Fertilization, Cleavage and Implation

Most of the livestock in Indonesia are still conventional or traditional farms, where the quality of the seeds, the use oftechnology and the skills of farmers are still relatively low, thus affecting the productivity and genetic quality oflivestock. Therefore it is necessary to have knowledge about livestock reproduction that discusses fertilization,cleveage and implantation. Fertilization or fertilization (singami) is the fusion of two gametes which can be a nucleusor nucleus cells to form a single cell (zygote) or fusion of the nucleus. the process starts with the preparation of ovumcells and spermatozoa; penetration; core incorporation; and early zygote cleavage. Fertilization phase is the meetingbetween sperm cells and ovum cells and will produce zygote. Zygote will perform cell division (cleavage). The zygotethen undergoes growth and development through stages, namely division, gastrulation, and organogenesis.Implantation or also known as oxidation is the process of implanting the embryo, which is the result of conception,into the uterine wall (endometrium) to further develop.

Strategies to reduce genetic mosaicism following CRISPR-mediated genome edition in bovine embryos

Genetic mosaicism is the presence of more than two alleles on an individual and it is commonly observed following CRISPR microinjection of zygotes. This phenomenon appears when DNA replication precedes CRISPR-mediated genome edition and it is undesirable because it reduces greatly the odds for direct KO generation by randomly generated indels. In this study, we have developed alternative protocols to reduce mosaicism rates following CRISPR-mediated genome edition in bovine. In a preliminary study we observed by EdU incorporation that DNA replication has already occurred at the conventional microinjection time (20 hpi). Aiming to reduce mosaicism appearance, we have developed three alternative microinjection protocols: early zygote microinjection (10 hpi RNA) or oocyte microinjection before fertilization with either RNA or Ribonucleoprotein delivery (0 hpi RNA or 0 hpi RNP). All three alternative microinjection protocols resulted in similar blastocyst and genome edition rates compared to the conventional 20 hpi group, whereas mosaicism rates were significantly reduced in all early delivery groups (~10–30% of edited embryos being mosaic depending on the loci) compared to conventional 20 hpi microinjection (100% mosaicism rate). These strategies constitute an efficient way to reduce the number of indels, increasing the odds for direct KO generation.

47 EFFECT OF THE TYPE OF CYTOPLASTS, SOURCE OF KARYOPLASTS, AND ACTIVATION PROCESS ON IN VITRO DEVELOPMENT OF BOVINE CLONE EMBRYOS

