Asian Pacific Journal of Reproduction

247-251

Authors: Nahid Yari, Sahabeh Etebary, Mohammad Ali Khalili, Ali Reza Talebi

Objective: To evaluate the impact of freezing–thawing on the human sperm head vacuoles and the potential value of motile sperm organelle morphology examination for selection of frozen-thaw spermatozoa. Methods: In 30 sperm samples from infertile men, analysis for conventional sperm parameters (motility, vitality, and normal morphology) and a morphological analysis at high magnification for vacuoles examination were done before cooling and after warming. For description of sperm head vacuoles, two hundred spermatozoa were examined and were classified into three groups according to presence and vacuole areas including no vacuole group (free of any vacuole), small vacuole group (occupy not more than 4% of the nuclear area), and large vacuole group (occupy more than 4% of the normal nuclear area). Results: Significant reduction of progressive motility and vitality was observed following cryopreservation (P<0.001). Also, normal morphology decreased significantly after cryopreservation (P<0.05). Spermatozoa with a vacuole free head had a significant reduction in cryopreservation group (P=0.013). The percentage of spermatozoa with small vacuoles increased slightly, but not significantly after cryopreservation (P=0.296). Conclusions: Motile sperm organelle morphology examination is a powerful research tool for investigating spermatozoa abnormalities such as vacuoles that are increased post cryopreservation.

257-263

Authors: Asmarinah, Tri Panjiasih Susmiarsih, Amalia Shari, Putri Ratri, Dwi Ari Pujianto, Endang Winiati Bachtiar

Objective: To express recombinant protein that comprises an important fragment of human sperm specific voltage dependent anion channel 3 (VDAC3) protein as a potential molecule for generation of antibody, which can affect sperm function, aiming at spermicide development. Methods: The produce of VDAC3 recombinant protein encoded by cDNA sequence of human VDAC3 exon 5-8, based on experimental design of VDAC3 knock-out mice study. And after the purification of various human sperm VDAC3 recombinant proteins, epitope has been predicted in our recombinant protein determined by ElliPro program. Polyclonal antibody was produced for 14 wk. Then anti-VDAC3-exon 5-8 recombinant antiserum was inoculated to human sperm. After the process, antibody VDAC3 protein in human sperm was incubation with anti-VDAC3 recombinant antibody. Finally evaluation the effect of VDAC3 antiserum to human sperm motility and plasma membrane integrity was proceeded. Results: Human VDAC3 recombinant protein was successfully over-expressed in Escherichia coli and purified by affinity chromatography method. Purified human sperm VDAC3 recombinant protein could stimulate immune response in rabbit producing an antibody against VDAC3. Anti-VDAC3 recombinant antibody recognized VDAC3 antigen in human sperm could decrease human sperm motility and membrane integrity significantly. Conclusions: Anti-VDAC3 recombinant polyclonal antibody that we produced in rabbit by ourselves could decrease sperm motility and sperm membrane integrity. The authors suggest this polyclonal antibody could be used as a candidate agent for male contraception in the future. Furthermore, the authors intend to explore the effect of this antibody into sperm function aiming at male contraceptive vaccine development.

268-278

Authors: Perumal P, Savino N, Sangma CTR, Khan MH, Ezung E, Chang S, Sangtam TZT

Objective: To analyse the seasonal effect on physiological parameters, reproductive profiles and in vitro fertility in breeding mithun bulls. Methods: A total of ten adult mithun bulls age of 5 to 6 years old with good body condition (score 5-6) were selected from ICAR-NRC on Mithun, Jharnapani, Nagaland, India. The seasons were categorised into winter, spring, summer and autumn seasons based on the meteorological data and sunshine hours. The physiological parameters, reproductive profiles and in vitro fertility parameters were assessed during different seasons in mithun under the semi-intensive system of management. Results: The statistical analysis revealed that these experimental parameters were differed significantly (P<0.05) among the seasons and in overall spring and winter seasons were more beneficial in mithun breeding programme, although, the breeding in mithun occurred throughout the year with variation. Conclusions: It is concluded that collection & preservation of mithun semen and artificial insemination in mithun species during the season of spring and winter has significant beneficial effect in terms of semen production, freezability and fertility for artificial breeding programme in mithun under the semi-intensive system.

