Asian Pacific Journal of Reproduction

Pages 1-12

Authors: P Perumal, JK Chamuah, AK Nahak, C Rajkhowa

Objective To assess the effect of melatonin (MT) at different seasons of the year on sperm motility, viability, total sperm abnormality, acrosomal membrane, plasma membrane and nuclear integrity, antioxidant profiles such as superoxide dismutase (SOD), catalase (CAT), glutathione (GSH) and total antioxidant capacity (TAC), enzymatic profiles such as aspartate amino transaminase (AST), alanine amino transaminase (ALT) and biochemical profiles such as malondialdehyde (MDA) production. Methods Total numbers of 80 ejaculates (20 ejaculates in each season) were collected twice a week from mithun bulls and semen was split into five equal aliquots, diluted with the tris egg yolk citrate (TEYC) extender. Group 1: semen without additives (control), group 2 to group 5: semen was diluted with 1 mM, 2 mM, 3 mM, and 4 mM of melatonin, respectively. These seminal parameters, antioxidant, enzymatic and biochemical profiles were assessed at liquid storage of mithun semen (5 °C). Simultaneously, retrospective study was conducted on birth rate of calf at different seasons from 2002 to 2012 from farm birth register of mithun farm. Results Inclusion of melatonin into diluent resulted in significant (P<0.05) decrease in percentages of dead spermatozoa, abnormal spermatozoa and acrosomal abnormalities at different seasons of the year as compared with control group. Additionally, spring season has highest seminal and antioxidant profiles followed by autumn and winter season, whereas lower values were observed in ejaculates collected from summer season. Similarly retrospective study revealed that highest birth rate was in winter followed by autumn, spring and summer season and breeding was occurred in spring, winter, summer and autumn season, respectively with gestation period of 270–290 days. Conclusions The result of present study indicates that the melatonin protects seminal and antioxidant profiles varied in different seasons, semen quality also varied from different seasons and was highest in spring and lowest in summer season. It was concluded from the present study that breeding takes place throughout year but the peak breeding season is from November to April (spring and winter season) in mithun.

Pages 17-21

Authors: Andrey Lyashenko

Objective To improve the indicators of motility, survival and fertilizing ability of spermatozoa by optimizing temperature factors and the duration of exposure at unfreezing straws. Methods Straws by volume 0.25 mL were thawed at water bath temperatures at 65 °C, 67 °C and 70 °C for 6–7 seconds and at 75 °C for 4–6 seconds. Impact of exposure time and temperature thawing in the water bath on motility and survival of spermatozoa were studied. Results Studies indicate that for the procedure of defrost water bath straws in seven seconds for temperature conditions of 65 °C, 67 °C and 70 °C, indicators of progressive motility and absolute survival rate were significantly higher than for the control group an average on 11.4 % (P<0.01). Optimum exposure time (6–7 seconds) and temperature range (65–70 °C) defrosting semen doses were defined. Conclusions Owing obtained the positive result, method of thawing was developed which increases the indicators of motility, survival and fertilizing capacity of bull semen.

Pages 26-31

Authors: Reda I. El-Sheshtawy, Gamal A. Sisy, Walid S. El-Nattat

Objective To examine the effect of different concentrations of trehalose or sucrose (50 or 100 or 200 mM) on post-thawed quality of bull semen, cryo-preserved in Tris-citric acid-egg yolk-fructose (TCYF). Methods Semen samples were diluted in TCYF extender, TCYF + trehalose (50, 100 and 150 mM/L) or TCYF + sucrose (50, 100 and 150 mM/L) to ensure 60 million motile spermatozoa mL-1, cooled slowly up to 5 °C and equilibrated for 4 h. Semen was packed into 0.25 mL polyvinyl French straws. The straws were placed horizontally on a rack and frozen in a vapor 4 cm above liquid nitrogen (LN2) for 10 minutes then dipped in liquid LN2. Frozen straws were thawed at 37 °C for 1 min. The parameters studied were sperm motility, sperm viability, sperm abnormality, sperm membrane integrity (HOST), percent of normal intact acrosome and DNA fragmentation. Results The output data demonstrated that addition of 50–100 mM of trehalose or sucrose/L TCYF after chilling at 5 °C had significantly (P<0.0001) ameliorated motility, membrane integrity, viability, abnormal morphology, and acrosome integrity % compared to control diluted semen while 50 mM of trehalose/L, and 50–100 mM of sucrose/L to TCYF diluent had significantly (P<0.0001) improved after thawing motility (43.00,% 45.00% and 41.00%, respectively), membrane integrity (67.40%, 67.80% and 69.40%, respectively), life sperm % (70.20%, 69.40% and 71.40% respectively), and acrosome integrity percentages (56.40%, 58.80% and 55.80% respectively) compared to the control tris-base diluent, while diminishing the abnormal sperm morphology (6.20, 3.80 and 3.80 respectively) and DNA fragmentation (3.60%, 3.80% and 3.80% respectively). Besides, the addition of 100 mM of trehalose/L to tris-base diluent has also a promising effect when added to the tris-base diluent concerning the above parameters. Conclusion It is finally concluded that the addition of 50–100 mM trehalose or sucrose/L to TCYF have a beneficial effect in chilling diluted bull semen, while the use of 50 mM trehalose or 50–100 mM sucrose had their benefits on freezing-thawing of extended semen.

Pages 37-40

Authors: Augustave Kenfack, Ferdinand Ngoula, Paul Désiré Womeni Dzeufiet, Omer Bébé Ngouateu, Tsambou Megnimeza Astride Martine, Judith Kegne Chombong, Guylène Marie Zambou Zeukeng, Isabelle Leinyuy Nyuysemo, Arthenice Jemima Noumamo Guiekep, Tah Patience Nain, Pierre Kamtchouing, Joseph Tchoumbou, Narcisse Bertin Vemo

Objective To study the effects of chlorpiryphos-ethyl (CE) on male fertility. Methods Rats were gavaged daily from 30 to 120 days old with four doses of CE (10.50; 5.25; 3.50 and 0.00 mg/kg) and thereafter followed up for 90 additional days. Following this period of observation, each male rat was submitted to a fertility test with two virgin females. Male were then sacrificed and organs collected while females were followed up till delivery. Results The weights of the testis and epididymis were lower (P<0.05) in rats treated with 10.50 mg/kg CE dose compared to controls. A decrease in the number of sperm per cauda epididymis was recorded in rat given the highest dose of CE with respect to the control value. The sperm motility was lower (P<0.05) in all CE-treated groups compared to the controls. Severe histological abnormalities were observed in testes of treated rats. The males exposed to the highest dose of CE did not produce any offspring. Conclusion The effects of CE on reproductive system were persistent 90 days after the end of exposure.

