Asian Pacific Journal of Reproduction

Pages 85-89

Authors: Lilaram, R Nazeer Ahmed

Objective In the present study, an attempt has been made to investigate the effect of ethanolic seed extract of Caesalpinia bonducella(C. bonducella) on fertility in female Wistar albino rats. Methods Pregnant rats were randomized into two groups containing eight animals in each group. Control group rats were orally administered with 1 mL/100g b.wt of distilled water while rats in second group received 300mg/kg of the seed extract from gestation day 1-7, once daily. The animals were sacrificed twenty four hours after the last dose on gestation day 12. Results A significant reduction in implantation index and a contrasting significant increase in resorption index, pre-implantation and post-implantation loss was recorded in the seed extract treated rats. Progesterone level declined significantly in rats treated with seed extract. O vary revealed degenerated corpora lutea in treated rats. The embryos were noticed with craniofacial deformities in extract treated group. Conclusions The results obtained in this study suggest that the seeds of C. bonducella possess antifertility activity probably due to its antiprogesterogenic hormonal properties that can modulate the reproductive function of the experimental rats.

Pages 93-98

Authors: Stanimir A Yotov, Anatoli S Atanasov, Yordanka Y Ilieva

Objective To determine the possibilities for induction of ovarian activity in Bulgarian Murrah buffaloes by hormonal treatment in the early postpartum period. Methods Twenty six clinically healthy animals, weighing 480-520 kg, 3-5 years of age, with normal parturition, without clinical signs of endometritis during the entire experimental period were divided into 2 groups: control (n=10) and experimental (n=16). Primiparous and multiparous buffaloes were proportionally allotted into both groups. By 5, 21, and 28 postpartum days, control animals were intramuscularly treated with 2 mL physiological saline, whereas experimental buffaloes received 500 mg PGF2 α by the 5th postpartum day, 100 μ g GnRH by the 21st postpartum day and a second prostaglandin dose 7 days later. All buffaloes were submitted to transrectal ultrasound examination on days 5, 21 and 28 after calving. Evaluation of ovarian activity was made on the basis of follicle type and detection of a newly formed corpus luteum. The percentage of animals with small, medium or dominant follicle and corpus luteum after either spontaneous or induced ovulation was determined. Insemination management included introduction of two proven fertile bulls into the herd at the start of the treatment. Pregnancy ultrasound checks were performed on postpartum days 58, 68, 120 and 150. The proportion of buffaloes with clinical signs of spontaneous or induced oestrus until the 35th postpartum day, pregnancy rates by the 35th and 90th postpartum days and service periods <60 days, 60-90 days and >90 days were registered. Results On the 5th day after calving, small follicles were predominating in the ovaries of buffaloes from both groups. Similar findings were established on the 21st day in the control group, whereas the share of experimental buffaloes with small follicles was significantly (P<0.01) lower. By the 21st day, there were no medium follicles in untreated animals, while in hormonally treated ones they were found out in 25% of cases. The proportion of experimental buffaloes with dominant follicle (56.3%) by the 21st day was statistically significantly higher (P<0.01) compared to that of controls (10%). Cumulative percentage of animals with detected corpus luteum (66.8%) until the 28th postpartum day differed considerably (P<0.01) from that in controls (10.0%). There was a substantial between-group difference (P<0.05) between the total percentage of buffaloes with clinical signs of oestrus and pregnancy rates until the 35th day after calving. The share of buffaloes with service period <60 days was higher (P<0.05) compared to the untreated group. The analysis of results demonstrated that the PGF2 α -GnRH-PGF2 α treatment on 5, 21 and 28 postpartum days, was able to induce ovarian activity and oestrus in Bulgarian Murrah buffaloes. The total proportion of animals with signs of oestrus (both spontaneous and induced) and pregnancy rates by the 35th postpartum day were significantly higher (P<0.05) in the treated group compared to controls. Conclusions The tested hormonal treatment protocol, combined with the presence of fertile bulls in the herd, could be successfully used for reduction of service period and calving interval in clinically healthy buffaloes.

Pages 105-109

Authors: Ma Regina R Abesamis, Miguel A Buluran, Gliceria B Ramos

Objective To investigate the effects of pregestational and gestational exposure to an isoflavone of sexually mature female mice on their reproductive health and on the postnatal development of their offspring. Methods Sexually-mature ICR female mice were randomly segregated into Control group 1 that was given the normal diet consisting of food pellets, Control group 2 that was given food pellets with additional corn oil of 2 mL/kg bodyweight. The three (3) isoflavone treatment groups were given 50 mg/kg body weight, 100 mg/kg body weight and 150 mg/kg body weight for low dose (LD), medium dose (MD) and high dose (HD) respectively. Treatments were administered 2 weeks prior to mating and gestation and thereafter until parturition. The delivered pups were weaned up until 21 days. On the 21st day, postnatal development were determined, excluding the birth weight of pups which was measured one day post-parturition. The dams were sacrificed as well and the markers of maternal health were determined. Results There were no significant differences found between the control groups and the treatment groups in terms of the markers of maternal reproductive health. For postnatal development, only HD group displayed a significantly higher mean AGD from the other groups. Conclusions The data implies that exposure to the isoflavone genestein, with the given dosages, does not impact the maternal reproductive health while the high dose brings about masculinization of the pups which implies that isoflavone exerts its action as an endocrine disruptor affecting postnatal development. This could be attributed to the decrease in estrogen due to the inhibition of aromatase, an enzyme involved in estrogen synthesis.