283-288

Authors: K.H. El-Shahat, M. I. Taysser, M.R. Badr, K.A. Zaki

Objective: To determine the effects of different concentrations of penicillamine, hypotaurine, and epinephrine (PHE) as well as incubation time on motility, hyperactivity and acrosome reaction (AR) of ram sperm in vitro. Methods: Freshly ejaculated spermatozoa from three ram were collected, pooled and subjected to swim up technique in modified sperm Tyrode's albumin lactate pyruvate medium supplemented with different concentrations of PHE (10, 20, 30, 40, 50, 75 and 100 mM/mL). Then best concentrations were compared and examined for motility, hyperactivity and AR. Results: A high concentrations of PHE (30, 40, 50, 75 and 100mM/mL) showed a significant increase in motility when compared to control immediately after dilution and exist for the first and second hour of incubation period. However, when longer incubation time were used, a significant (P<0.05) decrease in motility was achieved. Similar finding was observed in hyperactivity. Stained semen samples showed a maximum percentage of incomplete AR after 1 h incubation corresponding to 50 and 75 mM/mL of PHE; however, spermatozoa treated with 75 mM/mL had a higher tendency to undergo complete AR after further incubation up to 4 h. A dose dependent relationship was detected where the maximum value of total AR was shown in spermatozoa treated with 75 mM/mL PHE for 4 h. Conclusions: To obtain better motility, hyperactivity and AR, treatment of ram spermatozoa with 75 mM/mL PHE for 4 h before being used in insemination was considered the best concentration of PHE to be used in the process of in-vitro fertilization.

197-201

Authors: Hanieh Vaseghi, Asghar Mogheiseh, Mojtaba Kafi, Masood Sepehrimanesh, Mohammad Hossein Nooranizadeh

Objective: To study the effects of anestrus dog serum in rodents (contains high amount of FSH) compared with two common superovulatory programs. Methods: Rats (n=30) at diestrus phase were evenly divided into pregnant mare serum gonadotrophins (PMSG) group (administrated with 30 IU PMSG, and 48 h later following by 25 IU hCG), recombinant follicle stimulating hormone (rFSH) group (reducing dose every 12 h from 5 to 1 IU, then following by 25 IU hCG) and anestrus dog serum group (reducing dose every 12 h from 0.6 to 0.1 mL, then following by 25 IU of hCG). Mice (n=30) were also evenly divided into PMSG group (5 IU PMSG used at 13 pm, and 48 h later following by 5 IU of hCG), rFSH group (from 13 pm, reducing dose every 12 h from 2.5 to 0.5 IU, then following by 5 IU hCG) and anestrous dog serum group (from 13 pm, reducing dose every 12 h from 0.100 to 0.025 mL, then following by 5 IU hCG). Mice and rats placed with males for 24 h after last injection. Histology samples of ovaries were prepared and the numbers of corpus lutea were counted on day 14 after mating. Results: In mice, the differences among mean number of corpus lutea in all groups (P=0.01) and between FSH and dog serum group were significant (P=0.000 7). But no significant differences were found between the mean number of corpus lutea in dog serum and PMSG, or between FSH and PMSG groups. In rats, mean number of corpus lutea were significant differences among three groups (P=0.01), and between PMSG and dog serum groups (P=0.02). Conclusions: Superovulatory response in anestrus dog serum group is similar to PMSG group in mice, which is relatively similar to FSH in rats.