Pages 44-48

Authors: Ibrahim A. Abdelazim, Walid Farok Elsawah

Objective To detect the prevalence of metabolic syndrome (MS) among infertile women with polycystic ovary syndrome (PCOS). Methods Two hundred and twenty infertile PCOS women were included in this prospective cross section study. Diagnosis of PCOS was based on at least two of ESHRE/ASRM criteria and diagnosis of MS was based on at least three of NCEP ATP III criteria. A standard questionnaire was used to document length of menstrual cycles, personal and family history of medical disorders. Signs of androgen excess and insulin resistance were noted in the physical examination. Anthropometric measurements were done to measure waist circumference, hip circumference and body mass index (BMI) was calculated. Overnight fasting blood sample and a 75 gram oral glucose tolerance test, TSH, prolactin, total testosterone, SHBG and lipid profile levels were evaluated in all studied PCOS women. Trans-vaginal ultrasound was also done to measure; ovarian volume and number of follicles in both ovaries. Results The prevalence of MS in studied PCOS women was 30.5% (67/220). There is strong positive correlation between prevalence of MS and both age and BMI of the studied PCOS women. Logistic regression analysis showed that; the age > 25 and waist-hip ratio ⩾ 0.85 were powerful predictors for the prevalence of MS in PCOS women. Conclusion The prevalence of MS was 30.5% in the studied PCOS women. The age above 25 years and waist-hip ratio ⩾ 0.85 were powerful predictors for prevalence of MS in PCOS women.

Pages 54-60

Authors: Abdelhanine Ayad, Mourad Salaheddine, Kamal Touati, Mokrane Iguer-Ouada, Hama Benbarek

Objective To evaluate the effectiveness of norgestomet Crestar® for synchronization of oestrus and its effect on pregnancy rate in cows. Methods after a waiting period (∼90 days postpartum), Holstein-Frisian females kept in different dairy farms were inseminated for the detection of estrous behavour (n =133). Females were allocated randomly to treated (n =69) or control (n =64) groups. The treated animals received the Crestar® treatment during 9–10 days. After implant removal, the treated animals were artificially inseminated (AI) for the detection of standing estrous behaviour. Blood samples were collected on Days 0, 21 and 30 after service. Plasma progesterone (P4) and pregnancy-associated glycoprotein (PAG) levels were determinate by radioimmunoassay. Results the study showed a higher rate of heat synchronization in the treated group (94%) compared to the control group (86%). Out of 133 cows inseminated, thirteen (4 treated and 9 control) had a plasma progesterone concentrations higher than 1.0 ng/mL at service. The plasma PAG levels at Day 30 after AI were significantly lower in the treated group (7.13 ± 2.29) compared to the control group (8.37 ± 2.30). Pregnancy rate was 28% (16/57) and 42% (23/54) for treated and untreated group, respectively. There were no significant differences in the pregnancy rates of different parity in both groups. Conclusion The norgestomet Crestar® treatment carried out during 9–10 days did not improve reproductive performance in dairy cattle. However, these data demonstrate that this hormonal protocol can be used to obtain high oestrus cow rates to initiate correct timing of insemination.

Pages 65-69

Authors: R Asadpour, MH Asadi, R Jafari-Joozani, GH Hamidian

Objective To use PCR to investigate the presence of fetal SRY gene in the ovine cervical secretions and maternal blood plasma, and to assess predict fetal sex at different times of gestation in the ewe. Methods Fetal DNA was isolated from blood plasma and cervical secretions of 32 pregnant ewes during the 6 to 21 week of gestation. Overall, 15 male and 17 female fetuses were included in this study. After DNA extraction, the PCR amplified a 280 bp fragment from the X-chromosome and a 217 bp fragment from the Y-chromosome based on a sex-related polymorphism in the amelogenin locus. Results The presence of fetal Y-chromosome was confirmed in 11 out of 15 cervical mucus and 12 out of 15 blood plasma samples collected from sheep with male fetuses. The sensitivity and specificity of tests were 70% with false negative results. Conclusion This is the first report on validating the presence of fetal DNA material in the ovine cervical mucus and its potential usefulness for fetal sexing.

Pages 74-77

Authors: JE Rodríguez, A López, Instituto Sexológico Murciano

Objective To determine if they can produce increases in the IELT using a masturbator in subjects with premature ejaculation, producing improvements in the quality and satisfaction of the sexuality of the patient. Methods To carry out the study it will arrange of the utilization of a masturbation device aid for six weeks that they will use at least during 5 minutes without overcoming the half daily hour in the manual masturbation. The measures of results will be the differentiation of the basal and final IELT by means of the average of both results, obtaining the differential percentage of the above mentioned measures. Results There was obtained a sample of 9 heterosexual participants of ages included between 20 and 42 years. The time of latency increased from the first week of treatment producing up to 57 % of increase in the IELT in the masturbation and up to 79.9 % of improvement during the coitus. Conclusions The use of a masturbation aid (FLIP HOLE) has shown to be a useful tool in increase of the ejaculatory latency in the masturbation and intravaginal, that accompanies of an improvement in the communication of couple, frequency of coitus and sexual satisfaction, and could be an effective option for the treatment of E.P.

Pages 83-84

Authors: Sora Yasri, Viroj Wiwanitkit

The spermatogenic transmission of infectious disease is an interesting consideration in reproductive medicine. The problem can be serious and classified as sexually transmitted infection. The concern is on the new emerging viral infections because there is usually little information on those new viruses. In this short article, the authors specially review and discuss on Spermatogenic transmission of Marbug and ebola virus.

