Colony stimulating factor 2 protects the preimplantation bovine embryo from heat shock.

Heat stress can have severe deleterious effects on embryo development and survival. The present study evaluated whether CSF2 can protect the developmental competence of the bovine embryo following exposure to a heat shock of 41°C at the zygote and morula stages. In the first experiment, putative zygotes and 2-cell embryos were assigned to receive either 10 ng/ml CSF2 or vehicle, and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Heat shock reduced blastocyst development for embryos treated with vehicle but not for embryos cultured with CSF2. In the second experiment, day 5 embryos (morula) were treated with CSF2 or vehicle and then cultured for 15 h at either 38.5°C or 41°C and then at 38.5°C until day 7.5. Temperature treatment did not affect development to the blastocyst stage and there was no effect of CSF2 treatment or the interaction. Results indicate that CSF2 can reduce the deleterious effects of heat shock at the zygote or two-cell stage when the embryo is transcriptionally inactive.

Effect of eCG dose on ovarian haemodynamics, hormonal profiles and prolificacy rate when oestrus was induced during low-breeding season in Beetal goats.

The objectives of the experiment were to determine the effect of two doses of equine chorionic gonadotropin (eCG) in a standard synchronization protocol based on a short-term progesterone (P4 ) priming on ovarian structures and haemodynamics, concentrations of steroid hormones and prolificacy rate when oestrus was induced during low-breeding season (LBS) in Beetal dairy goats. We hypothesized that inclusion of eCG in a short-term P4 priming-based synchronization protocol would increase the blood perfusion to ovarian structures leading to enhance oestrous and ovulatory responses and prolificacy rate in goats. Forty-two multiparous acyclic goats were blocked by body condition and, within block, assigned randomly to receive saline as control (CON), low eCG (L-eCG; 300 IU) or high eCG (H-eCG; 600 IU) dose. Initially, a controlled internal drug release (CIDR) device was placed in the anterior vagina on d -8, followed by removal of CIDR on d -3, concurrent with the administration of PGF2α and eCG according to their respective treatments. Goats were monitored for oestrous response. B-mode and Doppler ultrasonography was performed with 12-h interval, starting from day -3 until natural breeding (day 0), and then on days 5, 10, 15 and 20 post-breeding to monitor follicular and luteal dynamics and blood flow, respectively. Blood was sampled at 0, 12, 24, 36 and 60 h after CIDR removal to quantify plasma concentrations of estradiol-17ß (E2 ), whereas plasma concentrations of P4 were assayed at days 5, 10, 15 and 20 after breeding. Pregnancy and prolificacy rates were determined at day 30 and 150 after breeding, respectively. Data were analysed with mixed-effects models, and orthogonal contrasts were used to evaluate the effect of treatment [Con vs. (½ L-eCG + ½ H-eCG)] and dose of eCG (L-eCG vs. H-eCG). Data are presented in sequence as CON, L-eCG, H-eCG (LSM ± SEM). The oestrous intensity score (152.9 vs. 182.7 vs. 186.5 ± 15.1; p = .02) was greater in eCG-treated goats as compared to CON. Administration of eCG reduced the intervals to standing oestrus (66.2 vs. 41.8 vs. 48.9 h ± 5.5; p = .05), breeding (70.2 vs. 44.4 vs. 45.4 h ± 4.5; p = .03) and ovulation (84.5 vs. 61.2 vs. 63.4 h ± 6.2; p = .05) compared with CON goats. The mean growth rate of pre-ovulatory follicle was greater (1.11 vs. 1.49 vs. 1.45 mm ± 0.08; p = .01) in eCG-treated goats resulting in an increased diameter of pre-ovulatory follicle (6.27 vs. 7.20 vs. 7.31 mm ± 0.07; p < .01) and corpora lutea (6.75 vs. 8.26 vs. 8.07 mm ± 0.42; p = .04) than CON. The mean follicular blood flow did not differ among treatments; however, the mean luteal blood flow was greater in L-eCG-treated goats (0.81 vs. 1.61 vs. 1.07 cm2 ± 0.12; p = .001). The mean concentrations of E2 (4.03 vs. 5.21 vs. 4.78 pg/ml ± 0.42; p = .04) and P4 (4.85 vs. 6.39 vs. 6.22 ng/ml ± 0.34; p = .04) were greater in eCG-treated goats. The twinning rate did not differ between treatments; nevertheless, prolificacy rate was greater (p = .04) in L-eCG-treated goats. Collectively, our data suggest that the administration of eCG improves the induction of oestrous and ovarian dynamics. Administration of L-eCG enhances prolificacy rate, therefore, a low dose of eCG might be practically beneficial to improve reproduction during LBS in acyclic Beetal dairy goats.