212-216

Authors: Irfan Yunianto, Nurul Ain Kamar Bashah, Mahanem Mat Noor

Objective: To investigate the antifertility properties of Centella asiatica L. ethanolic extract and identify the sperm proteomic changes in treated male rats. Methods: A total of 32 male Sprague-Dawley rats were divided into two groups: control group (n=16) received distilled water and treatment group (n=16) which received 300 mg/kg of Centella asiatica L. extract for 42 d. After the treatment period, the number of implantation sites was recorded and the sperm proteomic changes were analysed by 2D gel electrophoresis. In addition, the expression of protein spots was quantified by MALDI-TOF analysis. Results: Centella asiatica L. extract resulted in low number of implantation sites in the treatment group (100.00±2.82) compared to the control group (183.00±2.14). The percentage of infertile male rats in the treatment group was higher (43.75%) compared to the control group (18.75%). Proteomic analysis showed the expression of protein spots identified in the treatment group decreased with 234 spots compared to the control group with 282 spots. Conclusions: The results revealed that there was antifertility activity in the male rats with the administration of Centella asiatica L. ethanol extract and the identified proteins could provide understanding on the adverse effect of Centella asiatica in male reproductive system.

221-225

Authors: Fabio Diniz de Souza

Objective: To explore some possibilities of computer applications in medicine, and to discuss an online infertility clinical decision support system. Methods: Retrospective data were obtained from 52 couples, and then entered into the online tool. Both its results and the initial diagnoses obtained by the treating physicians were compared with the final diagnoses established by laparoscopy and other diagnostic tests (semen analysis, hormone analysis, endometrial biopsy, ultrasound and hysteroscopy). The initial hypothesis of the research was that the online tool’s output was statistically associated with the final diagnoses. In order to verify that hypothesis, a chi-square (氈2) test with Yates’ correction for continuity (P<0.05) was performed to verify if the online tool’s and the doctor’s diagnoses were statistically associated with the final diagnoses. Results: Four etiological factors were present in more than 50% of the couples (ovarian, tubal-peritoneal, uterine, and endometriosis). The statistical results confirmed the research hypothesis for eight out of the nine etiological factors (ovarian, tubal-peritoneal, uterine, cervical, male, vaginal, psychosomatic, and endometriosis; P<0.05). Since there were no cases related to the immune factor in the sample, further clinical data are necessary in order to assess the online tool’s performance for that factor. Conclusions: The online tool tends to present more false-positives than false negatives, whereas the expert physician tends to present more false-negatives than false-positives. Therefore, the online tool and the doctor seem to complement each other. Finally, the obtained results suggest that the infertility online tool discussed herein might be a useful research and instructional tool.

231-237

Authors: Mohamed El-Raey, Heba M El-Khaiat

Objective: To assess the anion gap (AG) validity in prediction the goat’s fertility before parturition. Methods: This study was carried out on two groups (20/20, aged 4–6 years and weighted 30–43 kg) of pluriparous pregnant Baladi goats. Animals were subjected to clinical examination, blood sampling to assess serum biochemical parameters in correlation with utrasonographic assessment of uterine horn involution (UTI) and placental dropping time after kidding. Unpaired student t–test was used to detect the significance of differences (P<0.05). Correlation coefficient was used to study the relationships among the different assessed parameters, analysis (r) was assigned at P<0.05 using the same statistics program. Results: Results revealed that there were highly significant differences (P<0.05) between normally and abnormally parturated goats concerning Cl–, K+, Na+, P, Mg2+, Total/Ca2+, albumin, glucose and AG. Goats suffered from dystocia having high levels of HCO3 –, cortisol and lactate. Additionally, there were significant differences (P<0.05) between normally and abnormally parturated goat does regarding the process of UTI starting from 3rd to 10th wk after parturition. Furthermore, there was a highly significant difference (P<0.05) between normally and abnormally parturated goat does concerning their placental dropping time; where, distressed/abnormally parturated goat having the longest time. Furthermore, there were highly strong correlation coefficient between Mg2+, K+, Na+, HCO3–, glucose, albumin, AG and corrected AG concentrations during goat’s prepartum period and UTI, placental dropping time. Conclusions: Reporting serum bio-chemical changes and AG during goat’s prepartum period could be used as diagnostic tool to judge the normality of parturition process, placental dropping, UTI and genital health, in other word, could be used as predictor for goat’s fertility prior to parturition.

