181 Equine androgenic embryos: ability of the equine sperm to develop in a heterospecific ooplasm

Androgenic haploid embryos were originally produced for the study of certain aspects of early embryo development. The generation of androgenic haploid embryos allows us to better understand the complementary parental contribution to embryonic development, and to examine the effects of haploid development on gene expression. Because mare oocytes for research are scarce, the generation of heterospecific androgenic embryos could be useful to study aspects of the biology of early embryo development, or to identify genes and their variations or mutations that are responsible for reproduction-related problems in mares and stallions, which is of interest for the breeding industry. Therefore, the aim of this work was to study the capability of equine sperm to induce embryonic development after injection into an enucleated oocyte from a different species. Porcine cumulus-oocyte complexes (COC) were obtained from abattoir ovaries and placed in 100-µL drops in vitro maturation (IVM) medium for 42h. Cumulus cells were removed with hyaluronidase and vortexing. Then, mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) with stallion frozen-thawed semen (according to Rodriguez et al. 2015). Immediately after the last injection, the zona pellucida of injected oocytes was removed with protease treatment, the oocytes were treated with cytochalasin B, and the metaphase II enucleated with a 20-µm micropipette. Finally, embryos were placed in culture medium (SOF) in plates with the well-of-the-well (WOW) system. As control treatment, non-enucleated pig oocytes were injected with stallion (CE) and boar (CC) semen. At Day 4, embryos were evaluated for cleavage and number of blastomeres, and stained with Hoechst 33342 to verify the presence of DNA in each blastomere under the UV light. Embryos were stored for future PCR studies to validate the presence of equine DNA. Data were analysed by chi-squared test to compare the cleavage of both controls with the androgenic embryos. From a total of 53 androgenic haploid embryos, the cleavage rate was 62% (33/53). Embryos were cleaved in 2 to 4 cells in 72.7%, 5 to 8 cells in 18.2%, and 9+ cells in 9.1% at Day 4. Presence of DNA in all blastomeres was observed in 60.6% (20/33) of the androgenic haploid embryos, while 21.2% (7/33) of the embryos had 10 to 50% of blastomeres with DNA, and 18.6% (6/33) of the embryos did not have DNA in their blastomeres. The ICSI control embryos cleaved in 45.3% (34/75) and 64.9% (98/151) for groups CC and CE, respectively. Cleavage rates in control CE were significantly higher than those in control CC (P<0.004). No statistical difference was observed in the control groups versus androgenic embryos. This preliminary results showed that a heterospecific ooplasm can be successfully used to allow an equine sperm DNA to decondense and to develop, even in absence of the female counterpart. Using this method, copies of a single sperm DNA can be produced to potentially evaluate individual aspects of early embryo development concerning the male contribution. This is the first report of successful androgenic embryos using a heterospecific oocyte to create copies of a horse sperm DNA.

Studies on the androgenesis in cultured anthers of Atropa belladonna L.

Embryological investigations were carried out on developing anthers of <i>Atropa belladonna</i> grown in natural conditions and on anthers which produced androgenic embryos in the <i>in vitro</i> culture. The anatomy of developing anthers was analized in details. Meiotic abnormalities were not detected and 36 bivalents were present at metaphase of meiosis I. About 90% of pollen grains were normally developed. Anthers inoculated at the tetrad or microspore stage and cultured on Linsmaier and Skoog medium with kinetin 4 mg/1 and IAA - 2 mg/1 produced androgenic embryos. Differences in the development of septum, in the morphology of pollen grains, formation of tapetum, development of proembryos and the occurrence of storage materials were recorded. The origin of autopoliploid plants from haploid cells is discussed.

Comparative Study of Antioxidant Status in Androgenic Embryos ofAesculus hippocastanumandAesculus flava

In vivo(leaves and seed embryos) andin vitro(androgenic embryos) antioxidant scavenging activity ofAesculus hippocastanumandAesculus flavamedical plants was examined. Here we report antioxidant enzyme activities of superoxide dismutase, catalase, guaiacol peroxidase and glutathione peroxidase, reduced glutathione quantity, flavonoids, soluble protein contents, quantities of malondialdehyde, and•OH radical presence in the investigated plant samples. Total antioxidant capacity of all the samples ofA. hippocastanumandA. flavawas determined using FRAP, DPPH, and NO•radical scavenger capacity. The leaves ofA. flavacollected from the botanical garden exhibited stronger antioxidant activity (higher activities of SOD, and higher quantities of GSH, TSH, TPC, and scavenging abilities of DPPH and NO•, and higher FRAP values and lowest quantities of•OH and MDA) thanin vitroobtained cultures. However, the leaves ofA. flavashowed higher antioxidant activity than the leaves ofA. hippocastanum, and therefore they have a stronger tolerance of oxidative stress. Androgenic embryos of both species had low amount of antioxidants due to controlledin vitroenvironmental conditions (T, photoperiod, humidity, nutritive factors, and pathogen-free). Our results confirmed that we found optimalin vitroconditions for producing androgenic embryos of bothAesculusspecies. Also, we assume that horse chestnut androgenic embryos can be used as an alternative source for large-scale aescin production.

Study of individual plant responsiveness in anther cultures of selected pepper (Capsicum spp.) genotypes

One of the key factors determining the effectiveness of pepper anther cultures is donor plant genotype. The stock material for androgenic embryos inductions is usually made up of hybrid forms, since the higher the degree of heterozygosity, the greater the chances of producing regenerates with unique genotypes. The aim of the presented research was to evaluate individual plant reaction in anther cultures for the C. annuum hybrid (‘ATZ1’ × ‘TG’)F2, interspecific hybrid (C. frutescens × C. chinense)F2 and androgenic DH line AT6. The effectiveness of androgenesis was determined individually for each plant as the percentage of the embryos produced compared to the total number of anthers cultured. In the hybrid (‘ATZ1’ × ‘TG’)F2, anthers of 19 out of 20 plants evaluated produced embryos at a rate of 0.5 to 16.5%. Anthers of the AT6 DH line formed embryos considerably less frequently. A positive reaction was recorded for 13 out of 20 plants and the effectiveness of androgenesis did not exceed 3% for this genotype. The lowest androgenic response was recorded for the hybrid (C. frutescens × C. chinense)F2, where embryo development was observed in only five out of 19 plants and the effectiveness of androgenesis did not exceed 2%. The ploidy level of the regenerates was defined cytometrically. The analysis revealed the presence of haploid and diploid plants among the regenerates of all the genotypes evaluated.