Vitrification improves in-vitro embryonic survival in Bos taurus embryos without increasing pregnancy rate post embryo transfer when compared to slow-freezing: A systematic meta-analysis.

Objectives were to use meta-analytic approaches to compare slow-freezing (SF) and vitrification (VF) methods of cryopreservation on in-vitro (n = 12,211) and in-vivo (n = 3473) survival of Bos taurus embryos. The literature was systematically reviewed and data from 40 manuscripts including 78 experiments, and comprising 183 treatment means, were used for the analyses. The in-vitro parameters included rates of re-expansion, hatching, and survival of blastocysts either at 24 h or 72 h post-thawing/warming and total number (TN) of embryonic cells, whereas in-vivo parameters evaluated pregnancy rate between 35 and 60 d post embryo transfer (ET). Mixed models were fitted using MIXED and GLIMMIX procedures of SAS. Additionally, classical meta-analytical statistics were also fitted using METAN and METAREG procedures of STATA. The final models included the fixed effects of methods of cryopreservation and random effects of the experiment. Rates (LSM ± SEM) of re-expansion (0.36 ± 0.07 vs. 0.48 ± 0.08), hatching (0.25 ± 0.05 vs. 0.42 ± 0.07), and survival (0.57 ± 0.09 vs. 0.76 ± 0.07) at 72 h post-thawing/warming were lower (P < 0.05) in SF than VF, respectively. The TN of embryonic cells (96.89 ± 7.15 vs. 117.83 ± 7.15) remained lower (P < 0.05) in SF than VF, however, the relative risk (RR) of pregnancy rate post ET remained similar (RR = 1.0, CI = 0.8-1.2; P > 0.05) between both methods. Collectively, VF technique has a short-term protective effect against cryodamage of preimplantation embryos, however, it might be dysregulating genes involved in pregnancy success post ET in cows.

An improved method for specific-target preamplification PCR analysis of single blastocysts useful for embryo sexing and high-throughput gene expression analysis.

Gene expression analysis in preimplantation embryos has been used for answering fundamental questions related to development, prediction of pregnancy outcome, and other topics. Limited amounts of mRNA in preimplantation embryos hinders progress in studying the preimplantation embryo. Here, a method was developed involving direct synthesis and specific-target preamplification (STA) of cDNA for gene expression analysis in single blastocysts. Effective cell lysis and genomic DNA removal steps were incorporated into the method. In addition, conditions for real-time PCR of cDNA generated from these processes were improved. By using this system, reliable embryo sexing results based on expression of sex-chromosome linked genes was demonstrated. Calibration curve analysis of PCR results using the Fluidigm Biomark microfluidic platform (Fluidigm, South San Francisco, CA) was performed to evaluate 96 STA cDNA from single blastocysts. In total, 93.75% of the genes were validated. Robust amplification was detected even when STA cDNA from a single blastocyst was diluted 1,024-fold. Further analysis showed that within-assay variation increased when cycle threshold values exceeded 18. Overall, STA quantitative real-time PCR analysis was shown to be useful for analysis of gene expression of multiple specific targets in single blastocysts.