Pages 114-118

Authors: Kullaiyan Sathiyadash, Thangavelu Muthukumar, Shanmugaraj Bala Murugan, Ramalingam Sathishkumar, Eswaranpillai Uma, Sarah Jaison, Perumalsamy Priyadharsini

Objective To develope a conservation strategies for orchid species. Methods Seeds of Acampae praemorsa (Roxb.) Blatt. Mc Cann (A. praemorsa) were obtained from mature pods collected from Velliangiri hills and germinated on Murashige and Skoog (MS) medium supplemented with various concentration of Benzylaminopurine (BAP). Results Maximum seed germination (85%) was observed on MS media supplemented with 2 mg/L BAP. Seed germination percentage increased with increasing concentrations of BAP (0.5 mg/L to 2 mg/L), but 3 mg/L of BAP inhibited seed germination. Variations observed were significantly (P<0.01) different for concentrations of growth regulators, days and their interactions. The embryos in the seeds swelled prior to germination. Fifty days after inoculation, well developed greenish porotocorm like bodies (PLBs) were formed and by 80th day, 85% (maximum) of the PLBs were observed in MS supplemented with 2 mg/L BAP. Eighty days after seed sowing, the germination rate decreased and the PLBs were transformed into first and second leaf stages in media supplemented with Naphthaleneacetic acid (NAA). Maximum seedling numbers (84%) were observed in MS medium supplemented with 1 mg/L NAA at 110th day. After 110 days, the seedlings were transferred to 0.6 g/L MS medium containing activated charcoal and similar concentration of growth regulators for root induction. After rooting, the seedlings were transferred to ex vitro conditions. Conclusions In present study all the mycorrhizal seedlings survived, because orchid mycorrhizal fungi enhance growth of orchid plantlets and present study gives an effective protocol for seed germination and plantlet regeneration from immature seeds which can be used for establishing A. praemorsa populations in Velliangiri Hills and elsewhere.

Pages 126-131

Authors: N Srivastava, SK Srivastava, SK Ghosh, Amit Kumar, P Perumal, A Jerome

Objective To evaluate the cryoinjury prediction of spermatozoa in relation to its cholesterol content at fresh and frozen-thaw stages. Methods Ejaculates (n=12) were processed for cryopreservation, acrosome integrity (fluorescent and giemsa stains), cryoinjury (distribution of non capacitated, capacitated and acrosome reacted, pattern F, B and AR, respectively of Chlortetracycline, CTC assay), in vitro fertiltiy (IVF) and cholesterol content of spermatozoa at fresh, pre-freeze and frozen-thaw stages were evaluated. Values were fitted in prediction equation to predict acrosome integrity (AI) and cryoinjury. Results Study indicated that cholesterol content of fresh spermatozoa can be used to predict cholesterol content of spermatozoa at pre-freeze and frozen-thaw stages of cryopreservation protocol with medium to high level of accuracy (P<0.05 and P<0.01, respectively). Cholesterol content of fresh spermatozoa can be used to predict AI, pattern B and AR and Penetration Index (PI) of IVF with medium level of accuracy (P<0.05) at frozen-thaw but not at pre-freeze stage. Similarly cholesterol content of frozen-thaw spermatozoa can be used to predict AI and pattern AR of frozen-thaw spermatozoa with medium level of accuracy (P<0.05). Conclusion This study revealed strong evidence that cholesterol content of fresh as well as frozen-thaw bull spermatozoa can be a good predictor of level of cryoinjury following preservation at ultra low temperature.

Pages 142-145

Authors: Wachyu Hadisaputra

Objective To evaluate clinical signs and symptoms and serum levels of interleukin-6 (IL-6), tumor necrosis factor- α (TNF- α), matrix metalloproteinase-2 (MMP-2), and vascular endothelial growth factor (VEGF) as non-invasive methods to diagnose endometriosis. Methods Eighty women scheduled to laparoscopy underwent blood sampling for measurement of IL-6, TNF- α, MMP-2, and VEGF. The diagnosis of endometriosis was established by laparoscopy using The American Fertility Society visual diagnosis. The presence or absence of endometriosis was correlated with clinical signs and symptoms and with serum levels of those substances. Results The sensitivity and specificity to detect endometriosis of infertility (OR 134.3) were 78 % and 98 %, dysmenorrhoea (OR 11.7) were 63 % and 88 %, and chronic pelvic pain (OR 13.0) were 28 % and 100 %. The presence of rectovaginal nodules had a sensitivity 25 % and specificity 100 %. (OR 11.3, 95 %). The sensitivity and specificity of biologic markers IL-6 (OR 2.5) were 68 % and 53 %, and TNF- α (OR 28.1) were 68% and 60 %. Conclusions History of infertility, dysmenorrhea, chronic pelvic pain, dyspareunia, cervical tenderness and rectovaginal nodule are clinical signs and symptoms suggesting endometriosis. IL-6 and TNF- α appears to be best serum markers for endometriosis.

