Comparison of embryo development between intracytoplasmic and Piezo-assisted sperm injection after treating mouse sperms by Ca2+ ionophore

BACKGROUND: The purpose of this study was to evaluate the efficiency of intracytoplasmic sperm injection (ICSI) and Piezo-assisted sperm injection after pretreatment with calcium ionophore (CaI) on the mouse embryo development. METHODS: In this study, the conventional ICSI and Piezo-ICSI procedures were used. The efficacy of the methods was examined after mouse matured oocytes were fertilized with or without CaI-treated sperms. RESULTS: Piezo-ICSI demonstrated significantly more favorable results, with a fertilization rate of 64% (conventional ICSI: 42%, P<0.001) and a cleavage rate of 73% (conventional ICSI: 58%, P<0.05). When the Piezo-ICSI procedure was performed with CaI-pretreated sperms, the cleavage rate significantly increased (92% vs. 73%, P<0.05). However, the fertilization rate did not change significantly (64% vs. 56%). CONCLUSION: The Piezo-ICSI accompanies with CaI-treated sperms is more efficient than the conventional ICSI method for fertilizing and thus obtaining more mouse embryos.     

Abscisic acid alleviates the deleterious effects of cold stress on ‘Sultana’ grapevine (Vitis vinifera L.) plants by improving the anti-oxidant activity and photosynthetic capacity of leaves

The effects of exogenous application of abscisic acid (ABA) on anti-oxidant enzyme activities and photosynthetic capacity in ‘Sultana’ grapevine (Vitis vinifera L.) were investigated under cold stress. When vines had an average of 15 leaves, 0 (control), 50, 100, or 200 µM ABA was sprayed to run-off on all leaves of each plant. Twenty-four hours after foliar spraying with ABA, half (n = 5) of the water-only control vines and half (n = 5) of each group of ABA-treated plants were subjected to 4°C for 12 h, followed by a recovery period of 3 d under greenhouse conditions (25°/18°C day/night). The remaining plants in each treatment group were kept at 24°C. Cold stress increased H₂O₂ and malondialdehyde (MDA) concentrations in vine leaves, whereas all foliar ABA treatments significantly reduced their levels. Chilled plants showed marked increases in their total soluble protein contents in response to each ABA treatment. ABA significantly increased the activities of superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase in cold-stressed grapevine leaves. In contrast, cold stress markedly decreased the rates of leaf photosynthesis (A) and evaporation (E), stomatal conductance (g_s), and chlorophyll concentrations in leaves, but increased intercellular CO₂ concentrations (C_i) in leaves. Treatment with all concentrations of ABA resulted in lower leaf A, E, and g_s values, but higher C_i values at 24°C. However, following cold stress, ABA-treated vines showed higher leaf A, E, and g_s values, but lower C_i values compared to control vines without ABA treatment. The application of 50–200 µM ABA allowed chilled vines to recover more quickly when re-exposed to normal temperatures, enabling the vines to resume their photosynthetic capacity more efficiently following cold stress. These results showed that, by stimulating anti-oxidant enzyme systems and alleviating cold-induced stomatal limitations, ABA reduced the inhibitory effect of cold stress on the rate of CO₂ fixation in ‘Sultana’ grapevine plants.     

Pregnancy in the Caspian Miniature Horse Using Frozen Semen Cryopreserved with the EquiPRO CryoGuard Freeze Medium and Customized Freezing Protocols

The primary goal of this project was to establish a protocol to freeze the sperm of the Caspian miniature horse in an attempt to start an intensive artificial insemination program to effectively increase the population of this breed, which has been listed as “Critical Rare Breed” by the American Livestock Breed Conservancy and is in danger of extinction. Commercially available equine freezing medium (EquiPRO CyoGuard Complete egg-yolk extender) was used for the initial setup of two different freeze protocols: slow and fast. The fast-freeze protocol had slightly better postthaw results and was used for a fertility demonstration. Five mares of proven fertility, aged 3 to 12 years, were used in the fertility trials, two of which resulted in pregnancy. This is the first report of pregnancy in the Caspian miniature horse using frozen semen, and the results seem to be a promising start to an extensive program to help this endangered breed, although further research on freezing protocols and conditions for this process are necessary to further improve the survival of semen and pregnancy rate.     