151-157

Authors: Deotima Sarkar, Arijit Chakraborty, Dakshayani Mahapatra, Amar K Chandra

Objective: To evaluate the effect of daily consumption of bamboo shoots (BS) on the morphological features and functional status of the female reproductive system in adult with respect to thyroid. Methods: Adult female rats were divided into control and experimental groups of six each. Control group was given normal diet while experimental group was fed BS by 1/3rd replacement of 180 g of their food i.e. 60 g of BS containing 35 g of goitrogens of cyanogenic origin such that each rat likely consumed 6 mg/100 g of body weight per day for a period of 45 d. Morphological features like changes in body weight and organ weight were noted. Key steroidogenic enzyme levels viz Δ5 3β hydroxysteroid dehydrogenase (HSD) and 17β HSD along with serum estradiol, estriol and progesterone levels were measured. Estrous cyclicity of the animals monitored regularly followed by histological analysis of thyroid, ovary and uterus at the end of experimentation. Results: Increase in body weight, thyroid gland weight and thyroid stimulating hormone, decrease in serum triiodothyronine and thyroxine, a decrease in ovarian as well as uterine weight and the activity of steroidogenic enzymes Δ5 3 β HSD and 17β HSD along with diminished serum estradiol, estriol and progesterone levels were noted; while histological plates showed prominent degenerative changes in both the ovary and uterus. Estrous cyclicity of the treated animals were irregular and almost stopped at diestrous stage of the cycle in the latter stage of the treatment as compared to control. Conclusions: Overall results indicates that BS rich in cyanogenic constituents induces biochemical hypothyroidism in the experimental animals that acts in corroboration to cause morphological and functional alteration of reproductive organs indicating its likely impact in fertility on continued use.

164-171

Authors: Maryam Owjfard, Manzarbanoo Shojaei Fard

Objective: To evaluate the effect of long-term exposure to radiofrequency radiations emitted from mobile jammers onrat's reproductive parameters. Methods: Eighty male rats were divided into neonatal group (n=20), immature group (n=30) and mature group (n=30). Neonatal rats were randomly divided into two subgroups. Immature and mature rats were randomly divided into three subgroups. Experimental rats were fixed in the restraining tube and were exposed to radiofrequency radiations emitted from mobile jammers at a distance of 100 cm, for 30 d (7 h/d). Sham group rats were also fixed in restraining tube but without radiations. The control group was allowed to move freely without being exposed to radiation. At the end of the experiment, weight of testes, sperm quality, histological testicular sections and serum testosterone levels were evaluated. Results: Long-term exposure to radiofrequency from mobile jammers could adversely affect neonatal rat fertility, but it did not have significant effects on male mature and immature rat's reproduction parameters. However, restraint stress induced by immobilizing them for a long-period could adversely affect male mature rat's reproductive parameters, but it had no significant effect on male immature rat's reproduction parameters. Conclusion: The effects of EMF exposure to be various based on the life stage.

176-180

Authors: Mohamed MM El-Sokary, Mohamed El-Raey, Karima GhM Mahmoud, Mahmoud EA Abou El-Roos, Gamal MS Sosa

Objective: To assess the effects of melatonin and/or cysteamineon in vitro maturation, culturing and post-warming of buffalo embryos. Methods: Buffalo oocytes were classified into control, cysteamine (50 μM), melatonin (10 ng/mL) and cysteamine (50 μM) + melatonin (10 ng/mL) treatment groups. In experiment 1, previous treatments were added during in vitro maturation and culturing of buffalo oocytes. Results: Cleavage and blastocyst rates were significantly (P<0.05) increased in melatonin treated group (70.5±0.9 and 12.8±1.0, respectively). However this effect was potentiated when combined with cysteamine (74.0±1.7 and 14.8±1.7, respectively). In experiment 2, the treatements were added in maturtaion, culturing as well as post-warming culture media. Embryos at 7 d were vitrified.Viability assessement directly after warming showed significant increase (P<0.05) in cysteamine, melatonin and their combination groups (76.8±2.8, 80.0±2.1 and 83.3±1.7, respectively) than control (65.8±2.4); but the viability after 24 h post-warming was the best in cysteamine + melatonin combination group (61.4±2.1). Conculsions: Enriching maturation, culturing and post-warming media of buffalo oocytes and embryos with melatonin and/or cysteamine have significantly beneficial effects on oocyte developmental competence as well as embryos vitrification procedure outcomes which in turn resulting in enhancement of commercial buffalo embryo production.

