EFFECT OF CRYOPROTECTANTS AND CRYOPRESERVATION METHODS ON THE QUALITY OF BUFFALO OOCYTES

This study was conducted to evaluate the effect of two types of cryoprotectants and two cryopreservation methods on viability and normal morphology of the immature and in vitro mature of post-thawed buffalo oocytes. The in vitro mature and immature oocytes were kept in the vitrification solution (VS1) for 5 min and the vitrification solution (VS2) for 15 sec. The results revealed that the proportion of buffalo oocytes found to be viable and normal morphology were significantly (P< 0.01) higher in using glycerol which is 76.14% and 55.1%; respectively. We conclude from the present study that the viability and normal morphology of post-thawed buffalo vitrified in vitro matured oocytes were significantly (P < 0.01) higher in glycerol than those obtained in dimethyl sulfoxide (DMSO) and in vitrification technique than rapid technique

Impact of xenotransplantation of sheep ovarian cortex and follicular fluid- enriched SMART medium on the morphology of recovered of sheep oocyte

Ovarian follicles and cortical tissue can survive viable after ovariectomy and use successfully for xenotransplantation or in vitro maturation. This study designed to assess in vitro maturation (IVM) of sheep oocytes recovered from immature follicles using simple medium for assisted reproductive techniques (SMART) medium enriched with follicular fluid (FF) using xenotransplanted ovarian tissue cortex inside the body of female mice injected with or without different hormonal stimulation protocols. In this study, follicular fluid (FF) aspirated from randomly sheep ovarian follicles. Seventy-five healthy and mature female mice were used for transplantation of sheep ovarian tissue (OT) on the inner side of the peritoneum. Later on, these female mice were classified into two groups. Group A: control (without medication). Group B: hormonal programs (hormonal stimulation). The last experimental group was Group C: the direct examination of sheep ovarian cortex group. The sheep ovarian cortex was recovered from female mouse then oocytes were collected by slicing for assessment and classified into three groups. Group1: oocytes incubated within SMART medium alone (control group). Group 2: oocytes incubated within SMART medium enriched with 5% FF, and Group 3: oocytes incubated within SMART medium enriched with 10% FF. The normal oocytes morphology was assessed post-xenotransplantation and parameters were statistically analyzed. No significant (P> 0.05) difference of normal oocytes morphology was seen between treated group (GB) and control group (GA). However, significant (P= 0.043) difference was observed in the percentage of normal oocyte morphology of recovered oocytes for the group (GC) in compare to (GA). Meanwhile, there was highly significant (P< 0.001) difference between GB and GC groups. Addition 5% FF to SMART medium of GB revealed significant (P= 0.044) difference in compare to the GA. Moreover, highly significant (P< 0.001) difference was observed in GC in compare to GA and GB compared to GC. Meanwhile, addition 10% FF to SMART medium of G3 revealed significant (P< 0.05) difference in the percentages of the normal and abnormal morphology of recovered oocytes. In conclusion, this study approved that normal oocyte morphology and maturation was valuable when using ovarian tissue grafts. In addition, the combination of SMART medium with 10% FF was revealed the best oocyte morphology and maturation

Improving outcome of in vitro sperm activation using non–liquefied versus liquefied semen of oligoasthenozoospermic patients

