322 EFFECT OF PLASMIN ON ACROSOME REACTION OF BUFFALO (BUBALUS BUBALIS) SPERMATOZOA IN VITRO

2010 ◽  
Vol 22 (1) ◽  
pp. 317
Author(s):  
I. Venditto ◽  
E. Mariotti ◽  
L. Boccia ◽  
M. Rubessa ◽  
M. De Blasi ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Proteolytic Enzymes ◽  
Critical Role ◽  
Bubalus Bubalis ◽  
Positive Control ◽  
Negative Control ◽  
Chi Square ◽  
Chi Square Test ◽  
Response Trial

Fertilization is a critical step of the in vitro embryo production (IVEP) technology in buffalo. It is known that proteolytic enzymes are involved in different steps of the fertilization process; among these, a critical role may be played by the plasminogen activator-plasmin system. It has been demonstrated that plasmin, the active enzyme of this system, induces acrosome reaction (AR) in bull spermatozoa (Taitzoglou IA et al. 2003 Andrologia 35, 112-116). The aim of this study was to investigate the effect of plasmin on the ability of buffalo sperm to undergo the AR. Frozen- thawed sperm from 4 buffalo bulls were treated by swim-up and incubated with 0.01 mM heparin for 4 h. At 0, 2, and 4 h, aliquots of spermatozoa were exposed for 10 min to 60 μg mL-1 of lysophosphatidylcholine (LPC), as positive control, and to 0.01 μg mL-1 of plasmin. This concentration was chosen after a preliminary dose-response trial. Another sample from each treatment was incubated with IVF medium (negative control). After 10 min, sperm motility was evaluated and sperm were fixed in 37% formaldehyde and stained with trypan blue-Giemsa for subsequent microscopic examination. The total number of sperm counted, over 3 replicates, was 1269 for the negative control, 1293 for LPC, and 1238 for plasmin, equally distributed among incubation times. Differences among groups were analyzed by chi-square test. After swim-up, acrosomal loss was observed only in 4% of the sperm. The addition of 0.01 μg mL-1 of plasmin for 10 min to buffalo spermatozoa at time 0 significantly (P < 0.01) enhanced (23%) AR compared with the control (7.8%), with the same efficiency of LPC (17.1%). After 2 h of incubation with heparin, both plasmin and LPC increased the AR compared to the control (24.4, 20.1, and 14.0%, respectively; P < 0.01). After 4 h, plasmin gave higher percentages of AR (27.2%) compared to both the control (21.0%; P < 0.05) and LPC (19.2%; P < 0.01). Another interesting result is the improved motility recorded with plasmin compared to both the control and LPC groups at 2 h of incubation (90, 75, and 75%, respectively; P < 0.05) and at 4 h of incubation (75, 60, and 60%, respectively; P < 0.05). Finally, no differences in sperm viability were observed between plasmin and the control, whereas a decreased viability was found when LPC was used at 0 h (96.2, 95.0, and 89.0%, respectively, for plasmin, control, and LPC; P < 0.05), at 2 h (85.0, 87.5, and 77.0%, respectively, for plasmin, control, and LPC; P < 0.01), and at 4 h (85.0, 93.3, and 81.1%, respectively, for plasmin, control, and LPC; P < 0.01). In conclusion, we found that addition of plasmin to capacitated sperm increases the percentage of acrosome-reacted spermatozoa and improves motility. Our results suggest that plasmin may play a role in events surrounding fertilization and suggest to evaluate in further studies whether the addition of plasmin during IVF improves the IVEP efficiency in buffalo.

311 MELATONIN PROMOTES IN VITRO SPERM CAPACITATION IN BUFFALO (BUBALUS BUBALIS)

2010 ◽  
Vol 22 (1) ◽  
pp. 311 ◽  
Author(s):  
S. Di Francesco ◽  
E. Mariotti ◽  
M. Tsantarliotou ◽  
A. Sattar ◽  
I. Venditto ◽  
...  
Keyword(s):  
Acrosome Reaction ◽  
Optimal Dose ◽  
Bubalus Bubalis ◽  
Positive Control ◽  
Negative Control ◽  
Sperm Capacitation ◽  
Chi Square ◽  
Melatonin Administration ◽  
Chi Square Test