Effect of pterostilbene on development, equatorial lipid accumulation and reactive oxygen species production of in vitro-produced bovine embryos.

The pterostilbene (PT) molecule is a phytoalexin with a reducing effect on reactive oxygen species (ROS) and with a capacity to block lipogenesis. However, the potential reducing effects of PT on equatorial lipid accumulation and ROS have not yet been elucidated for in vitro-derived bovine embryos. The present study evaluated the effects of concentrations of 3, 1, 0.33, 0.11 µM PT, and a vehicle group on the percentage of cleaved embryos, embryos with more than 6 cells, percentage of blastocyst on Day 7 and 8, percentage of transferable embryos on Day 7, the cell count and relative concentration of lipids. In the second experiment, the effects of 0.33 µM PT and a vehicle group within two different O2 environments (5% and 20%) were evaluated for ROS generation and the percentage of Day 8 blastocysts. In the first experiment, no significant differences were found between the treatments with PT and the vehicle group (p > .05) concerning the percentage of cleaved embryos and embryos with more than 6 cells. Lipid reduction was observed in the groups treated with PT versus the vehicle group (p < .05). The vehicle group showed a higher rate of blastocyst production on Days 7 and 8 (p < .05) and an increase in the percentage of transferable embryos on Day 7 compared to the PT treatment groups (p < .05). Cell counts were not significantly different between treatments with PT and the vehicle group (p > .05). In the second experiment, the O2 concentration did not significantly affect ROS generation (p > .05); however, the groups treated with PT (0.33 µM) had a reduction in ROS (p < .05). The O2 concentration also did not significantly affect the rate of blastocyst production on Day 8 (p = .7696). Future research should be conducted to ascertain whether the reduction of lipids could enhance the cryopreservation and post-thaw viability of PT-treated embryos.

Transcriptional metabolic reprogramming implements meiotic fate decision in mouse testicular germ cells.

Nutrient starvation drives yeast meiosis, whereas retinoic acid (RA) is required for mammalian meiosis through its germline target Stra8. Here, by using single-cell transcriptomic analysis of wild-type and Stra8-deficient juvenile mouse germ cells, our data show that the expression of nutrient transporter genes, including Slc7a5, Slc38a2, and Slc2a1, is downregulated in germ cells during meiotic initiation, and this process requires Stra8, which binds to these genes and induces their H3K27 deacetylation. Consequently, Stra8-deficient germ cells sustain glutamine and glucose uptake in response to RA and exhibit hyperactive mTORC1/protein kinase A (PKA) activities. Importantly, expression of Slc38a2, a glutamine importer, is negatively correlated with meiotic genes in the GTEx dataset, and Slc38a2 knockdown downregulates mTORC1/PKA activities and induces meiotic gene expression. Thus, our study indicates that RA via Stra8, a chordate morphogen pathway, induces meiosis partially by generating a conserved nutrient restriction signal in mammalian germ cells by downregulating their nutrient transporter expression.

Disruption of CSF2RA in the bovine preimplantation embryo reduces development and affects embryonic gene expression in utero.

The hypothesis that CSF2 plays a role in the preimplantation development of the bovine embryo was tested by evaluating consequences of inactivation of CSF2RA (the functional receptor in the embryo) for development of embryos in utero. CRISPR/Cas9 was used to alter sequences on exon 5 and intron 5 of CSF2RA, Control embryos were injected with Cas9 mRNA only. Embryos > 16 cells at day 5 after insemination were transferred to synchronized recipient females in groups of 7 to 24. Embryos were flushed from the uterus two days later. The proportion of recovered embryos that developed to the blastocyst stage was lower for knockout embryos (39%) than for control embryos (63%). RNA sequencing of individual morulae and blastocysts indicated a total of 27 (morula) or 15 (blastocyst) differentially-expressed genes (false discovery rate <0.05). Gene set enrichment analysis indicated that the knockout affected genes playing roles in several functions including cell signaling and glycosylation. It was concluded that signaling through CSF2RA is not obligatory for development of the bovine preimplantation embryo to the blastocyst stage but that CSF2 signaling does enhance the likelihood that the embryo can become a blastocyst and result in specific changes in gene expression.