As the recipient cytoplast plays a key role in nuclear reprogramming after somatic cell nuclear transfer (SCNT), the aim of this study was to compare the type of cytoplast/karyoplast [metaphase II (MII) oocyte, early zygote, somatic cells] and the chemical (CA) or sperm-mediated/spontaneous activation (SA) on in vitro development of bovine SCNT embryos produced by handmade cloning (HMC). After 17 h of in vitro maturation, a group of cumulus–oocyte complexes (COCs, n = 945) was manually bisected following zona removal and segregated as enucleated (MII hemi-Cyt) or non-enucleated (MII hemi-Kar). Another group of COCs was in vitro-fertilized, and, 4 h after the onset of IVF, zona-free zygotes with 2 polar bodies (n = 490) were manually bisected under fluorescent light to obtain IVF hemi-Cyt and IVF hemi-Kar. A somatic cell (SC) culture from an adult cow was used for HMC procedures (SC Kar). In 5 replications, experimental groups were composed of: zona-intact MII oocytes (parthenote control, PG); zona-intact zygotes (IVF control); MII Cyt + MII Cyt + SC Kar (SCNT control); IVF Cyt + MII Cyt + SC Kar (G1); MII Cyt + IVF Kar (G2); IVF Cyt + IVF Kar (G3); IVF Cyt + IVF Cyt + SC Kar (G4); and MII Cyt + MII Kar (G5). Following reconstruction and electrofusion, groups G1 to G5 were further divided into 2 sub-groups each, 1 being chemically activated (ionomycin/6-DMAP) along with the control groups PG and SCNT, whereas the others were cultured to verify sperm-mediated (G1 to G4) or spontaneous (G5) activation. Embryos were in vitro-cultured in the WOW system for 7 days. Cleavage (Day 2) and blastocyst (Day 7) rates were compared by the chi-square and Fisher tests, respectively. Cleavage rates in G1-SA, G2-SA, and G3-SA were lower than in their CA counterparts, which were similar to controls (Table 1). Such decrease in cleavage in G1-SA and G2-SA may be caused by the manipulation process rather than by sperm-mediation, since the observed rates were very similar to the G5-SA group. Cleavage in G3 and G4 were also similar to controls, most likely due to the fusion of 2 sperm-activated IVF hemi-Cyt. Blastocyst rates were generally higher in CA than in SA sub-groups except for G4, for which SA benefited from 2 sperm-activated cytoplasts. The lower blastocyst yield in SA sub-groups may reflect at least 2 possible mechanisms: an increased level of heteroplasmy (G1 and G2), potentially caused by an insufficient sperm-activated IVF hemi-Cyt or by a blocking effect imposed by the M-phase-derived hemi-Cyt, and/or a disruption in karyokinetic events caused by the manipulation in sperm-activated IVF hemi-Kar (G2 and G3). In G4, both mechanisms were probably attenuated by the use of 2 sperm-activated IVF hemi-Cyt and a SC-kar, analogous to conditions in the SCNT and G5 groups. Table 1.Effect of cytoplast type and activation process on in vitro development of bovine SCNT embryos This study was supported by a grant from CAPES/Brazil.

An X chromosome effect responsible for asymmetric reproductive isolation between male Drosophila virilis and heterospecific females

Reproductive isolation between closely related species is expressed through uncoordinated courtship, failed fertilization, and (or) postzygotic barriers. Behavioural components of mating often form an initial barrier to hybridization between species. In many animals, females are responsible for mating discrimination in both intra- and interspecific crosses; males of Drosophila virilis group represent an exception to this trend. Using overall productivity tests, we show that a lower proportion of D. virilis males sire progeny when paired with a heterospecific female ( Drosophila novamexicana or Drosophila americana texana ) for 2 weeks. This suggests male mate discrimination or some other kind of asymmetrical incompatibility in courtship and mating or early zygote mortality. We used males from D. virilis – D. novamexicana and from D. virilis – D. a. texana backcross populations to map chromosome effects responsible for male reproductive isolation. Results from the analysis of both backcross male populations indicate a major X chromosome effect. Further, we conduct a male behavioural analysis to show that D. virilis males significantly fail to continue courtship after the first step of courtship, when they tap heterospecific females. The combined results of a major X chromosome effect and the observation that D. virilis males walk away from females after tapping suggest that future studies should concentrate on the identification of X-linked genes affecting the ability of males to recognize conspecific females.

First evidence of a calcium transient in flowering plants at fertilization

We report here the first evidence of a transient elevation of free cytosolic Ca2+ following fusion of sperm and egg cell in a flowering plant by the use of an in vitro fertilization system recently developed in maize. Imaging changes in cytosolic Ca2+ at fertilization was undertaken by egg cell loading with the fluorescent Ca2+ indicator dye fluo-3 under controlled physiological conditions. The gamete adhesion step did not induce any cytosolic Ca2+ variation in the egg cell, whereas the fusion step triggered a transient cytosolic Ca2+ rise in the fertilized egg cell, lasting several minutes. This rise occurred after the establishment of gamete cytoplasm continuity. Through these observations, we open the way to the identification of the early signals induced by fertilization in flowering plants that give rise to the calcium transient and to investigations of the role of Ca2+ during egg activation and early zygote development in plants, as has been reported for other better characterized animal and algae systems.