Melatonin, the main hormone secreted by the pineal gland, plays many roles in reproduction. In ram spermatozoa, melatonin administration increases plasminogen activator activity (Tsantarliotou MP et al. 2007 Theriogenology 69, 458-465), known to be involved in sperm capacitation and acrosome reaction (Taitzoglou IA et al. 1996 Mol. Androl. 8, 187-197). The aim of this study was to evaluate the efficiency of melatonin to induce buffalo in vitro sperm capacitation, indirectly assessed by estimating the capability of spermatozoa to acrosome-react. Frozen-thawed semen from 4 different bulls was pooled and treated by swim-up in order to select only the motile population. Spermatozoa (n = 829) were assessed immediately after swim- up separation, to evaluate the incidence of acrosomal loss in non-treated cells (time 0). The remaining spermatozoa were incubated in the absence of capacitating agents (negative control; n = 513), in the presence of 0.01 mM heparin (positive control; n = 775), 10 μM melatonin (n = 684), 100 μM melatonin (n = 751), and 1 mM melatonin (n = 650), for 2 h. Sperm were then exposed for 15 min to 60 μg mL-1 of lysophosphatidylcholine, an agent known to induce acrosome reaction (AR) only on capacitated spermatozoa. Trypan blue was used first to differentiate live from dead spermatozoa and the dried smears were then fixed in 37% formaldehyde and stained with Giemsa for acrosome evaluation by microscopic examination. The percentage of acrosome-reacted spermatozoa in each group was used to assess the efficiency of capacitation under different incubation conditions. The experiment was repeated 4 times. Differences among groups were analyzed by chi-square test. Acrosomal loss was observed only in 2.1% of the sperm population at time 0. Interestingly, sperm treatment with both heparin and the different concentrations of melatonin resulted in a significantly higher incidence of AR compared to the negative control (24.4, 20.5, 20.0, 23.6 v. 8.0% for the positive control, 10 μM melatonin, 100 μM melatonin, 1 mM melatonin, and the negative control, respectively; P < 0.01). These results demonstrated that melatonin determines capacitation of buffalo spermatozoa in vitro. Furthermore, the effect of melatonin was comparable to that of heparin, that is, the capacitating agent currently used in the IVF system. The capacitating effect was observed at all the tested concentrations, and viability was not affected. This suggests to extend the range of concentrations to test in future studies, in order to identify the optimal dose. Moreover, considering the seasonality of the species and the great differences in fertility attitude of buffalo bulls, it would be interesting to investigate the capacitation effect of melatonin in relation to both the season and the bull.

145 EFFECT OF RELAXIN ON FERTILIZING ABILITY OF BUFFALO SPERM

2014 ◽  
Vol 26 (1) ◽  
pp. 186 ◽  
Author(s):  
A. R. Elkhawagah ◽  
V. Longobardi ◽  
G. A. Sosa ◽  
G. Albero ◽  
A. Salzano ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Positive Control ◽  
Negative Control ◽  
Cleavage Rate ◽  
Chi Square ◽  
Chi Square Test ◽  
Live Sperm ◽  
Student’S T ◽  
Fertilizing Capability