253-259

Authors: Jillian L. Sia Aleli, Gliceria B. Ramos, Miriam P. de Vera

Objective To assess the protective potential of α-tocopherol on ethanol-induced dysmorphogenesis in 10.5 embryonic day (ED) mouse embryos. Methods Forty female mice were randomly assigned into control (CON), positive control (ETOH), low-, medium and high-α-tocopherol-supplemented-Ethanol groups (LTOC, MTOC, HTOC, respectively). CON received drinking water without ethanol, ETOH, LTOC, MTOC and HTOC groups received 20% ethanol in drinking water. The supplemented groups were given respective dosages of α-tocopherol, 0.410, 0.819 and 1.640 mg/g body weight, at 14 days before mating until the 9th day of gestation. The 10.5 ED embryos were assessed for embryo weight, head- and crown-rump length, and morphological scoring of brain and sensory vesicles, flexion and somites. The embryo yield was assessed by counting the number of full-term developed embryos from the bulging implantation sites while resorption was assessed by counting the bulging implantation sites but without formed embryos. Results The weight and head- and crown-rump length of the embryos from the α-tocopherol supplemented groups were comparable to the control. These were significantly higher than that of positive control (P < 0.05). Overall morphological scores of the hindbrain and sensory vesicles were significantly higher in the supplemented and control groups than that of the positive control (P < 0.05). The number of full-term developed embryos was neither affected by ethanol alone nor with supplementation with α-tocopherol. Resorption was significantly lower in the supplemented groups than that of positive control (P < 0.05). Conclusion The medium and high dosages of α-tocopherol exhibited a protective effect on ethanol-induced dysmorphogenesis.

264-268

Authors: Deepika Jaiswal, Sameer Trivedi, Neeraj K. Agrawal, Kiran Singh

Objective To analyze global DNA methylation along with DNA methyltransferases (DNMTs) expression at transcript level in patients with impaired spermatogenesis to dissect its role in pathophysiology of human male infertility. Method The content of Global methylated cytosine (mC) was determined using ELISA system (Imprint Methylated DNA Quantification Kit, Sigma–Aldrich) in 31 testicular biopsies showing impaired spermatogenesis and 8 with normal spermatogenesis. Real-time reverse transcription-polymerase chain reaction was done to analyze DNMTs (DNMT1, DNMT3A, DNMT3B and DNMT3L) mRNA levels in biopsies with different testicular phenotypes. Results There was significant increase in levels of global methylation in different impaired testicular phenotypes as compared to normal. Expression analysis revealed significantly increased expression of DNMT1 and its positive correlation with global DNA methylation. Conclusion In conclusion, increased levels of global methylation in impaired cases might be the one of the contributing factors for aberrant gene expression in infertile patients.

275-287

Authors: S. Thangapandiyan, S. Miltonprabu

Objective To explore the ameliorative potential of Epigallocatechin gallate (EGCG) by evaluating markers of oxidative stress, apoptosis, and inflammation and antioxidant competence in Fl intoxicated rats. Methods The animals were divided in to four groups that is control, EGCG alone, NaF, and EGCG with NaF. Group III animal were exposed to Fl as sodium Fluoride (NaF) (25 mg/kg BW) for 4 weeks. After the completion of the treatment, the testis tissues has been removed and used for the experimental observations. Results Pre-administration of EGCG to Fl intoxicated rats showed a significant normalization in the levels of steroidogenic enzymes, testosterone, sperm functions, oxidative stress markers and antioxidant status. The altered levels of proinflammatory cytokines and apoptotic markers were also relapsed in close proximity to control. In addition, EGCG significantly improved antioxidant status and reduced the oxidative stress and pathological changes in testes. The mRNA and protein analysis also substantiated that EGCG pre-treatment markedly enhanced the expression of Nrf2 and its target genes HO-1, NQO1 and γGCS and suppressed the expression of Keap1 in testis. Conclusion Altogether, our findings supports that EGCG attenuates Fl toxicity in testis through Nrf2 activation.

297-304

Authors: Gulfam Ahmad, Muhammad Arslan

Objective The present study was undertaken as a part of a continuing search for proteomic based approaches for the diagnosis of ovarian cancers. Methods The study comprised four groups of women with: i): malignant ovarian cysts, ii) benign ovarian cysts, iii): breast cancer (positive controls) and, iv) healthy females (negative controls). Serum and cystic fluids were processed for gel electrophoresis (SDS-PAGE). Serum and cystic follicular fluid estradiol and testosterone concentrations were measured by ELISA. Results Proteomics analysis revealed the presence of a uniquely expressed protein with MW of 35 kDa in the serum and cystic fluid of patients with malignant ovarian cysts. On the other hand a protein of 100 kDa was not expressed in malignant ovarian patients whereas it was differentially expressed in the cystic fluid and serum of other three groups. Although mean values of estradiol levels were discernibly higher in patients with benign ovarian cysts as compared to those with malignant ovarian cysts and of positive and negative controls, difference was not significant. Mean concentrations of estradiol were significantly higher in cystic fluid aspirated from benign cysts as compared to that from malignant cysts. Conclusions Taken together these data indicate a uniquely expressed protein of 35 kDa in patients with malignant ovarian cysts that may serve as a specific protein biomarker for the differential diagnosis of the ovarian cancer. However, a much larger sample of subjects is required to validate and confirm these findings.

309-312

Authors: N. Faria, J. Simões

Objective To determine the incidence of uterine torsion and their association with live births after vaginal delivery at pasture. Methods A total of 119 veterinary-assisted dystocia, occurred in Holstein-Friesian cows, were accessed between September 2012 and February 2013 from Azores islands (Portugal). The general health status of dam, cause of dystocia, obstetric treatment choice, number and viability of fetus were evaluated. Results The uterine torsion represented 24.4% (29/119) of total assisted dystocia. The general health status was normal or slowly affected, at delivery time, for 96.6% (28/29) of the cows with uterine torsion. Vaginal delivery after rolling cows, cesarean section or euthanasia were performed in 72.4% (21/29), 24.1% (7/29) and 3.5% (1/29) of dams with uterine torsion, respectively. Singletons were observed in 96.6% (28/29) and 86.7% (78/90) of cows with uterine torsion and remaining dystocia, respectively. After vaginal delivery, the occurrence of singleton live births was more probable to occur in cows with uterine torsion (17/21) than the remaining cows (37/70) of control group (odds ratio = 3.79; 95% interval of confidence from 1.16 to 12.41; P < 0.05). Conclusions A high frequency of uterine torsion was observed in Holstein-Friesian cows with normal or slowly affected general health status at delivery time at pasture. The singleton live births prevailed and, in general, their occurrence by vaginal delivery after uterus reposition were most likely to occur in cows with uterine torsion than dams presenting other dystocia.