186-190

Authors: Kanoknan Somnuk, Pim Wannapakorn, Waraporn Raksapol, Bunlue Kornmatitsuk, Sudsaijai Kornmatitsuk

Objective: To establish fetal age equations based on ultrasound measurements in cross-bred Holstein cows in Thailand. Methods: The animals were bred for 30-120 d before pregnancy diagnosis using transrectal ultrasonography with real-time B-mode and specify 5 or 7.5 MHz linear array transducer. Parameters examined included crown rump length (CRL), trunk diameter (TrD) and eye diameter (ED). Results: One hundred and two cows had been examined, 80 of them were pregnant and contained at least one measurable characteristic as defined in the methods. Regression analysis and curve estimation were implied. The most frequently visualized parameters were TrD (42/80; 52.50%) and CRL (41/80; 51.25%) whereas ED (13/80; 16.25%) was least frequently detected. In our study, the equations to estimate fetal age (indicated as Y) from CRL and TrD were established with very high correlation coefficients as follows, Y = 22.679+12.005 (CRL)-1.042 (CRL)2, R2 = 0.950, P<0.001 and Y = 14.583+29.878 (TrD)-3.759 (TrD)2-0.225 (TrD)3, R2 = 0.950, P<0.001. The equation from ED was Y=107.582-98.928 (ED)+61.116 (ED)2-9.221 (ED)3 but with very low correlation coefficient (R2 = 0.673, P=0.021). Comparing between our equations (CRL and TrD) and the ones embedded in the commercially available ultrasounds, the higher fetal age was estimated based on the same value of parameters (P<0.05). In conclusion, the most reliable equations created in our study were from CRL and TrD. Conclusions: Comparing with others, Thai cross-bred fetus clearly showed slower growth rate and thus, to use our CRL’s and TrD’s equations for estimating fetal age seems more reliable than to use the ones embedded in the commercial ultrasounds.

104-111

Authors: Ali SAEED. H. Alchalabi, Erkihun Aklilu, Abd Rahman Aziz, Hasliza Rahim, Suzanna H. Ronald, Mohd F. Malek, Mohd Azam Khan

Objective: To evaluate the teratogenic effect of mobile phone radiation exposure during pregnancy on embryonic skeletal development at the common used mobile phone frequency in our environment. Methods: Sixty female Sprague-Dawley rats were distributed into three experiment groups; control and two exposed groups (1 h/day, 2 h/day exposure groups) (n=20/ each group) and exposed to whole body radiation during gestation period from day 1- day 20. Electromagnetic radiofrequency signal generator was used to generate 1 800 MHz GSM-like signals at specific absorption rate value 0.974 W/kg. Animals were exposed during experiment in an especial designed Plexiglas box (60 cm × 40 cm × 30 cm). At the end of exposure duration at day 20 of pregnancy animals were sacrificed and foetuses were removed, washed with normal saline and processed to Alizarin red and Alcian blue stain. Skeleton specimens were examined under a stereo microscope and skeleton's snaps were being carefully captured by built in camera fixed on the stereo microscope. Results: Intrauterine exposure to electromagnetic radiation lead to variation in degree of ossification, mineralization, formation of certain parts of the skeleton majorly in head and lesser in other parts. Deformity and absence of formation of certain bones in the head, ribs, and coccygeal vertebrae were recorded in skeleton of foetuses from exposed dams compare to control group. Conclusions: The electromagnetic radiation exposure during pregnancy alter the processes of bone mineralization and the intensity of bone turnover processes, and thus impact embryonic skeleton formation and development directly.