The aim of this work was to evaluate the effect of relaxin, known to improve fertility parameters of frozen-thawed sperm in other species (Miah et al. 2006 J. Reprod. Dev. 52, 773–779; Miah et al. 2007 Anim. Sci. J. 78, 495–502), on buffalo sperm motility, capacitation, and fertilizing capability. Frozen-thawed sperm from 2 bulls (4 replicates each) were separated by Percoll, diluted to a 20 × 106 mL–1 concentration and incubated in TALP medium in the absence of capacitating agents (negative control), in the presence of 10 μg mL–1 of heparin (positive control) and 100 ng mL–1 of relaxin for 2 h. Following incubation, sperm were exposed for 15 min to 60 mg mL–1 of lysophosphatidylcholine, a fusogenic agent known to induce the acrosome reaction only on capacitated sperm. To evaluate acrosome-reacted (AR) live sperm, cells were fixed and stained with Trypan blue-Giemsa (Kovacs and Foote 1992 Biotech. Histochem. 67, 119–124) and evaluated (800 sperm counted/group). Sperm motility was examined by a phase contrast microscope, whereas the fertilizing capability was evaluated by heterologous IVF. Abattoir-derived bovine oocytes (n = 258, 86 per group) were in vitro matured and fertilized according to standard procedures (Rubessa et al. 2011 Theriogenology 76, 1347–1355) with buffalo sperm in the absence of capacitating agents and in the presence of 10 μg mL–1 of heparin and 100 ng mL–1 of relaxin. Twenty hours after IVF, presumptive zygotes were denuded and cultured in SOF for 24 h, when cleavage rate was evaluated and confirmed by fixation with absolute ethanol overnight and staining with 2.5 μg mL–1 of Hoechst 33342 after zona removal by pronase (2 mg mL–1) digestion. The differences in the percentages of AR sperm and cleavage among groups were analysed by a chi square test and those in sperm motility by Student's t-test. Acrosomal loss was observed in 10.8% of the sperm after thawing, which may indicate freezing-induced capacitation, and, hence, this value was detracted from the percentages of AR recorded following incubation. After 2 h of incubation, 100 ng mL–1 of relaxin significantly (P < 0.05) increased the percentages of live AR sperm (P < 0.05) compared with the negative control (31.3 ± 2.2 and 25.8 ± 2.8, respectively), with intermediate results in the positive control (27.0 ± 2.2). Motility was significantly improved (P < 0.05) when sperm were exposed to 100 ng mL–1 of relaxin compared with both the negative and positive control (73.7 ± 2.4, 60.0 ± 4.1, and 60.0 ± 7.1, respectively). A significant (P < 0.05) improvement of cleavage rate was recorded both in the positive control (71.5 ± 4.8) and in the group treated with 100 ng mL–1 of relaxin (70.7 ± 0.5) compared with negative control (52.1 ± 1.5). In conclusion, these preliminary results indicate that relaxin at the concentration of 100 ng mL–1 improves sperm motility, capacitation, and the IVF capability of buffalo sperm.

62 EXPOSURE TO ETHYLENE GLYCOL AND DIMETHYL SULFOXIDE CAUSES ACTIVATION AND SPINDLE ANOMALIES IN BUFFALO (BUBALUS BUBALIS) OOCYTES

2009 ◽  
Vol 21 (1) ◽  
pp. 131 ◽  
Author(s):  
M. De Blasi ◽  
E. Mariotti ◽  
M. Rubessa ◽  
S. Di Francesco ◽  
G. Campanile ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Embryo Development ◽  
Sucrose Solution ◽  
Bubalus Bubalis ◽  
Meiotic Spindle ◽  
Blastocyst Development ◽  
Chi Square ◽  
Chi Square Test ◽  
Simple Exposure

Despite the increasing interest, buffalo oocyte cryopreservation is still inefficient, especially in terms of blastocyst development after IVF. The aim of this work was to evaluate chromatin and spindle organization of buffalo in vitro-matured oocytes after vitrification/warming by cryotop and after their simple exposure to cryoprotectants (CP). An overall amount of 251 COC was selected and matured in vitro. In the vitrification group, COC were first exposed to 10% ethylene glycol (EG) + 10% DMSO for 3 min, and then to 20% EG + 20% of DMSO and 0.5 m sucrose, loaded on cryotops, and plunged into liquid nitrogen within 25 s. Oocytes were warmed into a 1.25 m sucrose solution for 1 min and then to decreasing concentrations of sucrose (0.625 m, 0.42 m, and 0.31 m) for 30s each. In order to test CP toxicity, COC were simply exposed to the vitrification and warming solutions. Two hours after warming, oocytes were fixed and immunostained for microtubules using a method previously described (Messinger SM and Albertini DF 1991 J. Cell Sci. 100, 289–298), stained for nuclei with Hoechst, and examined by fluorescence microscopy. Fresh in vitro-matured oocytes were fixed and stained as controls. Data were analyzed by chi-square test; results are shown in Table 1. The percentages of MII oocytes in the control and vitrification groups were greater than in the toxicity group, in which a greater percentage of telophase II stage oocytes were found compared with both the control and vitrification groups, indicating occurrence of activation. Of the MII oocytes, both exposure to CP and vitrification procedures gave greater percentages of oocytes with abnormal spindle and abnormal chromatin configuration compared with the control. An unexpected datum was the evidence of a significant percentage of spontaneously activated oocytes in the toxicity group. We speculate that the lack of activation in the vitrification group may be related to the slowing down of metabolic activity subsequent to thermal shock, and hence, that activation after vitrification may occur later than 2 h post-warming. In conclusion, the simple exposure to CP causes activation of the COC and damage to the cytoskeleton similar to that induced by the whole vitrification protocol. The damages to the meiotic spindle and DNA fragmentation may lead to aneuploidy incompatible with subsequent embryo development and account for the poor embryo development currently recorded in buffalo. Table 1.Chromatin and spindle organization in oocytes vitrified and exposed to cryoprotectants

scholarly journals A Case-Control Study on the Relationship Between Cholecystectomy and Cholangiocarcinoma in China