Expression of the herpes simplex virus type 2 glycoprotein D in baculovirus expression system and evaluation of its immunogenicity in guinea pigs

Background: Herpes simplex virus type 2 (HSV-2) is highly prevalent and major cause of genital herpes in humans. The life-long nature of infection and the increasing prevalence of genital herpes imply that vaccination is the best strategy for controlling the spread of infection and limiting HSV disease. HSV glycoprotein D (gD) is one of the most important viral immunogen which has an essential role in virus infectivity and induction of immune responses. Methods: HSV-2 DNA was extracted and used as template in polymerase chain reactions to amplify gD2 gene. The PCR product was confirmed by restriction enzyme analysis, cloned into a cloning vector and then sequenced. The Bac-to-Bac expression system was used to express HSV-2 gD in insect cells. The expressed protein was used as subunit vaccine to immunize guinea pigs after confirmation. Results: The expressed protein was confirmed with SDS-PAGE and Western-blot analysis. In Western-blot analysis, two major protein bands, with approximate molecular weights of 52-55 and 41-43 kDa corresponding to the glycosylated and non-glycosylated forms of gD2 protein, were observed, respectively. Immunization with the recombinant gD2 could elicit humoral responses in guinea pigs as measured by neutralization test and ELISA, and offered high protection against induced HSV-2 genital disease. Conclusion: The baculovirus expression of heterologous genes permits proper folding, post-translational modification and oligomerization in manners that are often identical to those that occur in mammalian cells. Expression of proteins under the control of the strong polyhedrin promoter, allowing high level protein production, can be used as subunit vaccine.     

Relationship among vegetative growth and nutrient elements in the scion of different Persian lime accessions and its effect on WBDL phytoplasma

Journal of Plant Diseases and Protection - Nutrient elements have a significant role in plant physiology and metabolism, and the concentration of nutrients in the plant depends on plants genetic...     

Immune responses against excreted/secreted antigens of Toxoplasma gondii tachyzoites in the murine model

In the present study, excretory secretory antigens (ESA) of Toxoplasma gondii were evaluated in immunization of 8-10 week inbred female Balb/c mice. Tachyzoites of the parasite were cultured in cell-free incubation medium (RPMI-1640), and then supernatant of the medium was loaded on an ion-exchange chromatography column. Two fractions (ESA-F(1) and ESA-F(2)) were collected from the column. For immunization of the mice, 50 were allocated into 5 groups of 10. The first, second, third, and fourth groups were immunized, twice with total-ESA, ESA-F(1), ESA-F(2) or toxoplasma lysate antigen (TLA), respectively. The fifth group was selected as a negative control group (non-immunized). The virulent RH strain of Toxoplasma gondii was used to challenge. Delayed-type hypersensitivity responses (DTHs) were measured by intra-footpad injection measuring induration at timed intervals. Lymphocyte transformation tests (LTTs) were done on lymph node cells using [3H] thymidine incorporation as an indication of reactivity. Peritoneal macrophages from sensitized mice were stimulated and nitric oxide was measured by Griess method. The ESA-F(1) and ESA-F(2) fractions were separated on poly acrylamide gel electrophoresis (PAGE) and SDS-PAGE. ESA-F(1) had 4 bands on PAGE and 14 bands on SDS-PAGE. ESA-F(2) had one band on PAGE and two bands on SDS-PGE. Sensitized mice showed DTH and lymphocyte transformation responses to total-ESA, ESA-F(1), and ESA-F(2) and peritoneal macrophages produce nitric oxide following stimulation. In challenge experiments, all non-immunized mice died within 10 days, whereas immunized mice survived for longer time periods (P<0.05). The highest survival rate was observed in mice that immunized with ESA-F(2). We suggest that these antigens especially ESA-F(2) should be of value for the development of new strategies for immunization against toxoplasmosis.