116-120

Authors: Reni Kurniati, Didik Huswo Utomo, Sri Rahayu, Widodo, Sutiman Bambang Sumitro

Objective: To evaluate the role of ZP3R in the species-specific fertilization mechanism. Methods: ZP3/ZP3R protein sequences of Mus musculus, Rattus norvegicus, and Cavia porcellus were downloaded from UNIPROT. Percentage of amino acids that was calculated by using the SIAS program. Protein sequences modeled was established by using the Modeller 9.14 program and glycosylation of the ZP3 using GlyProt program. Docking simulation of the ZP3R-ZP3 was performed between the same species and different species with PatchDock program. Results: Comparison of the ZP3R and ZP3 structure between species showed that ZP3 in these three species was more similar than ZP3R. Docking simulations of protein showed that changes in the pattern of the ZP3-ZP3R domain for interaction on cross-species compared to the same species. Changes in the pattern of binding ZP3R-ZP3 made spermegg binding was not functional and could inhibit cross-fertilization. Conclusions: ZP3R-ZP3 interaction is species-specific, and the role of ZP3R is greater than ZP3 in determining the species-specific recognition stage and sperm-egg binding.

128-132

Authors: El-Sheshtawy RI, El-Nattat WS

Objective: To explore the effect of Diospyros kaki on cattle spermatozoa during chilling and cryopreservation. Methods: Five milliliter of blended Persimmon (Diospyros kaki) flesh was added to 45 mL TCF to obtain 10% stock solution. Kaki enriched extender (KEE) was prepared by adding to TCF in concentrations 0.0/5.0 mL (control, 0%), 0.5/4.5 mL (1%), 1/4 mL (2%), 1.5/3.5 mL (3%), 2.0/3.0 mL (4%), 2.5/2.5 mL (5%), 3.0/2.0 mL (6%), 3.5/1.5 mL (7%), 4.0/1.0 mL (8%), 4.5/0.5 mL (9%) and 5.0/0.0 mL (10%) to obtain a final volume 5 mL in each tube. Whole egg yolk was added to each tube to obtain KEE with 20% egg yolk (KEEY), all tubes were centrifuged to get rid of debris. Semen was added to the supernatants in other tubes. Extended semen was subjected to evaluation (motility, alive sperm and intact sperm membrane (HOST) %) in both chilled and cryopreserved semen. Conception rate was carried out. Results: Sperm motility was significantly (P<0.000 1) kept high after 11 d of chilling with the concentration 1%, 2%, 3%, 4%, 5% as compared to the control (41.67±1.67, 41.67±1.67, 40.00±0.00, 41.67±1.67 and 41.67±1.67, respectively) and also non-significantly kept high at the other concentrations up to 9 d of chilling. Addition of KEE had significantly (P<0.003 3) improved post thawing sperm motility % with the concentrations 1, 2, 3, 4, 5 and 6% as compared to the control (51.67±5.27, 55.00±3.16, 48.33±1.05, 45.00±3.96, 57.00±2.50, 55.00±5.00 and 43.33±5.11 respectively).While the other concentrations exhibit no effect. Addition of KEE maintained alive sperm%, abnormalities% and % of intact spermatozoa membranes (HOST %) as good as the control with all concentrations of kaki used in our study. The conception rate upon using frozen semen in insemination showed higher conception rate in concentrations of 2%, 4% and 6 % KEE in cattle. Conclusion: It could be concluded that some concentrations of Diospyros kaki improved bull semen quality post-chilling and post-freezing.