2020 ◽  
Author(s):  
Donglie Zhu ◽  
Hang Fu ◽  
Zelong Yang ◽  
Mingzuo Jiang ◽  
Yanjie Ren ◽  
...  
Keyword(s):  
Negative Control Group ◽  
Positive Control ◽  
Negative Control ◽  
Control Group ◽  
Case Group ◽  
Hepatic Hemangioma ◽  
Chi Square ◽  
Chi Square Test ◽  
History Of ◽  
The Difference

Abstract Aims: The present study aimed to explore the correlation between cholecystectomy and cholangiocarcinoma, and to provide preliminary clinical basis for precise cholecystectomy in China.Methods: We conducted a retrospective analysis of 9744 patients with cholangiocarcinoma, colon cancer, pancreatic cancer, femoral fracture, and hepatic hemangioma diagnosed in Xijing hospital from August 2008 to August 2018. They were divided into three groups: case group (1749 cases of cholangiocarcinoma), positive control group (3137 cases of colon cancer and 1950 cases of pancreatic cancer), negative control group (1794 cases of femoral fracture and 1114 cases of hepatic hemangioma). We collected the general information (gender, age), past medical history, cholecystectomy history from the patients, and these data were analyzed by chi-square test and logistic regression analysis. Results: The cholecystectomy rate of the case group was significantly higher than that of the positive control group and the negative control group by chi-square test (p<0.025). The cholecystectomy rate and the history of cholecystolithiasis were analyzed by logistic multivariate regression analysis. The OR values of cholecystectomy rate were 1.553 (95%CI: 1.311-1.840) and 3.181 (95%CI: 2.561-3.951), respectively, and the difference was statistically significant (p<0.000). The OR values of the history of cholecystolithiasis were 2.460 (95%CI: 2.093-2.890) and 5.426 (95%CI: 4.325-6.809), respectively, and the difference was statistically significant (p<0.000). In case group, the difference between cholecystectomy and cholecystolithiasis was statistically significant (p<0.000) by chi-square test. Conclusions: In conclusion, cholecystectomy is one of the risk factors of cholangiocarcinoma and the patients who undergo cholecystectomy have a higher risk of cholangiocarcinoma than the control groups. Cholecystectomy should be conducted with caution and the precise surgical treatment of gallbladder diseases is advocated.

scholarly journals Prostaglandin E2 induces ovulation in prepubertal mice

2021 ◽  
Vol 58 ◽  
pp. e182745
Author(s):  
Jéssica de Souza Andrade ◽  
Juliana Pavan Zuliani ◽  
Jaswant Singh ◽  
Sulamita da Silva Setúbal ◽  
Renata Reis da Silva ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prostaglandin E2 ◽  
Positive Control ◽  
Negative Control ◽  
Cox1 Gene ◽  
Gene Expressions ◽  
Chi Square ◽  
Chi Square Test ◽  
Phosphate Buffered Saline ◽  
Cox2 Expression

The objective of this study was to determine the ability of prostaglandin E2 (PGE2) to induce ovulation and expression of PGE2 receptor (EP2 and EP4) and COX genes (COX-1 and COX-2) in the ovary and pituitary of prepubertal mice. The positive control consisted of the application of 5 μg of gonadotropin-releasing hormone (GnRH, n = 29); the negative control applied 0.5 mL of phosphate buffered saline (PBS, n=31); the treatment tested the application of 250 μg of PGE2 (n = 29), making a total of 89 prepubertal mice (BALB/c). Mice were euthanized 14 to 15 h after treatments to detect ovulation and tissue collection. A Chi-square test was used to compare the proportion of animals ovulating. Gene expressions and number of ovulation were analyzed by one-way ANOVA and Tukey’s test was used to compare means among groups. A greater proportion of mice (P < 0.001) ovulated after receiving GnRH (89.7%, 26/29) compared to PGE2 group (58.6%, 17/29). However, the proportion was higher compared to those treated with PBS (0%, 0/31). Ep2 gene expression in the pituitary was > two-fold higher (P < 0.05) in the PGE2 group compared to the PBS and GnRH groups. Further, PGE2 stimulated Cox1 (2.7 fold, P < 0.05) while GnRH stimulated Cox2 expression (6.5 fold, P < 0.05) in the pituitary when compared to the PBS group. In conclusion, our results support the hypothesis that PGE2 can induce ovulation in prepubertal mice with a concomitant increase in Ep2 and Cox1 gene expression in the pituitary gland.