Objective: To evaluate the efficacy of in vitro sperm activation (ISA) using non-liquefied versus liquefied asthenozoospermic semen samples for improvement of sperm parameters. Materials and Methods: Fifty six oligoasthenozoospermic (OA) patients (age range: 22-44 years; mean: 32.089 years) were enrolled in this study. OA patients were classified according to type of infertility. Also, duration of infertility was recorded. Fifty six semen samples were collected, and seminal fluid analyses were done involving macroscopic and microscopic examinations were performed according to WHO methodology. Direct swim–up technique was used to separate the motile spermatozoa from seminal plasma. Minimum Essential Medium Eagle (MEME) enriched with 5% Human serum albumin (HSA) was used. One mL of either non–liquefied or liquefied semen was layered beneath 1 mL of MEME enriched with 5% HSA, and placed for incubation in an air incubator at 37 oC for 30 minutes. Then, one drop (10 μL) from upper layer of culture medium was taken using automatic pipette to be examined under high power field (40 X) for assessment of sperm parameters. Results: According to type of infertility, infertile patients were classified into patients with either primary infertility (no. 46; 82.15 %) or secondary infertility (no. 10; 17.85 %). In contrast to other parameters, significant (P<0.05) reductions were noticed in the percentages of sperm motility and progressive sperm motility for OA patients with primary infertility as compared to OA patients with secondary infertility. All sperm parameters were significantly (P<0.001) enhanced after in vitro activation of liquefied and non-liquefied semen samples when compared to pre-activation. In the present study, best results were achieved for non-liquefied semen samples as compared to liquefied semen samples. Conclusion: It was concluded that the outcome of ISA was enhanced in regard to sperm parameters when using non-liquefied semen of OA patients. Furthermore, some components of seminal plasma of OA patients may be have harmful effects on certain sperm functions when in vitro incubated for longer periods. Further study is recommended to investigate the effect of in vitro sperm activation from non-liquefied semen on successful rate of artificial insemination husband

The detrimental effect of cigarette smoking on semen parameters and sperm plasma membrane integrity in infertile patients undergoing intra-uterine insemination

The present study was designed to assess the effect of cigarette smoking on semen parameters, hypo-osmotic swelling (HOS) test (%) and outcome of intra-uterine insemination (IUI). In this study, one hundred infertile males were involved and according to cigarette smoking were divided into 55 smokers and 45 non smoker infertile couples. From each male, semen samples were collected and the sperm parameters including sperm concentration, sperm motility, progressive sperm motility, normal sperm morphology, and HOS-test were evaluated according to standard World Health Organization (WHO) criteria. For IUI, the sperm prepared using direct swim-up technique through incubation for 30 minute in 5% CO2 at 37ºC. The results of the present study demonstrated that the sperm parameters, HOS-test (%) and IUI outcomes for non smoker infertile males was higher than smokers. In addition, the results of sperm parameters and HOS-test (%) for smokers were more deviated from normal range of criteria of WHO than non smokers. Non significant differences (P>0.05) in the sperm HOS-test were assessed between non smokers and smokers.From the results of the present study, it was concluded that the cigarette smoking have several impacts on sperm functions and integrity of plasma membrane, as well as sperm fertilizing ability. Further studies are recommended to assess the effect of cigarette smoking on DNA damage and embryo quality after IVF-ET

Effect of Cumulus cell co-culture and Protein Supplement on Success of in vitro Fertilization and Development of Pre-implanted Embryos in mice

Successful oocyte fertilization and normal embryonic development of mice were considered the most important diagnostic criteria for the safety of materials and tools used for human in vitro fertilization and embryo transfer (IVF-ET). Therefore, we studied the influence of cumulus cells co-culture and protein supplement within culture medium on percentages of in vitro fertilization (IVF) and normal development of early stages of mouse embryo later. Oocytes were collected and treated with hyaluronidase to remove cumulus cells. Oocytes were divided into four groups namely: Group-1: Oocytes incubated within modified Earl’s medium (MEM) supplied with 10% inactivated bovine amniotic fluid as a protein source and cumulus cells; Group-2: Oocytes incubated with MEM supplied with cumulus cells only; Group-3: Oocytes incubated with MEM supplied with 10% inactivated bovine amniotic fluid only; and Group-4: Oocytes  incubated with MEM free of both protein source and cumulus cells. For IVF, 5-6 oocytes were incubated with active spermatozoa under paraffin oil for 18-20 hours at 37° oC in 5% CO2. Percentages of IVF and embryonic development were then recorded. Best results for IVF and normal embryonic development were achieved from oocytes of Group-1 when compared to the other groups. As compared to Group-1, the percentage of IVF for Group-2 and Group-3 were decreased insignificantly and significantly (P<0.002), respectively. Significant (P<0.01) reduction in the percentages of IVF and normal embryonic development were reported in Group-4 as compared to Group-1. Therefore, it was concluded that the presence of cumulus cells co-culture and bovine amniotic fluid as a protein source within culture medium may have an important role on the fertilizing capacity of spermatozoa and oocytes and normal development of pre-implanted mouse embryo later