318-322

Authors: J. Silva, D. Rocha, I. Cunha, L. Rui Sales, F. Neto, M.C. Fontes, J. Simões

Objective To evaluate/predict the offspring serum profile of antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV) in an affected intensive herd, whereas sows are vaccinated, and determine the better sample time using pigs as sentinels. Methods Serum samples were collected from a total of 66 offspring pigs from a PRRSV endemic herd, whereas only sows were vaccinated. Six animals per grouped age were randomly selected with 0 (at perinatal time), 1, 2, 3, 6, 9, 12, 15, 18, 21 and 24 weeks of age. Individual and three pooled samples were tested by ELISA and PCR, respectively. Results The proportion of seropositive animals was 75.0% (18/24), 33.3% (6/18) and 95.8% (23/24) at farrowing (0–3 weeks), nursery (6–12 weeks) and growing/finishing (15–24 weeks) phases, respectively. It was 46 times more likely (P < 0.01) to observe seropositive pigs on the growing/finishing than on nursery phase. European PRSSV strain was detected on serum of 6, 9 and 15-weeks-old pigs. Polynomial fit degree 4 regression between sample-to-positive (S/P) ratios and age of pigs (r = 0.78; R2 = 0.60; P < 0.001) estimated a quickly decrease of maternal antibodies, above 0.4 S/P value (cut off), until the 6th week following by a progressive increment of humoral immunity until 21st week of age, when it declines. Conclusions The results indicate that the piglets can present viremia early at nursery, and the 18th – 21st weeks are the best time to sample offspring pigs, as sentinel, from PRRSV contaminated intensive herds.

334-336

Authors: Achour Radhouane, Ben Aissa Imen, Neji Khaled

Twin pregnancy consisting of a complete hydatidiform mole coexisting with a live fetus is a rare condition with an incidence of 1 in 22 000 to 1 in 100 000 pregnancies. Clinical information is limited and management is difficult due to the risk of pregnancy complications such as fetal death, vaginal bleeding, preeclampsia, hyperthyroidism, and the risk of persistent gestational trophoblastic disease. Thus, the report described about the change of size and implantation site of the molar mass coexisting with a live fetus is rare especially about sonographic findings such as echo patterns. Recently we experienced a case of complete hydatidiform mole with a healthy infant delivered at term.

Pages 169-177

Authors: Ansoumane Kourouma, Peng Duan, Hady Keita, Aidogie Osamuyimen, Suqin Qi, Chao Quan, Tingting Yu, Kedi Yang

Objective To explore the mechanism by which nonylphenol (NP) interferes with male infertility through evaluation of its effects on epididymal sperm of adult male rats. Methods Twenty four Sprague–Dawley (SD) rats were used as epididymal sperm donors. Previously rats were administrated with NP (0, 2, 10 and 50 mg/kg) body weight respectively in corn oil every forty-eight hours by intraperitoneal injection for 30 days. Computer assisted sperm analysis (CASA) was used to determine parameters of sperm. The sperm morphology examination was conducted with a high resolution microscope. Results Results indicated that exposure to NP has no effect on body weight, while testes weights were significantly decreased. Computer assisted sperm analysis (CASA) showed significant decline in the percentage of motile spermatozoa (P < 0.001), STR and LIN (P < 0.01), significant increase in ALH (P < 0.001), while significant decline in BCF (P < 0.001) respectively. Plasma LDH was significantly increased while; plasma γ-GT activity was significantly decreased. H2O2 production and malondialdehyde (MDA) were significantly increased. The Plasma CAT, GSH-Px and SOD activities were significantly decreased. Conclusions This concludes that NP leads oxidative stress in the epididymal sperm of rats. Moreover, NP can disrupt sperm motility and alterations in the sperm morphology.

Pages 185-191

Authors: Nishant Shinde, Akhilesh S. Chauhan, Sanjay K. Gupta, Surendra H. Bodakhe, Devi Prasad Pandey

Objective To evaluate the antifertility potential of curcumin and andrographolide combination in female rats by studying its effect on implantation and estrus cycle. Methods Female Sprague Dawley rats (180 ± 30 g) were used for study. They were divided into groups and were treated as per schedule. The antifertility effect of curcumin and andrographolide was evaluated by measuring their effect on implantation and estrus cycle of rats. Also their antigonadotropic and antiestrogenic effects were evaluated using suitable screening method. Results The combination significantly reduced the number of implants and the size of the litters in rats compared to the normal control group. The combination also significantly altered the durations of each phase of estrus cycle and synergized the effect to decrease the number of ovarian follicles. Conclusion The results indicate that the combination produces greater antifertility effects and thus can play a vital role in fertility control.

Pages 198-204

Authors: Abhinandan Ghosh, Kishalay Jana, Bhabani Prasad Pakhira, Adrija Tripathy, Debidas Ghosh

Objective To explore the anti-fertility efficacy of aqueous-ethanolic (1:1) extract of fruits of Terminalia chebula (T. chebula). Methods Aqueous-ethanolic (1:1) extract of fruit of T. chebula was administered orally at a dose of 60 mg/0.5 mL distilled water/day for 28 days. Different parameters were studied including body weight, relative weight of reproductive organ, sperm motility, sperm count, testicular cholesterol, plasma testosterone, testicular androgenic key enzymes such as Δ5, 3β-HSD and 17-β-HSD, bio-markers of oxidative stress, toxicity study and histological analysis of the tissues. Results The treated group showed a significant diminution in spermatogenic profile. On the other hand testicular cholesterol showed a significant elevation in T. chebula treated group and plasma testosterone was decreased significantly in comparison to control. The above said androgenic key enzymes were exerted a significant diminution in extract treated group. Anti-oxidative enzymes such as catalase and superoxide dismutase showed a significant reduction and a significant elevation in conjugated diene and Thiobarbituric acid reactive substance was noted in treated group. GOT and GPT study of liver and kidney showed a non-significant change which confirmed the non-toxic nature of T. chebula. Histological study of testis of treated group exhibited significant reduction in seminiferous tubular diameter. Conclusion The results of present experiment suggested that the aqueous-ethanolic (1:1) extract of fruit of T. chebula exerted a significant anti-spermatogenic effect in male rat.