284 FERTILIZING CAPACITY OF BUFFALO (BUBALUS BUBALIS) SPERM CO-CULTURED WITH OVIDUCT EPITHELIAL CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 299 ◽  
Author(s):  
E. Mariotti ◽  
S. Di Francesco ◽  
M. De Blasi ◽  
C. Siniscalchi ◽  
M. V. Suárez ◽  
...  
Keyword(s):  
Production Efficiency ◽  
Penetration Rate ◽  
Bubalus Bubalis ◽  
Cleavage Rate ◽  
Chi Square ◽  
Chi Square Test ◽  
Group A ◽  
Group B ◽  
Bovine Oocytes

The overall in vitro embryo production efficiency in buffalo is hampered by the poor IVF efficiency. The aim of this work was to evaluate whether the fertilizing ability of buffalo sperm is improved by the presence of bovine oviductal cells (BOEC) during IVF. Because of limited availability of buffalo oocytes, this was assessed by heterologous IVF. Bovine oviducts were obtained at a local abattoir from cows that were in the preovulatory phase of a normal estrous cycle. BOEC recovered from 5 oviducts as previously described (Gualtieri and Talevi 2000 Biol. Reprod. 62, 1754-1762) were pooled and plated in 100 μL drops of TCM-199 + 10% FCS, 100 U mL-1 penicillin, 100 μg mL-1 streptomycin and 0.25 μg mL-1 amphotericin B under mineral oil. Medium was changed every 48 h up to Day 6, when cell confluence and cilia activity were optimal. On day of IVF the medium was removed from the drops and replaced with TALP supplemented with 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin (IVF medium). Frozen-thawed sperm from an IVF-tested buffalo bull, treated by Percoll gradients, were used for all IVF groups (2 × 106 sperm mL-1). In vitro-matured bovine oocytes (n = 409), over 3 replicates, were distributed in 4 fertilization groups: (A) IVF medium alone (control); (B) BOEC monolayer + IVF medium; (C) sperm preincubated for 6 h in IVF medium; and (D) sperm preincubated for 6 h with BOEC + IVF medium. After 20 h of coincubation at 38.5°C and 5% CO2 in air, putative zygotes were denuded, washed, and cultured in SOF medium. Forty-eight hours after IVF, cleavage rate was evaluated, and cleaved and uncleaved oocytes were fixed in 60% methanol and stained with DAPI for nuclei examination under fluorescence microscope. Data were analyzed by chi-square test. Although cleavage rate was not different among groups (46.2, 55.8, 50.0, and 50.0% for A, B, C, and D, respectively), the monospermic penetration rate increased (P < 0.01) in group B (79.3%) compared with group A (69.6%), with intermediate values in groups C (75.2%) and D (76.0%). Interestingly, the percentage of advanced embryos (>4 cells) was higher (P < 0.01) in groups C and D (47.9 and 37.1%, respectively) than in group A (12.1%), whereas group B (21.0%) was only different from group C. We demonstrated that the fertilizing capacity of buffalo sperm, evaluated as oocyte penetration rate after heterologous IVF, is enhanced by the presence of BOEC. This suggests that IVF of buffalo oocytes on BOEC monolayer may improve the IVF efficiency in buffalo. The higher incidence of advanced embryos in both groups with preincubated sperm may be accounted for by an earlier accomplishment of capacitation, leading to anticipated oocyte penetration. However, because the penetration rate in these groups was not improved compared with the control, we hypothesize that sperm viability may have decreased and hence that shorter incubation times should be tested in further studies.

scholarly journals Prevalence of various types of allergens in ocular allergic conditions of patients from Pokhara, Nepal

2014 ◽  
Vol 6 (1) ◽  
pp. 6-23 ◽  
Author(s):  
Sachet Prabhat Shrestha ◽  
Amod K Pokhrel ◽  
Pushpa Malla ◽  
Srijana Thapa Godar
Keyword(s):  
Skin Prick Test ◽  
Allergic Conjunctivitis ◽  
Allergic Diseases ◽  
Positive Control ◽  
Negative Control ◽  
Test Reaction ◽  
Chi Square ◽  
Prick Test ◽  
Chi Square Test ◽  
Skin Prick Tests