Effects of the SLICK1 mutation in PRLR on regulation of core body temperature and global gene expression in liver in cattle.

The SLICK1 mutation in bovine PRLR (c.1382del; rs517047387) is a deletion mutation resulting in a protein with a truncated intracellular domain. Cattle carrying at least one allele have a phenotype characterized by a short hair coat (slick phenotype) and increased resistance to heat stress. Given the pleiotropic nature of prolactin, the mutation may affect other physiological characteristics. The liver is one organ that could potentially be affected because of the expression of PRLR. The mutation is a dominant allele, and heterozygous animals have a similar hair coat to that of animals homozygous for the mutation. Present objectives were to determine whether inheritance of the SLICK1 mutation affects liver gene expression and if animals homozygous for the SLICK1 allele differ from heterozygotes in liver gene expression and regulation of body temperature during heat stress. In one experiment, rectal and ruminal temperatures were less for Holstein heifers that were heterozygous for the SLICK1 allele compared with wildtype heifers. There were 71 differentially expressed genes in liver, with 13 upregulated and 58 downregulated in SLICK1 heterozygotes. Among the ontologies characteristic of differentially expressed genes were those related to immune function and fatty acid and amino acid metabolism. In a prospective cohort study conducted with adult Senepol cattle, body temperature and hepatic gene expression were compared between animals heterozygous or homozygous for the SLICK1 mutation. There were no differences in ruminal temperatures between genotypes, rectal temperature was higher in animals homozygous for the SLICK1 mutation, and there was only one gene in liver that was differentially expressed. It was concluded that inheritance of the SLICK1 allele can exert functional changes beyond those related to hair growth although changes in liver gene expression were not extensive. Results are also consistent with the SLICK1 allele being dominant because there were few differences in phenotype between animals inheriting one or two copies of the allele.

Regulation of NANOG and SOX2 expression by activin A and a canonical WNT agonist in bovine embryonic stem cells and blastocysts.

Bovine embryonic stem cells (ESC) have features associated with the primed pluripotent state including low expression of one of the core pluripotency transcription factors, NANOG. It has been reported that NANOG expression can be upregulated in porcine ESC by treatment with activin A and the WNT agonist CHIR99021. Accordingly, it was tested whether expression of NANOG and another pluripotency factor SOX2 could be stimulated by activin A and the WNT agonist CHIR99021. Immunoreactive NANOG and SOX2 were analyzed for bovine ESC lines derived under conditions in which activin A and CHIR99021 were added singly or in combination. Activin A enhanced NANOG expression but also reduced SOX2 expression. CHIR99021 depressed expression of both NANOG and SOX2. In a second experiment, activin A enhanced blastocyst development while CHIR99021 treatment impaired blastocyst formation and reduced number of blastomeres. Activin A treatment decreased blastomeres in the blastocyst that were positive for either NANOG or SOX2 but increased those that were CDX2+ and that were GATA6+ outside the inner cell mass. CHIR99021 reduced SOX2+ and NANOG+ blastomeres without affecting the number or percent of blastomeres that were CDX2+ and GATA6+. Results indicate activation of activin A signaling stimulates NANOG expression during self-renewal of bovine ESC but suppresses cells expressing pluripotency markers in the blastocyst and increases cells expressing CDX2. Actions of activin A to promote blastocyst development may involve its role in promoting trophectoderm formation. Furthermore, results demonstrate the negative role of canonical WNT signaling in cattle for pluripotency marker expression in ESC and in formation of the inner cell mass and epiblast during embryonic development. This article has an associated First Person interview with the first author of the paper.