Pages 216-220

Authors: Wei-Siong Lim, Mary-Shi-Ying Gan, Melissa-Hui-Ling Ong, Choy-Hoong Chew

Objective To investigate the effect of different concentrations of lauric acid on Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) expression in IFN-γ stimulated human monocytic THP-1 cell line. Methods THP-1 cell were cultured using Roswell Park Memorial Institute medium supplemented with 10% fetal bovine serum. THP-1 monocytes were firstly differentiated into macrophages by using phorbol-12-myristate-13-acetate. IFN-γ response test was performed and total cellular RNA was extracted using TRI Reagent® LS before q-RT-PCR was carried out. Subsequently, IFN-γ treated THP-1 macrophages were stimulated with increasing doses of lauric acid for another 24 h, before q-RT-PCR. Results The mRNA expression levels of ICAM-1 and VCAM-1 were normalized to β-actin and relatived to the untreated cells. The expressions of ICAM-1 and VCAM-1 were significantly induced in cells treated with 10 ng/mL of IFN-γ. This showed that IFN-γ could up-regulate inflammatory process and may cause atheroma formation. MTT assay was carried out to investigate the effect of lauric acid on undifferentiated and differentiated THP-1 cells. Although lauric acid did not have any significant impact on undifferentiated and differentiated THP-1 cell viability, the normalized fold expressions of ICAM-1 and VCAM-1 in IFN-γ-treated THP-1 macrophages were decreased significantly in a dose dependent manner with the presence of increasing doses of lauric acid. Conclusions This study successfully proved that lauric acid was able to antagonize the up-regulatory effect of IFN-γ on ICAM-1 and VCAM-1 expressions in THP-1 macrophages. This indicates that lauric acid may be an anti-inflammatory therapeutic and prophylaxis agent for atherosclerosis.

210-215

Authors: Ibukun P. Oyeyipo, Yinusa Raji, Adeyombo F. Bolarinwa

Objectives To evaluate the effects of inhibiting nitric oxide synthase as a means of intervention in nicotine-induced infertility in male rats. Methods Forty-eight male and thirty female Wistar rats (180–200 g) were randomly assigned to six groups and treated orally for 30 days with saline (control), nicotine (0.5 mg/kg, 1.0 mg/kg) with or without NG Nitro-l-Arginine Methyl Ester (l-NAME, 50 mg/kg). Treated male rats were cohabited with untreated females in ratio 1:2 for fertility studies. Sperm analysis was done by microscopy. Results There was a significant decrease in the epididymal sperm motility and count after nicotine treatment. However, the percentage of abnormality significantly increased in nicotine treatment groups. Fertility studies revealed that nicotine reduced libido in male rats and decreased litter weight and number delivered by the untreated female during the experiments. Co-treatment with l-NAME effectively reversed the nicotine-mediated alterations in the sperm functional parameters, fertility indexes and hormone when compared to nicotine only. Conclusion Taken together, the present data indicate the abilities of l-NAME to ameliorate nicotine-induced spermatotoxic effects in male rats via a mechanism dependent on the circulating testosterone level.

228-235

Authors: Boubakari Ibrahimou, Hamisu M. Salihu, Muktar H. Aliyu, Gary English, Getachew Dagne

Objective To assess whether conversion from twin to singleton pregnancy following the demise of a co-twin influences survival. Methods This retrospective study compared the risk for neonatal, post-neonatal and infant death for converted co-twins versus unconverted co-twins using the US matched multiple file dataset for the period 1995–2000. We also examined the same risks for converted versus same-quantile co-twins, hazard ratios (HR) and 95% confidence intervals (CI) were computed using Cox Proportional Hazards models. Results The risk for neonatal (HR = 0.18, 95% CI: 0.09–0.34 and HR = 0.69, 95% CI: 0.50–0.96) and infant death (HR = 0.22, 95% CI: 0.12–0.42 and HR = 0.57, 95% CI: 0.42–0.77) were significantly lower for converted twins than for unconverted twins and same-quantile twins, respectively. For black compared to white, the risk for post-neonatal death increased by 89% (HR = 1.89, 95% CI = 1.03, 3.48), and 79% (HR = 1.79, 95% CI = 1.53, 2.09) for converted vs. unconverted and converted vs. same-quantile, respectively. For converted black, the risk for neonatal death decreased by 17% (HR = 0.83, 95% CI = 0.73–0.93) as compared to unconverted. Conclusions Risks for all mortality types were lower among converted co-twins than their unconverted or same-quantile counterparts. The lower neonatal and higher post-neonatal mortality among black require future research.

242-243

Authors: Natalie K. Binder, Natalie J. Hannan, David K. Gardner

Objective To determine the efficacy of embryo outgrowth on fibronectin as a low cost, high throughput alternative to embryo transfer to model embryo attachment and the initial stages of implantation. Methods Following in vitro embryo culture, embryo quality was assessed via embryo transfer or embryo outgrowth with metabolic assessment. Results This study shows that blastocysts attach to fibronectin at the same rate that they implant in vivo, and that the carbohydrate utilisation of embryos that successfully outgrow is comparable to those that are able to develop into a fetus. Conclusions Embryo outgrowth is a suitable alternative endpoint to embryo transfer.

251-252

Authors: Achour Radhouane, Basly Mohamed, Ben Aissa Imen, Ferjaoui Aymen, Neji Khaled

Congenital cervical atresia and hypoplasia are rare abnormalities that generally require reconstructive or extirpative procedures to relieve outflow tract obstruction. Infertility is a common sequel, and only four previous pregnancies have been reported. We report a case of successful pregnancy after in-vitro fertilization in a 32-year-old patient with congenital cervical atresia diagnosed at the age of 28 years. She was referred to our unit and had a successful pregnancy after in-vitro fertilization. Caesarean section was performed at 38 weeks gestation. A healthy male baby weighing 3650 g was safely delivered.

