was evaluated based on the growth rate, indicators of stress conditions, good-quality cotyledonary embryo yield, and embling survival in a greenhouse. The proliferation rate in suspension was found equal to on semisolid media, but with a remarkable genotypic variation. Embryogenic tissue matured directly without pre-treatments from suspension onto semisolid media produced lower numbers of good-quality embryos than tissue matured from semisolid media. Rinsing the suspension-grown tissue with hormone-free liquid media before maturation improved embryo yield, bringing it closer to that of semisolid-grown tissue. Decreasing 6-benzylaminopurine and 2,4-dichlorophenoxyacetic acid concentrations in suspension proliferation media to 0.5 or 0.1 times those in semisolid media did not affect tissue growth and did not improve embryo production. The hydrogen peroxide (H2O2) content and guaiacol peroxidase activity were elevated in suspension cultures compared with semisolid medium, which had the same plant growth regulator content. In one experiment out of four, the greenhouse survival of germinants was lower when proliferation was carried out in full strength suspension than on semisolid media; in other experiments the survival rates were equal.

The Effects of Heat Shock Protein 70 Addition in the Culture Medium on the Development and Quality of In Vitro Produced Heat Shocked Bovine Embryos

The aims of the present study were to examine the effects of HSP70 addition in the in vitro culture medium of day 3 embryos on their developmental competence and quality. Bovine oocytes (n = 1442) were in vitro matured, inseminated and cultured for the first two days according to standardized methods. The presumptive zygotes were randomly allocated in three experimental groups: Control, C (embryos cultured at 39 °C throughout the culture period), group C41 (temperature was raised to 41 °C from the 48th to 72nd h post insemination (p.i.) and then it returned at 39 °C for the remaining culture period), and group H41 (the temperature modification was the same as in C41 and during heat exposure, HSP70 was added in the culture medium). Cleavage and embryo yield were assessed 48 h p.i. and on days 7, 8, 9, respectively and gene expression in day 7 blastocysts was assessed by RT-PCR. Blastocyst yield was the highest in group C39; and higher in group H41 compared to group C41. From the gene expression analyses, altered expression of 11 genes was detected among groups. The analysis of the orchestrated patterns of gene expression differed between groups. The results of this study confirm the devastating effects of heat stress on embryo development and provide evidence that HSP70 addition at the critical stages can partly counterbalance, without neutralizing, the negative effects of the heat insult on embryos, acting mainly through mechanisms related to energy deployment.

Immunization Against Inhibin Promotes Fertility in Cattle: A Meta-Analysis and Quality Assessment

Superovulation and embryo transfer techniques are important methods in cattle breeding. Combined with traditional superovulation protocols, immunization against inhibin can further improve follicular development and embryo yield. The aim of this study is to determine the efficacy of immunization against inhibin in improving the fertility of cattle through meta-analysis and to provide better clinical veterinary practice guidance. Three English databases (PubMed, EMBASE, Web of Science) were searched for research articles of immunizations against inhibin influence on cattle fertility. Literature screening, data extraction, and meta-analysis were conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. In addition, the Systematic Review Center for Laboratory animal Experimentation (SYRCLE) risk-of-bias (RoB) tool was used to assess the risk of bias of the included animal studies. Potentially relevant studies (317) were identified, and finally 14 eligible studies (all in English) were included. The results of meta-analysis revealed that immunization against inhibin has significant effects on improving the number of ovulations [mean difference (MD) = 0.44, 95% confidence interval (CI) = (0.31, 0.56)], embryos and unfertilized ova [MD = 4.51, 95% CI = (2.28, 6.74)], follicles of the three size categories, the incidence of multiple ovulations [OR = 22.50, 95% CI = (8.13, 62.27)], and the conception rate [OR = 2.36, 95% CI = (1.26, 4.40)]. Moreover, it improved the production of embryos [grades 1 embryos: MD = 3.84, (3.54, 4.15); grade 2 embryos: MD = −0.73, (−0.89, −0.57); grade 3 embryos: MD = −0.50, (−0.75, −0.25); degenerated embryos: MD = 1.16, (−0.51, 2.82); transferable embryos: MD = 2.67, (2.03, 3.31)] and the number of corpora lutea [MD = 1.25, 95% CI = (0.79, 1.71)]. In the above indicators, the differences between the two groups were statistically significant (all p < 0.0001). Additionally, according to the quality evaluation results, the risk of bias in the included studies is relatively high. The quality evaluation of the results of the included studies showed that the risk of bias mainly concentrated in the selective, performance, detection, and reporting of bias aspects.

P–281 Is it worth to go to blastocyst when there are less than 4 embryos on day 3?

Study question Are pregnancy rates similar with blastocyst transfer compared to D3 transfer for patient with a poor embryo yield. Summary answer In poor prognosis patients, more D3 embryos are needed to result in similar outcome compared to single blastocyst and it increases the multiple pregnancy risk. What is known already Good prognosis patients benefit from a blastocyst transfer rather than cleavage-stage embryo because day 3 morphology has limited predictive value for subsequent developmental. A Cochrane meta-analysis in 2016 found a higher live birth rate per transfer in the blastocyst group compared to cleavage-stage and no difference in miscarriage and multiple pregnancies. However, in unselected patients, studies have yielded conflicting results and especially in poor prognosis patients at risk of transfer cancellation. A threshold of four good embryos on the third day has been previously correlated with blastocyst yield and live birth rate compared with cleavage-stage embryo transfer. Study design, size, duration We analyzed the outcome of 1115 cycles with less than 4 embryos during 2019–2020 and compared the results between two groups of D3 and D5 transfers. Participants/materials, setting, methods Amongst 1115 study cycles, in 691 cycles a D3 transfer was performed and in 424 cycles a D5 transfer was performed. We compared transfer cancellation rates, mean number of transferred embryos and ongoing pregnancy rates between the two groups and also in subgroups with female age <37 and female age >37. The statistical analyses were done by Chi square and t-test for paired samples. Main results and the role of chance In the overall study population, the mean female age was 36.3 ± 4.3 years, the mean number of obtained embryos was 2.4 ± 1.0, the mean number of transferred embryos was 1.4 ± 0.8. 17.2% of the cycles resulted in transfer cancellation (6.2% in D3 transfer group and 35.0% in D5 transfer group). After D3 transfer the ongoing pregnancy rate (OPR) per transfer was 21.5% compared to 39.7% in D5 transfers (p < 0.05). A similar pattern was observed in subgroups of age <37 years and >37 years with OPR per transfer significantly higher when D5 transfer was performed. Notably more embryos were transferred on D3 compared to D5 (mean number 1.4 for D3 and 1 for D5). Nonetheless, OPR were similar per cycle in both groups and subgroups of different ages. Limitations, reasons for caution A prospective randomized controlled trial is needed to confirm these results that are consistent with previously reports on retrospective and observational studies. Wider implications of the findings: In poor prognosis patients with low embryo yield, D3 and D5 transfers result in similar OPR per cycle. Transferring at blastocyst stage is not inferior to D3, despite the high cancellation rate, and appears safer permitting a single embryo transfer to avoid multiple pregnancy. Trial registration number Not applicable

Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.