Introduction: Ocular allergic conditions are mostly recurrent and the drugs prescribed, especially corticosteroids, have serious side effects. Therefore, when maximal tolerated topical and systemic medications are unable to control allergic conjunctivitis, a skin prick test for allergens should be conducted and patients should be taught to avoid these allergens. Objective: To find out the prevalence of common allergens inciting ocular allergic diseases in Nepal. Subjects and methods: A total of 13,376 skin prick tests were performed on 76 patients suffering from different chronic recurrent ocular allergic conjunctivitis with 176 common allergens (pollens, fungi, insects, dusts, danders, fabrics/ feathers, food, parthenium leaves, tobacco and mite). Buffer saline was used as a negative control while histamine acid phosphate was used as a positive control. Grading of the skin prick test reaction was done by comparison to the histamine positive control. Only markedly positive reactions were considered positive. Relevant data were entered into the excel spreadsheet and analyzed with the Stata-12 commercial package. The association between allergic conditions and socio-demographic, environmental and other co-variates were tested by the chi-square test. Results: The common offenders found in the study were mite (42.11 %) followed by fabrics/ feathers (20.39 %), dusts (18.18 %), pollen (17.05 %), non-juicy food (15.02 %), dander (13.60 %), juicy food (11.64 %) and fungus (9.87 %), and tobacco (6.58 %), parthenium leaves (5.26 %) and insects (3.17 %) were less common offenders. Conclusions: All ocular allergy patients should undergo skin prick tests to find out the allergens causing their allergy and then receive specific immunological treatment (SIT). DOI: http://dx.doi.org/10.3126/nepjoph.v6i1.10759 Nepal J Ophthalmol 2014; 6 (2): 6-23

124 CO-CULTURE WITH BOVINE INTACT CUMULUS - OOCYTE COMPLEXES DURING IN VITRO FERTILIZATION OF BUFFALO DENUDED OOCYTES COMPLETELY RESTORES THEIR FERTILIZING AND DEVELOPMENTAL COMPETENCE

2011 ◽  
Vol 23 (1) ◽  
pp. 167
Author(s):  
M. De Blasi ◽  
M. Rubessa ◽  
L. Boccia ◽  
S. Di Francesco ◽  
M. V. Suárez Novoa ◽  
...  
Keyword(s):  
Cumulus Cells ◽  
Blastocyst Stage ◽  
Negative Control ◽  
Developmental Competence ◽  
Control Group ◽  
Chi Square ◽  
Oocyte Vitrification ◽  
Vitro Fertilization ◽  
Chi Square Test

Removal of cumulus cells is necessary for several technologies such as vitrification, intracytoplasmic sperm injection, and nuclear transfer. However, it is known that the presence of cumulus cells during IVF of buffalo oocytes is fundamental for fertilization and embryo development (Gasparrini et al. 2007 Anim. Reprod. Sci. 98, 335–342; Nandi et al. 1998 Theriogenology 50, 1251–1262). The aim of this work was to evaluate whether co-culture with intact bovine cumulus–oocyte complexes (COC) during IVF would restore the developmental competence of denuded buffalo oocytes. Due to the scarce availability of buffalo ovaries, the somatic support was provided by bovine cumulus cells. Abattoir-derived COC were matured in vitro according to our standard procedures (Gasparrini et al. 2006, Theriogenology, 65, 275–287) and randomly distributed in 3 fertilization groups: 1) a control group of COC (n = 122), 2) a negative control of denuded oocytes (DO; n = 119), and 3) DO co-cultured with in vitro matured bovine COC (DO+COC; n = 103) in a 1:1 ratio (3 bovine COC + 3 denuded buffalo oocytes/50 μL drop). Fertilization was carried out with frozen–thawed spermatozoa from a tested bull in TALP medium supplemented by 0.2 mM penicillamine, 0.1 mM hypotaurine, and 0.01 mM heparin at 38.5°C under a controlled gas atmosphere of 5% CO2 in humidified air. After fertilization the zygotes were cultured in SOF medium including essential and nonessential amino acids and 8 mg mL–1 BSA, at 38.5°C under humidified 5% CO2, 7% O2, and 88% N2, up to the blastocyst stage. On Day 5 and on Day 7 (Day 0 = IVF) cleavage and blastocyst rates were respectively recorded. Data were analysed by chi-square test. As expected, cleavage and blastocyst rates were lower (P < 0.01) in DO (36.1 and 9.2%, respectively) compared with the control (67.2 and 27.1%, respectively). However, co-culture during IVF (DO+COC) significantly increased (P < 0.01) both parameters compared with DO, giving cleavage (70.9%) and blastocyst (27.2%) rates similar to the control. The results of this study demonstrated that co-culture with bovine intact COC during IVF of buffalo denuded oocytes completely restores their fertilizing capability and blastocyst developmental competence. We conclude that this may be a suitable strategy for preserving the developmental competence of oocytes devolved to technologies, such as oocyte vitrification, that require cumulus removal.