Pages 1-7

Authors: Roberto Puglisi, Anna Pozzi, Roberta Vanni, Donatella Balduzzi, Irina Lagutina, Silvia Colleoni, Giovanna Lazzari, Cesare Galli, Graziella Bongioni, Andrea Galli

Objective To investigate the telomere length in bovine offspring produced by a cloned and control bull, and the telomerase activity in embryos produced with the same technology. Methods Five daughters of a control and five daughters of a bull cloned using a fibroblast of the control were produced by IVF using sperm of the two bulls. Blood samples of the offspring were collected at 2, 6, and 12 months of age and the relative telomere length (RTL) was assessed by flow cytometry. At same time the body growth, hematological profile, and clinical biochemistry of the same progeny was extensively surveyed, and results have been reported in a previous work. Thereafter, the telomerase activity was assessed using a real time PCR quantitative assay in groups of embryos produced with the same technology. Results The offspring of the clone exhibited a modest, but significant (P<0.05), shortening of the telomeres (21.36%, 20.56% and 20.56%) compared to that of the control (23.78%, 23.53% and 22.43%) as mean values determined at 2, 6 and 12 months, respectively. Shortening of telomeres in respect to the age was not significant. No statistical difference was reported between telomerase activity assessed in 144 cloned (3.4−03 ± 2.4−03 amoles/μL) and 80 control (2.1−03 ± 1.8−03 amoles/μL) embryos. Conclusions The results have revealed a moderate shortening of telomeres in the offspring of the clone with respect to control. However, this study did not evidence differences in the two progenies that suggest welfare problems during the first year of life.

Pages 13-17

Authors: Vijetha Bajjalli Thippeswamy, Siddartha Shankar Layek, Arumugam Kumaresan, Tushar Kumar Mohanty, Ashok Kumar Gupta, Atish Kumar Chakravarty, Ayyasamy Manimaran, Shiv Prasad

Objective To study the effects of different levels of exotic inheritance on ejaculate quality in bulls and its passage through different generations. Methods Data on semen production traits and ejaculate quality were obtained for 38 crossbred bulls and grandsire-sire-progeny relationship in relation to semen quality was studied. The bulls were classified into three groups based on the level of exotic inheritance viz. F1, 50.0%-62.5% exotic germplasm and >75% exotic germplasm. Results Results of the present study indicated that about 40% of the ejaculates obtained from the crossbred bulls were rejected from further processing due to poor ejaculate quality. The F1 bulls produced significantly higher proportions (57.00±10.00) of poor quality ejaculates compared to the interse mated bulls. The age at first semen collection in crossbred bulls ranged from 567 to 1 010 days with an average of 738.89±18.18 days while the mean age at first semen freezing was 865.72±34.60 days. Conclusions It may be inferred that the “acceptable quality semen producing ability” decreased from grandsire through sire to male progeny and among the increasing exotic genetic levels of CB cattle, F1 bulls produced significantly higher “low grade ejaculates” that were unfit for cryopreservation.

Pages 23-29

Authors: Sharanabasappa A Patil, M Sujaya, Saraswati B Patil

Objective To investigate the in vivo aphrodisiac activity of various extracts obtained from aerial parts Cocculus hirsutus (C. hirsutus). Methods We evaluated whether oral administration of C. hirsutus has spermatogenic activity in male rats. Petroleum ether, chloroform and alcohol extract of aerial parts (stem and leaf) of C. hirsutus were administered at the dose level of 25 mg/100 g body weight to adult male albino rats for 30 days. Results The above said extracts have stimulated the spermatogenic activity and accessory reproductive organs performance in albino rats. Out of the three extracts administered, alcohol extract showed highly stimulant spermatogenic effects in mature male albino rats. Alcohol extract showed potent androgenic activity when treated in immature Albino rats. Males treated with the extract displayed more frequent and vigorous anogenital sniffing and mounting as compared to untreated animals. The increased spermatogenesis in extract treated groups was confirmed by change in histoarchitecture as evidenced by increase in number of spermatogonia, spermatocyte, spermatids and caudal spermatozoa. After subjecting to preliminary phytochemical screening, the alcohol extract showed positive tests for steroids, saponins, oils and fats, phenolic compounds and tannins. Conclusion C. hirsutus exhibited remarkable increase in spermatogenic activity. These findings support the folk use of this plant as an aphrodisiac.

Pages 35-40

Authors: M.H. Jan, G.K. Das, F.A. Khan, J. Singh, S.T. Bashir, S. Khan, J.K. Prasad, S. Mehrotra, M.C. Pathak, G. Singh, M. Sarkar

Objective To investigate changes in follicular fluid concentrations of reactive oxygen species (ROS) and total antioxidant capacity (TAC) and degree of oxidative damage to follicular cells, using protein carbonyl (PC) as marker of oxidative stress, were investigated during reproductive acyclicity in buffalo. Methods Follicular fluid was aspirated from follicles grouped into three classes depending upon their diameter [small (5.0–7.0 mm), medium (7.1–10.0 mm), and large (>10.0 mm)]. Progesterone and estradiol were estimated to determine functional status (P:E ratio) of the follicles. Results Acyclic buffaloes had greater concentrations of ROS (P<0.001) and PC (P=0.0412) and lower concentrations of TAC (P=0.0280) than cyclic buffaloes. An interesting novel finding was the complete absence of low P:E functionally active follicles in acyclic buffaloes. Results indicated a pronounced follicular fluid oxidant-antioxidant imbalance and oxidative damage to follicular cells during acyclicity in buffalo. Conclusion In conclusion, this study provided evidence about role of oxidative stress in pathogenesis of reproductive acyclicity.

Pages 46-52

Authors: Enid Simon Chiwanga, Gileard Massenga, Pendo Mlay, Joseph Obure, Michael Johnson Mahande

Objective To compare maternal outcome of multiple versus singleton pregnancies at a tertiary hospital in Tanzania. Methods A case control study was designed using maternally linked data from Kilimanjaro Christian Medical Centre (KCMC) medical birth registry for the period of 2000–2010. A total of 822 multiple gestations (cases) were matched with 822 singletons (controls) with respect to maternal age at delivery and parity. The odds ratio (ORs) with 95% confidence intervals (CIs) for adverse maternal outcome between singleton and multiple gestations were computed in a multivariable logistic regression model. Results Of the 33 997 births, there were 822 (2.1%) multiples. Compared with singletons, women with multiple gestations had increased risk for preeclampsia (OR 2.6; 95%CI: 1.7–3.9), preterm labour (OR 5.6; 95%CI: 4.2–7.4), antepartum haemorrhage (OR 1.6; 95%CI: 1.1–2.3), anaemia (OR 2.0; 95%CI: 1.6–2.6) and caesarean section (OR 1.5; 95%CI: 1.4–1.7). In addition, there were six maternal deaths among women with multiple gestations, of which all were attributed to postpartum haemorrhage. This accounted for a case fatality rate of 15.8%. Conclusions Multiple gestations are associated with adverse maternal outcomes. Close follow-up and timely interventions may help to prevent poor outcomes related to multiple gestations. These findings suggest the needs for clinicians to counsel women with multiple gestations during prenatal care regarding the potential risks.

