Ionophore application for artificial oocyte activation and its potential effect on morphokinetics: a sibling oocyte study

Purpose To evaluate whether ionophore application at the oocyte stage changes the morphokinetics of the associated embryos in cases of artificial oocyte activation. Methods In a prospective sibling oocyte approach, 78 ICSI patients with suspected fertilization problems had half of their MII-oocytes treated with a ready-to-use ionophore (calcimycin) immediately following ICSI (study group). Untreated ICSI eggs served as the control group. Primary analyses focused on morphokinetic behavior and the presence of irregular cleavages. The rates of fertilization, utilization, pregnancy, and live birth rate were also evaluated. Results Ionophore-treated oocytes showed a significantly earlier formation of pronuclei (t2PNa) and a better synchronized third cell cycle (s3) (P < .05). The rate of irregular cleavage was unaffected (P > .05). Ionophore treatment significantly improved the overall rates of fertilization (P < .01) and blastocyst utilization (P < .05). Conclusion Ionophore application does not negatively affect cleavage timing nor is it associated with irregular cleavage.

Methodological approaches for vitrification of bovine oocytes

Summary Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

Reproductive biology of Hybopsis amblops, the Bigeye Chub, in the Flint River of Alabama

Background. The purpose of this study was to establish a reproductive schedule and examine reproductive traits that shape fecundity of the Bigeye Chub, Hybopsis amblops Cyprinidae, in the Flint River system of north Alabama. Methods. Life history traits associated with reproduction, growth, and maturation were assessed. Fish collections were made monthly from August, 2013, through July, 2014. Results. The Bigeye Chub in Alabama primarily spawns in April and May as indicated by gonadosomatic index (GSI), ovarian condition and clutch size. Average GSI values began to rise in February, peaked in April and May at over 13% for females and 1.6% for males, and showed a steep decline from May to June for both sexes. Average clutch size was highest in April at 812. Diameter of the most mature oocyte stage averaged 0.74 mm, relatively small compared to other cyprinids found in the Flint River. Discussion. The Bigeye Chub’s relatively large clutch size as a measure of fecundity places the species intermediate between opportunistic and periodic in the trilateral life history scheme of Winemiller and Rose. The species is apparently responding to a flow regime with a defined seasonality as well as predictability of flow and resources.

Reproductive biology of Hybopsis amblops, the Bigeye Chub, in the Flint River of Alabama

Background. The purpose of this study was to establish a reproductive schedule and examine reproductive traits that shape fecundity of the Bigeye Chub, Hybopsis amblops Cyprinidae, in the Flint River system of north Alabama. Methods. Life history traits associated with reproduction, growth, and maturation were assessed. Fish collections were made monthly from August, 2013, through July, 2014. Results. The Bigeye Chub in Alabama primarily spawns in April and May as indicated by gonadosomatic index (GSI), ovarian condition and clutch size. Average GSI values began to rise in February, peaked in April and May at over 13% for females and 1.6% for males, and showed a steep decline from May to June for both sexes. Average clutch size was highest in April at 812. Diameter of the most mature oocyte stage averaged 0.74 mm, relatively small compared to other cyprinids found in the Flint River. Discussion. The Bigeye Chub’s relatively large clutch size as a measure of fecundity places the species intermediate between opportunistic and periodic in the trilateral life history scheme of Winemiller and Rose. The species is apparently responding to a flow regime with a defined seasonality as well as predictability of flow and resources.

Reproductive biology of Hybopsis amblops, the Bigeye Chub, in the Flint River of Alabama

Background. The purpose of this study was to establish a reproductive schedule and examine reproductive traits that shape fecundity of the Bigeye Chub, Hybopsis amblops Cyprinidae, in the Flint River system of north Alabama. Methods. Life history traits associated with reproduction, growth, and maturation were assessed. Fish collections were made monthly from August, 2013, through July, 2014. Results. The Bigeye Chub in Alabama primarily spawns in April and May as indicated by gonadosomatic index (GSI), ovarian condition and clutch size. Average GSI values began to rise in February, peaked in April and May at over 13% for females and 1.6% for males, and showed a steep decline from May to June for both sexes. Average clutch size was highest in April at 812. Diameter of the most mature oocyte stage averaged 0.74 mm, relatively small compared to other cyprinids found in the Flint River. Discussion. The Bigeye Chub’s relatively large clutch size as a measure of fecundity places the species intermediate between opportunistic and periodic in the trilateral life history scheme of Winemiller and Rose. The species is apparently responding to a flow regime with a defined seasonality as well as predictability of flow and resources.