Aktivitas Antifungi Air Perasan Bawang Merah (Allium ascalonicum L.) Terhadap Candida albicans Secara In Vitro

2017 ◽  
Vol 9 (2) ◽  
pp. 71
Author(s):  
Nurhasanah Nurhasanah ◽  
Fauzia Andrini ◽  
Yulis Hamidy
Keyword(s):  
Candida Albicans ◽  
Antifungal Activity ◽  
Allium Sativum ◽  
Fungicidal Activity ◽  
Positive Control ◽  
Negative Control ◽  
Randomized Design ◽  
Significant Difference ◽  
Completely Randomized Design

Shallot (Allium ascalonicum L.) has been known as traditional medicine. Shallot which has same genus with garlic(Allium sativum L.) contains allicin that is also found in garlic and has been suspected has fungicidal activity toCandida albicans. It is supported by several researches. Therefore, shallot is suspected has antifungal activity too.The aim of this research was to know antifungal activity of shallot’s water extortion againsts Candida albicans invitro. This was a laboratory experimental research which used completely randomized design, with diffusion method.Shallot’s water extortion was devided into three concentrations, there were 50%, 100% and 200%. Ketoconazole 2%was positive control and aquadest was negative control. The result of this research based on analysis of varians(Anova), there was significant difference between several treatments and was confirmed with Duncan New MultipleRange Test (DNMRT) p<0,05, there was significant difference between 100% shallot’s water extortion with othertreatments, but there was no significant difference between 50% shallot’s water extortion with 200% shallot’s. Theconclusion was shallot’s water extortion had antifungal activity againsts Candida albicans with the best concentration100%, but it was lower than ketoconazole 2%.

scholarly journals 46 Effect of the addition of α-tocopherol to in vitro maturation media of bovine oocytes

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 2-3
Author(s):  
Theisy P Acosta Pérez
Keyword(s):  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Control Group ◽  
Chi Square ◽  
Cumulus Expansion ◽  
Minimum Concentration ◽  
Maturation Rate ◽  
Chi Square Test ◽  
System Data

Abstract α-tocopherol is known to be a powerful antioxidant, in this regard, it was added to bovine oocyte in vitro maturation media to evaluate its effect on oocyte maturation. Oocytes (n = 624) aspirated from ovaries of slaughtered cows were classified by quality and divided in four categories according to cytoplasm appearance and cumulus cells layers. Oocytes were washed in TCM-199 supplemented with fetal bovine serum (FBS) and FSH, then distributed in maturation media (TCM-199 supplemented with FBS, FSH and gentamicin). Three experimental groups of α-tocopherol (50, 100 and 200 mM) and a control group without α-tocopherol were used. Maturation was carried 22 h at 38.5°C in a 5% CO2 atmosphere. Oocytes were examined to determine cumulus expansion as categorical data (expansion or no expansion), as well as cumulus expansion Index (CEI). For CEI determination oocytes were graded 0 to 4, being 0 those with null expansion and 4 those with a noticeable cell expansion, then the number of oocytes were multiplied by the grade given and a sum of the totals was obtained, the new total was divided by the total of oocytes in the group and the result obtained corresponded to the CEI of the group. Results were analyzed with Chi Square test (for maturation rates) and an ANOVA (for the CEI) using the SAS system, data are presented as mean ± standard error. There was no statistical difference between control and α-tocopherol groups (P &gt;0.05). Numerically, the control group showed a higher maturation rate (100%) and obtained a higher CEI (2.44±0.20), followed by the 50 mM group (98.16%; 2.39±0.13), the groups 200 mM (97.40%; 2.00±0.14) and 100 mM (96.25%; 2.06±0.24) were the lowest. The addition of the minimum concentration (50 mM) of α-tocopherol to the maturation media could improve maturation rates without exposing oocytes to toxic effects.

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