Pages 57-63

Authors: Rafiqul Islam, Harendra Kumar, Binsila B. Krishnan

Objective To determine the leukocyte profile in periparturient cows and their relation with the development of postpartum reproductive diseases (PRD). Methods Forty one advanced pregnant cows at 240 days of gestation were selected and critically observed daily during the periparturient period. Blood sampling was done for each experimental animal on 15 days prepartum (−15 d), calving day (0 d), 15 days (15 d) and 30 days (30 d) post partum (pp) for determination of total leukocyte (TLC) and differential leukocyte count (DLC) and thorough gynaecological examination was performed on 0 d, 15 d, 30 d and 45 d pp for diagnosis of PRD like retained placenta (ROP), clinical metritis (CM), clinical endometritis (CE), cervicitis (CT) and delayed involution of uterus (DIU). Results The percentage of cows suffered from various post partum reproductive diseases was 41.46% (17/41). In normal cows, a significant increase in TLC from −15 d to 0 d followed by significant decline has been observed at 15 d and 30 d. The cows suffering from ROP (25.52±2.49), CM (26.57±3.17) and CE (25.09±2.95) showed significantly (P<0.01) higher neutrophil% than normal cows (15.05±1.25) on day 15 pp. The normal cows showed significant higher lymphocyte% during the periparturient period than the cows with PRD on post partum days (15 d and 30 d). The neutrophil% and neutrophil: lymphocyte ratio increased from −15 d to the 0 d in all the groups of cows followed by a general decrease during the post partum days. Conclusions The neutrophil% and neutrophil: lymphocyte ratio was higher in the cows with PRD during periparturient period. Thus, the increased neutrophil percentage and neutrophil: lymphocyte ratio observed at day15 postpartum is an indication of PRD in cows. The significant rise in TLC on the day of calving than prepartum and post partum days only in normal cows is physiological. It is also clear that cows suffering from ROP most likely to suffer from other post partum reproductive diseases.

Pages 71-76

Authors: Sneha Panchal, Ramtej J. Verma

High fluoride content is known to cause dental and skeletal abnormalities. In addition, present review indicates that sodium fluoride consumption caused increased number of r=esorptions and dead foetuses. Various skeletal anomalies such as wavy ribs, presence of 14th ribs, lacking 6th sternebrae and incompete ossification of skull occur. All these changes could be due to oxidative stress caused by fluoride consumption. Fluoride-induced changes could be successfully ameliorated by cotreatment with vitamins and calcium.

Pages 80-82

Authors: Seema Dayal, Alok Kumar, SP Singh, Archana Verma

A 37-year old man presented with 5 months history of left scrotal mass and had underwent left orchidectomy following a presumptive diagnosis of testicular tumour. Histopathological diagnosis of testicular tuberculosis was subsequently made, which revealed a case of isolated TB orchitis. It signifies that the careful evaluation of patients with testicular mass is extremely significant for diagnostic accuracy, optimal treatment and avoiding unnecessary surgery in case of testicular tuberculosis. Simultaneously it will also help in early detection of testicular malignancies and vital for the clinicians to avoid negligence charges.

Pages 253-257

Authors: Vy Phan, Eva Littman, Dee Harris, Antoine La

Objective To carry out a detailed investigation of the impact of aneuploidy on blastocyst quality. Methods 1 257 cleavage-stage embryos from 203 patients underwent IVF treatment with aneuploidy screening were investigated. Comprehensive chromosome analysis involved the use of microarray comparative genomic hybridisation (aCGH) method on single blastomere biopsied on day 3 of embryo development. Embryo morphology was assessed and recorded on days 3 and/or 5–6 post-fertilisation. Morphologic grade A-C was assigned to the blastocysts with grade A being the highest grade and grade C the lowest. Results Among the 1 257 embryos that were analyzed, 474 (38%) were euploid, and 783 (62%) were aneuploid. 315/474 (66.5%) of euploid embyos developed into blastocysts, while 57/783 (32.8%) of aneuploid embryos grew to blastocysts (P<0.01). At the blastocyst stage, the best quality embryos (grade A) were in their majority (164/241 or 68%) euploid. Of the 219 fair quality blastocyts (grade B) 129 were euploid (59%) and 22/112 (20%) of poor quality blastocysts (grade C) were euploid. Conclusion Blastocyst morphology showed a significant link to aneuploidy (P<0.05). However, grading morphology alone can not replace preimplantation aneuploidy screening. Morphology screening or other markers for embryo competence, combined with preimplantation genetic screening for 24 chromosomes may produce the best results.

Pages 263-267

Authors: Etik Khusniyati, Alfie Ardiana Sari, Yuyun Yueniwati, Noorhamdani Noorhamdani, Tatit Nurseta, Kusnarman Keman

Objective To investigate whether Vigna unguiculata (V. Unguiculata, VU) able to reduced oxidative stress in cardiac and modulate the aorta estrogen receptor-β of ovariectomized rats. Methods Thirty female Wistar rats were divided into 5 groups (n=6); control (sham) group; ovariectomy group (OVX); OVX + VU1 (0.5 mg/kg); OVX + VU2 (2.5 mg/kg); and OVX + VU3 (5 mg/kg). The administration VU was started 28 days after surgery following 30 days later. Cardiac malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured colorimetrically. Estrogen receptor-β in the aorta was analyzed immunohistochemically. Results Level of MDA was significantly higher in the OVX group compared to the control group (P< 0.05), but the level of SOD was significantly lower. The level of MDA was significantly lower in OVX + VU compared with OVX group (P<0.05), to reach the level at a control group in OVX + VU2. Administration of VU significantly increases the level of SOD compared with OVX group (P<0.05), to reach the level at a control group in third dose of VU (P>0.05). The level of estrogen receptor-β was significantly decreased in the OVX group compared to the control group (P<0.05). OVX + VU3 could significantly increase the level of estrogen receptor-β compared to OVX group (P<0.05), to reach a level in the control group (P>0.05). Conclusions V. unguiculata is an alternative therapy in decreasing cardiac oxidative stress in ovariectomized rats. Besides, high dose of V. unguiculata also able to increase aorta estrogen receptor-β expression in ovariectomized rats.