136-139

Authors: Mohammad Rahim Ahmadi, Abdollah Derakhshandeh, Sadegh Shirian, Yahya Daneshbod, Maryam Ansari- Lari, Saeid Nazifi

Objective: To determine the possibility of presence of bacterial biofilm in the uterus of repeat breeder cows and to evaluate the effect of mucolytic agent in cleanup of uterus from biofilm. Methods: Twenty repeat breeder cows were selected from a large commercial dairy farm near Shiraz, Fars province, southern Iran. Uterine secretion samples were collected before and after uterine lavage with dimethyl sulfoxide (DMSO) 10% solution and periodic acid Schiff (PAS) staining was used to detect bacterial biofilm in uterine samples. After sampling, all cows were treated with two doses of PGF2α and intrauterine infusion of Cefquinome sulphate. Artificial insemination (AI) was performed after that. Results: Bacterial biofilms were found in 12 out of 20 animals (60%) in the first sampling with sterile saline lavage (before DMSO) and in 7 cows (35%) after DMSO lavage. Fourteen cows (70%) became pregnant after AI. This evidence showed the presence of bacterial biofilm in the uterus of dairy cows for the first time. Although non-significant, decrease in biofilm detection after DMSO lavage may suggest the potential ability of mucolytic agent for cleaning the uterus from bacterial biofilm. Also, high pregnancy rate after antibiotic treatment in the present study might be attributed to improved effect of antibiotic following lavage of uterine by DMSO. Conclusions: These findings should be investigated in future researches with more sample size.

144

Authors: Beuy Joob, Viroj Wiwanitkit

none

54-57

Authors: Dian Aby Restanty, Setyawati Soeharto, I Wayan Agung Indrawan

Objective: To prove the effect of administered orally lead acetate exposure on Bax expression and the apoptosis index of granulosa cells on antral follicle female albino rats Wistar strain (Rattus norvegicus). Methods: Post-test only control group, using female albino rats Wistar strain aged (10-12) wk (reproductive age) weighing (100-200) g. Twenty-four animal samples were classified into one control group and three groups exposed to lead acetate in doses of 30, 100, and 300 ppm, respectively. Lead acetate was administered orally through a feeding tube over 30 d. Bax expression of granulosa cells on antral follicle was checked using immunohistochemistry, and apoptosis index were examined using Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). Results: Lead increases apoptosis index on doses of 300 ppm. The increase of the Bax expression granulosa cell on antral follicle was not statistically significant. Conclusion: The exposure of lead acetate administrated orally can increase apoptosis index of granulosa cell on antral follicle although it doesn't affect Bax expression.

68-76

Authors: Islam El-Sayed El-Seadawy, Walid Said El-Nattat, Magda Mohamed El-Tohamy, Samy Ali Hussein Aziza, Yakot Abdelfattah El-Senosy, Ahmed Said Hussein

Objective: To maintain semen quality of male rabbits during chilled storage by enrichment the tris based diluent with different concentrations of propolis ethanolic extracts. Methods: Total phenolic and total flavonoid contents, as well as antioxidant activity was determined in propolis ethanolic extract (PEE). The extract was analysed by HPLC for separation and identification of target metabolites. Semen was collected from 10 rabbit bucks, pooled, then divided into five aliquots (each of 500 μL) and diluted each in 5 mL Tris-citric acid-glucose-egg yolk extender (TCGY). The 1st aliquot served as control while PEE was added at concentration of 0.8, 1.2, 1.6 and 2.0 mg/5 mL tris extender in the aliquot 2, 3, 4 and 5 respectively. Diluted semen samples were subjected to cooling at 4 ℃ for 72 h. Sperm motility, sperm viability, sperm abnormality, sperm membrane integrity and acrosome integrity were evaluated in chilled semen allover the chilling period. Results: The resluts revealed presence of a considerable amount of total phenolic compounds (98.67 mg GAE/g extract) and total flavonoids (70.16 mg CE/g extract) which were parallel to an antioxidant activity assessed as ABTS, DPPH and FRAP (198.65, 180.18 and 306.17 mM TE/g extract respectively). The dominant phenolic acid was chlorogenic acids (3.959 mg/g extract). Other compounds were found in less amounts rosmarinic acid (3.959 mg/g extract), myrcetin (1.946 mg/g extract), kaempferol (1.089 mg/g extract) and apeginin-7-glucoside (1.113 mg/g extract). Obtained results clearly demonstrated that the addition of (1.2-1.6) mg PEE in the chilled extended rabbit semen proved to be beneficial for maintaining semen characteristics compared to control and the addition of 0.8 and 2 mg PEE. Conclusions: The enrichment of rabbit semen tris-basic extender with (1.2- 1.6) mg PEE/5 mL tris-extender (as the best and safe concentrations) maintain the sperm characteristics in good condition all over 72 h of chilling.