Evaluation of Seminal Fluid Parameters, Reproductive Hormones and Testicular Biopsy for non-obstructive Azoospermic Patients with Sertoli cell only Syndrome

The aims of this study were (i) to determine the seminal fluid parameters, levels of serum reproductive hormones, testicular size and diameter of seminiferous tubules depending on multiple testicular biopsies from non-obstructive azoospermic (NOA) patients, and (ii) to assess the effect of smoking on concentrations of serum reproductive hormones. Thirty four NOA patients were involved in the present study. The selection of patients was based on an average of 2 pelleting seminal analysis, reproductive hormones profile and diagnostic testicular biopsy.  A detailed questionnaire was used to obtain a thorough history from the patients. The results of this study showed that the mean age of NOA patients with Sertoli cell only syndrome (SCOS) was 33.875 years, and 47.05 % of NOA patients have had 2-4 years duration of infertility. Macroscopic examination of seminal fluid appeared to have normal values as reported by WHO. Non-significant (P>0.05) difference was observed for testicular size and diameter of seminiferous tubules between right and left testis. High and abnormal concentrations of serum FSH were registered for NOA patients with SCOS. However, non-significant (P>0.05) differences were reported for concentrations of serum reproductive hormones between smokers and non smokers. In conclusion, testicular biopsy is the useful and predictable diagnostic tool for NOA patients with SCOS. Further studies are needed to explain the etiology, management and prognosis of NOA patients with SCOS

The effect of lead exposure of mice during pregnancy on the morphology of epididymal and testicular spermatozoa of their offspring

The aims of this study were to assess the differences in the percentages of abnormal morphology between the epididymal and testicular spermatozoa of mature male offspring mice whose mothers were injected with various doses of lead acetate during gestation. Seventy two healthy female mice were divided into three major groups according to the number of injections involving 1, 2 or 3 injections at 8th day; 8th and 13th days; and 8th, 13th and 18th days of gestation period, respectively. Each major group was subdivided into four minor groups according to of the dosage of lead administration of (0, 25, 50 and 100) mg/Kg. The percentages of abnormal morphology of epididymal and testicular spermatozoa were studied and the data were statistically analyzed. The results of the present study proved that an increased number of injections and/or dose of lead acetate injected to the mothers during gestation cause an elevation in the percentage of abnormal morphology of both epididymal and testicular spermatozoa of the male mice offspring. In conclusion this study demonstrated that lead acetate when exposed prenatally have toxic effects on the sperm abnormal morphology in the offspring male mice most likely by interfering with the phase(s) of spermatogenesis and/or spermiogenesis

The effect of lead exposure of mice during pregnancy on the concentration and motility of epididymal and testicular spermatozoa in offspring mature male mice

Introduction: The objective of this study was to investigate the effects of lead on concentration and motility of spermatozoa recovered from epididymis and testes in mature male offspring whose mothers were exposed to different doses and concentrations of lead acetate during gestation period. Materials and Methods: Seventy two healthy mature female mice were divided into three major groups according to the number of injections involving 1, 2 and 3 injections. Each major group was subdivided into four minor groups according to the concentration dose of (0, 25, 50 and 100) mg/Kg of lead acetate. Sperm concentration, percentage of motility and grade of activity were microscopically examined and statistically analyzed. Results: A significant reduction in the sperm functions were seen in relation to an increased in the number of injections and/or concentration of lead acetate dose as compared with the control groups. Conclusion: The toxic effects of lead acetate may interfere with spermatogenesis and metabolism of spermatozoa