Pages 275-281

Authors: Jacklyne Silva Barbosa, Márcia Marly Winck Yamamoto, Matheus Antonio Souto de Medeiros, Eloisa Helena Kubiszeski, Camila Regis Banhara, Sebastião Freitas de Medeiros

Objective To compare the clinical and endocrinological features of infertile Brazilian woman with or without endometriosis. Methods This is a cross-sectional comparative study including infertile patients without an established indication for in vitro fertilization or intracytoplasmatic sperm injection at a tertiary center for reproductive medicine. A complete investigation of the cause of female infertile included videolaparoscopy for pelvic cavity and peritoneal factor evaluation. Results Average patient age was (31.6±4.6) years. Sixty-nine percent patients presented with dysmenorrhea, 38% with bowel disturbances, and 21% with deep dyspareunia. Endometriosis was found in 76% of patients, and 91% had primary infertility. Dysmenorrhea was the only symptom that was more prevalent in infertile women with endometriosis. Compared to those without, patients with endometriosis had higher levels of follicle-stimulating hormone (FSH), prolactin (PRL), thyroid-stimulating hormone (TSH), and carcinogen antigen-125 and lower levels of luteinizing hormone (LH), estradiol, progesterone, and free thyroxin. Conclusions Endometriosis is highly prevalent in the Brazilian population and, dysmenorrhea is the only clinical symptom associated with the diagnosis of endometriosis. Infertile patients with endometriosis have higher levels of FSH, PRL and TSH than infertile women without endometriosis.

Pages 288-294

Authors: Azhar Mohammad Nomair, Nisreen K Aref, Ferzana Rizwan, Omaima H. Ezzo, Nada Hassan

Objective To compare serum leptin levels in obese women with polycystic ovary syndrome (PCOS) and normal ovulatory obese subjects in Saudi Arabia, and to evaluate the interrelationship between leptin concentration, sex hormones, and insulin resistance. Methods The present study was conducted on 40 women with PCOS (34.30 ± 2.08 years, body mass index (BMI) 34.84 ± 4.77 kg/m2, mean ± SD) and 16 obese women as control group (28.10 ± 4.61 years, BMI 33.59 ± 1.23 kg/m2). Diagnostic criteria for PCOS based on the presence of two out of three traits including oligo –and/or anovulation, clinical and/or biochemical signs of hyperandrogenism and the presence of polycystic ovaries on ultrasound scan. Concentrations of testosterone, progesterone, prolactin, gonadotrophins, glucose, insulin, and leptin were measured in the baseline fasting blood sample. Serum leptin concentrations were measured by enzyme linked immunosorbant assay. Results Serum leptin levels in PCOS patients were significantly higher than that in the control group (P=0.005) independently of BMI, and were significantly different between insulin resistant and non-insulin resistant obese PCOS (P=0.044), In PCOS patients there was a positive correlation between leptin and BMI (P=0.049), and there was no correlation between leptin and other hormonal indices in PCOS patients. Conclusions The study revealed that the body mass index and insulin resistance are the two main factors governing serum leptin levels.

Pages 299-304

Authors: Sutrisno Sutrisno, Mariyani Mariyani, Ita Herawati, Evi Rinata, Jehanara Jehanara, Yuyun Yueniwati, Nurdiana Nurdiana, Noorhamdani Noorhamdani, Sanarto Santoso

Objective To investigate whether genistein is anti-inflammation and anti-angiogenesis in endometriosis cells. Methods Primary endometriosis cells were divided into seven groups, including control group and genistein-treatment group at dose 5 until 50 μmol/L. The times of incubation is 6, 24, and 48 hours. Levels of TNF-α, IL-6, IL-1β, MMP-2, and VEGF-A in supernatant cells were determined using the enzyme linked immunosorbent assay. Results The level of IL-1β was significantly lower in genistein-treatment at dose 20 until 50 μM compared to the control group (6 hours of treatment) (P<0.05). In 24 hours of treatment, the level of IL-1β was significantly lower in genistein-treatment start at all doses compared to control group (P <0.05). For 48 hours of incubation, the level of IL-1β was significantly lower in of genistein-treatment group at dose 10 until 50 μM than that to control group (P<0.05). The level of TNF-α and IL-6 were significantly decreased in genistein-treatment compared to control group in all duration of treatment (P<0.05). Level of VEGF-A were significantly lower in genistein-treatment at dose 20 until 50 μM compared to control group (P<0.05). The level of MMP-2 was significantly lower in the genistein-treatment at dose 10 and 30 μM compared to control group in 6 hours of treatment (P<0.05), at dose 10, 40, and 50 μM (24 hours of treatment), and all doses (48 hours of treatment). Conclusion In conclusion, genistein act as anti-inflammatory and anti-angiogenesis in primary culture cells of endometriosis.

Pages 311-316

Authors: R Saidi, D Khelef, R Kaidi

Objective To determine: species distribution of 35 Enterobacteriaceae isolates involved in bovine mastitis; and antimicrobial susceptibility pattern of all isolates from geographical areas in Algeria. Methods Enterobacteriaceae species identification was performed based on conventional biochemical techniques and using the API 20 E test. The antibiotic susceptibility was determined according to the National Committee for Clinical Laboratory Standards guidelines (NCCLS). In total, 35 Enterobacteriaceae species: Escherichia coli (E. coli) (n=5), Klebsiella oxytoca (K. oxytoca) (n=5), Enterobacter cloacae (E. cloacae) (n=4), Klebsiella ornithinolytica (K. ornithinolytica) (n=4), Kluyvera spp (n=4), and Hafnia alvei (H. alvei) (n=3), were the most commonly identified. Enterobacteriaceae isolates were the most resistance to Amoxicillin+Clavulanic Acid (74.28%), Ampicillin (28.57%) and Amoxicillin (28.57%) followed by Tetracycline (20%) and Céfodiazin (14.7%). Conclusions We conclude that Enterobacteriaceae species from bovine milk presented significantly distinct antimicrobial resistance profiles, evaluated by phenotypic test, which has implications for treatment and management decisions.

Pages 320-321

Authors: Beuy Joob, Viroj Wiwanitkit

The Ebola virus 2014 outbreak is the big viral epidemic and cause several deaths. The infection widely spread in Africa and causes infection in thousands of people. The important concern is reproductive medicine is the infection in the pregnant. Here, the authors try to summarize on the information and evidence on this issue and also discuss on the perspective。