Surubim-do-Paraíba oocytes viability after being exposed to different cryoprotectants

ABSTRACT: To know the non-toxic cryoprotectants to fish oocytes is of extreme importance for tests that aim to increase oocyte resistance to cold, thus allowing more advanced studies in cryopreservation. Therefore, commonly used cryoprotectants such as methanol, dimethyl sulfoxide, ethylene glycol, propylene glycol, sucrose and fructose were studied. Immature oocytes from the initial to vitelogenic (diameter <1.7 mm) and mature (diameter >1.8 mm) stages of Steindachneridion parahybae were evaluated. Four distinct experiments were performed, three using immature oocytes and one using oocytes at the mature stage. For each oocyte stage, the best maintenance solution to be used: Hank or 50% L15 and; viability after baths for 30min (room temperature) at cryoprotectant concentrations ranging from 0.25 to 4M were evaluated. Different tests were used to evaluate oocyte viability: in vitro maturation followed by observation of germinal vesicle breakdown (only for immature oocytes), Trypan Blue staining (all stages) and fertilization and hatching rates (mature stage only). Results showed that the toxic effect of cryoprotectants on oocytes generally increases with increasing concentrations. Sensitivity of oocytes to cryoprotectants increases according to the stage of development, with mature oocytes being more sensitive. Sucrose, fructose, methanol, propylene glycol and dimethyl sulfoxide can be used as cryoprotectants for S. parahybae oocytes.

Oocyte stage-specific effects of MTOR determine granulosa cell fate and oocyte quality in mice

MTOR (mechanistic target of rapamycin) is a widely recognized integrator of signals and pathways key for cellular metabolism, proliferation, and differentiation. Here we show that conditional knockout (cKO) of Mtor in either primordial or growing oocytes caused infertility but differentially affected oocyte quality, granulosa cell fate, and follicular development. cKO of Mtor in nongrowing primordial oocytes caused defective follicular development leading to progressive degeneration of oocytes and loss of granulosa cell identity coincident with the acquisition of immature Sertoli cell-like characteristics. Although Mtor was deleted at the primordial oocyte stage, DNA damage accumulated in oocytes during their later growth, and there was a marked alteration of the transcriptome in the few oocytes that achieved the fully grown stage. Although oocyte quality and fertility were also compromised when Mtor was deleted after oocytes had begun to grow, these occurred without overtly affecting folliculogenesis or the oocyte transcriptome. Nevertheless, there was a significant change in a cohort of proteins in mature oocytes. In particular, down-regulation of PRC1 (protein regulator of cytokinesis 1) impaired completion of the first meiotic division. Therefore, MTOR-dependent pathways in primordial or growing oocytes differentially affected downstream processes including follicular development, sex-specific identity of early granulosa cells, maintenance of oocyte genome integrity, oocyte gene expression, meiosis, and preimplantation developmental competence.

Reproductive biology of Hybopsis amblops, the Bigeye Chub, in the Flint River of Alabama

Background. The purpose of this study was to establish a reproductive schedule and examine reproductive traits that shape fecundity of the Bigeye Chub, Hybopsis amblops Cyprinidae, in the Flint River system of north Alabama. Methods. Life history traits associated with reproduction, growth, and maturation were assessed. Fish collections were made monthly from August, 2013, through July, 2014. Results. The Bigeye Chub in Alabama primarily spawns in April and May as indicated by gonadosomatic index (GSI), ovarian condition and clutch size. Average GSI values began to rise in February, peaked in April and May at over 13% for females and 1.6% for males, and showed a steep decline from May to June for both sexes. Average clutch size was highest in April at 812. Diameter of the most mature oocyte stage averaged 0.74 mm, relatively small compared to other cyprinids found in the Flint River. Discussion. The Bigeye Chub’s relatively large clutch size as a measure of fecundity places the species intermediate between opportunistic and periodic in the trilateral life history scheme of Winemiller and Rose. The species is apparently responding to a flow regime with a defined seasonality as well as predictability of flow and resources.

MicroRNA expression in bovine preimplantation embryos

We profiled 98 mature microRNAs (miRNAs) using a stem-loop reverse transcription polymerase chain reaction assay array based on human miRNAs. We demonstrated that one, but not two, base-pair changes in the miRNA recognition sequence at the 3′ end only marginally affected copy number estimates. Absolute levels of miRNAs were measured in matured cattle oocytes, eight-cell embryos and normal and parthenogenetic blastocysts and Day-14 trophoblast. Most miRNA concentrations were below the expected functional threshold required for effective repression of moderately to highly abundant target RNA. In oocytes and peri-embryonic genome activation embryos, miRNA 320, a member of the Dgcr8/Drosha-independent class of miRNAs, was expressed at greater than 1000 copies per embryo. miRNAs were more abundant at the eight-cell than the oocyte stage. miRNA concentrations per cell increased from the eight-cell to the blastocyst stage. Both the number of miRNA species and their expression levels were reduced in trophoblast tissue at Day 14. The parthenogenetic samples were more related in their miRNA expression profiles to each other than to their wild-type (in vitro-produced cultured) counterparts. miRNAs 299 and 323, which have been shown to be maternally expressed in other species, were also more than 4-fold overexpressed in the cattle parthenogenetic samples.