Determinants of survival of the bovine blastocyst to cryopreservation stress: treatment with colony stimulating factor 2 during the morula-to-blastocyst transition and embryo sex.

BACKGROUND: Colony-stimulating factor 2 (CSF2) is an important maternal regulator of embryonic development. Earlier research indicates that CSF2 can regulate genes involved in cellular stress responses and block apoptosis. Here, we tested whether addition of 10 ng/mL CSF2 at day 5 of development would increase the survival of blastocysts harvested at day 7 and subjected to vitrification. Additional objectives were to determine whether embryo sex affected survival or whether effects of CSF2 interacted with sex. RESULTS: Survival after vitrification was measured as the percent of warmed blastocysts that re-established a blastocoele after culture and that underwent hatching from the zona pellucida. In the first experiment, blastocysts were vitrified, warmed, cultured for 24 h, and DNA embryo sexing performed by PCR. There was no effect of CSF2, sex, or the interaction on the percent of blastocysts that re-expanded or that were hatching or hatched. In the second experiment, vitrified blastocysts were warmed and cultured for 24, 48, and 72 h. Treatment with CSF2 increased (P = 0.021) the percent of blastocysts that re-expanded as compared to the vehicle group (overall, 77.8 ± 4.7% vs 73.3 ± 4.7%). Percent re-expansion was highest at 24 h and declined slightly thereafter (P = 0.024). Although the interaction was not significant, the effect of CSF2 was greater at 48 and 72 h than at 24 h because CSF2 reduced the incidence of embryos collapsing after re-expansion. Furthermore, the proportion of re-expanded blastocysts at 24 h that experienced blastocoel collapse by 72 h was lower (P = 0.053) for CSF2 (3.6%; 7/195) than for vehicle (8.2%; 16/195). The percent of warmed blastocysts that were hatching or hatched increased with time (P < 0.0001) but there was no effect of CSF2 or the interaction with time on hatching. CONCLUSION: Treatment with CSF2 from day 5 to 7 of development did not cause a significant effect on the percent of blastocysts that re-established the blastocoele after 24 h of culture but CSF2 reduced the collapse of the blastocoele that occurred for a portion of the embryos that had experienced re-expansion at 24 h. Thus, CSF2 can provide protection to a proportion of blastocysts from cryodamage caused by vitrification. Further work is needed to evaluate whether CSF2 increases survival of vitrified blastocysts after embryo transfer.

Meta-analysis to determine effects of treatment with FSH when there is progestin-priming on in-vitro embryo production using ovum pick-up in Bos taurus cows.

Treatments with follicle stimulating hormone (FSH) to enhance ovarian follicular development before ovum pick-up (OPU) are important for improving in-vitro embryo production (IVEP) procedures in cows, however, their procedural efficacy needs to be evaluated. The objective of the present study was to use meta-analytic methods to determine the effects of FSH treatments prior to OPU when there is progestin-priming on ovarian functions and IVEP in parous Bos taurus cows (n = 243). The literature was systematically reviewed and data from eight experiments, with 23 treatment means including 448 OPU sessions, were used for analyses. All eight experiments included a group of cows in which there was no FSH treatment (CON), and treatment with FSH before OPU to conduct IVEP. Mixed models were fitted using MIXED and GLIMMIX procedures of SAS. Additionally, classical meta-analytical statistics were also fitted using METAN and METAREG procedures of STATA. Models included fixed effects of treatment (i.e., CON and FSH) and random effect of the experiment, and data were weighted using the inverse of the SEM squared to account for precision of each experiment. Number of medium sized follicles, quality of oocytes, and transferable embryos were greater (P < 0.05) in FSH-treated as compared with cows of the CON group, without indications of publication bias or heterogeneity of data. Taken together, the results from the present study lead to the recommendation for treatments with FSH prior to OPU for IVEP when there was progestin-priming imposed to obtain a large number of transferable embryos for economic sustainability of commercial embryo transfer programs in parous non-lactating Bos taurus cows.