81-84

Authors: El-Sheshtawy RI, El-Nattat WS

Objective: To explore the effect of silymarin on bull spermatozoa during cooling and cryopreservation. Methods: Pooled bull semen were diluted by Tris-Citrate-Fructose egg yolk diluents, purified silymarin powder (obtained from the milk thistle silybum marianum), purchased from Unipharma, Al Obour city, Egypt, was soaked in Tris-citric acid-fructose diluent for 48 h at 10 ℃ making a stock solution (70 mg/ mL), from this stock solution we obtained concentrations of 0.18 mg/mL, 0.36 mg/mL, 0.54 mg/mL, 0.72 mg/mL, 0.90 mg/mL in addition to the control (0.0 mg/mL) reaching a final volume of 5 mL in each tube. Egg yolk was added to each tube to obtain silymarin enriched semen extender (SEE) with 20% egg yolk, cooled slowly up to 5 ℃ and equilibrated for 4 h. Semen was packed into 0.25 mL polyvinyl French straws (IMV, France). After equilibration periods, the straws were placed horizontally on a rack and frozen in a vapor 4 cm above liquid nitrogen for 10 min and were then dipped in liquid nitrogen. Extended semen was subjected to evaluation (motility, alive %, abnormality %, intact sperm membrane (HOST)% and conception rate) in both chilled and frozen semen. Results: Table 1 revealed that Sperm motility of the concentrations 2, 3 and 4 after 8 d of chilling were significantly (P<0.02) higher than control. Sperm motility of the concentration 2 (45.00%±2.89%) after 9 d of chilling was higher than control and the other concentrations. Addition of SEE in concentration 1 and 2 gave post thawing sperm motility as high as the control (47.50±2.81 and 45.00±2.58, respectively) while other concentration have lower effects on motility as compared to the control. Addition of silymarin improved post thawing alive% and was significantly higher (P<0.0 001) than the control. SEE decreased significantly (P<0.0 001) the % of post thawing abnormal sperm in concentration 3 and 4 (11.83±0.65 and 16.00±0.58, respectively). SEE improved significantly (P<0.018) the % of post thawing intact spermatozoa membranes (HOST%) in concentrations 2, 4 and 5 (71.17±0.83, 71.83±0.91 and 75.00±3.42 respectively) (Table 2). Conclusion: It could be concluded that silymarin as a natural additive to semen extenders improved preservability in both chilled and frozen bull semen.

89-92

Authors: Samaher Alfaraj, Fatima Alzaher, Sahar Alshwaiaer, Anwar Ahmed

Objective: To compare the pregnancy rate between day 3 and day 5 transfer regardless grades of embryos and number of transferred embryo. Methods: Retrospective cohort, a total of seven hundred and four patients met our inclusion criteria, with 411 had day 3 embryo transfer and 293 had day 5 embryo transfer. The patients who were older than 40 years old were excluded. Embryo transfer was carried out in all patients in both transfer groups. Results: Both clinical pregnancy rate and implantation rate did not show any statistically significant difference between the day 3 and day five transfer groups. These were 44% vs. 45% with P=0.82 and 19% vs. 19% with P=0.99 respectively. An increase of miscarriage rate with day 5 embryo transferred compare with day 3 (12.0% vs. 4.4%, P=0.01), but no significant difference was found about biochemical pregnancy rate (P=0.52). Conclusions: Transferring embryo at day 5 may not provide any additional benefit over day 3 transfers to patients. In addition, it increases the risk of miscarriage. Further studies of this issue needed for confirming our findings.