Pages 151-158

Authors: Eric Blyth

Objective This paper considers the potential contribution that assisted reproductive technology (ART) may make to population replenishment in countries that have experienced extended periods of below-population-replacement Total Fertility Rates (TFR), by focusing on the specific situation of Singapore, which has recorded ‘ultra-low’ TFRs for many years. Methods The factors contributing to ultra-low TFRs in Singapore, the economic and social consequences of endemic below-population-replacement fertility rates and remedial measures initiated by the government are critically analysed, focussing specifically on the government's subsided ART provisions of the ‘Marriage and Parenthood’ package. In addition the paper provides a close analysis of available contemporary data regarding ART and ART outcomes both in Singapore and internationally. Results Despite limited public accessibility to data concerning ART outcomes in Singapore, it is possible to make some assessment of the potential contribution of publicly-funded ART provision and the possible extension of access to elective oocyte preservation to population replenishment. Conclusions Subsidised ART can-at best-make a marginal contribution to government population policy.

Pages 163-165

Authors: Shiv Prasad, Beerendra Singh, Sumit Singhal, FA Khan, JK Prasad, HP Gupta

This report puts on record production of the first viable calf following ovum pick-up in vitro embryo production (OPU-IVEP) technique in buffalo (Bubalus bubalis) in India. A total of 313 transvaginal OPU sessions were performed in six elite Murrah buffaloes and 1 171 follicles were aspirated. From these follicles, a total of 729 immature oocytes were recovered. The recovered oocytes were graded as A (59, 8.1%), B (111, 15.2%), C (293, 40.2%) and D (262, 35.9%) following standard procedures. Maturable grade oocytes (A, B, and C; 473, 68.9%) were subjected to in vitro maturation in Medium 199 supplemented with 10% FBS, 0.5 μg/mL FSH, 10 IU/mL LH, 1 μ g/mL estradiol-17 β, 20 ng/mL EGF and 50 μM cysteamine in CO2 incubator at 39 °C temperature, 5% CO2 and high humidity. The matured oocytes (427 out of 473; 90.3%) were co-incubated with 1×106 /mL buffalo sperms in modified synthetic oviductal fluid (mSOF) containing 10 ng/mL heparin for 22 hrs for in vitro fertilization. After fertilization the presumptive zygotes were stripped off of remaining cumulus cells and cultured in mSOF for 7 days to study embryonic development. A total of 278 (65.1%) oocytes cleaved after 24-36 hrs of fertilization. Out of the cleaved oocytes, 249 (89.6%), 216 (77.7%), 167 (60.1%), 139 (50.0%) and 73 (26.3%) developed up to 4-cell, 8-cell, 16-cell, morula, and blastocyst stages, respectively. A total of 29 embryos were transferred into 27 synchronized recipient buffaloes. Two of these buffaloes were found pregnant at 90 days. One of them aborted at 4.5 months of gestation (male fetus) and the other gave birth to a live male calf weighing 25 kg. Currently, the calf is normal and has attained the age of 4.5 years.

136-141

Authors: Ibrahim A Abdelazim, Mohannad Lutfi Abu faza, Mohammed Al-Kadi

Abstract Objective To evaluate the relation between matrix metalloproteinase-2 (MMP-2) expression and the clinical and/or pathological parameters of the epithelial ovarian cancers (EOCs). Methods Forty-two (42) patients with EOCs diagnosed after histopathological examination of the specimens were included in this study. The pathological specimens were additionally stained by immunoperoxidase technique for MMP-2 using a monoclonal antibody against activated MMP 2. The staining intensity of MMP-2 was correlated with the clinical and pathological parameters of the studied cases, including patient's age, surgical stage, histological grade, omental, and lymph node metastasis. Results The studied cases of EOCs were classified according to the intensity or the degree of MMP-2 expression, as; seven cases (16.7%) negative, eighteen cases (42.8%) weak, seven cases (16.7%) moderate and ten cases (23.8%) intense for MMP-2 staining. There was a significant positive correlation between MMP-2 expression and the histological grades and the surgical stages of the studied EOC (r <1, P<0.05), while, there was no significant relation between MMP-2 expression and the histopathological types of the studied EOCs. MMP-2 expression was significantly high in EOCs with ascites, omental, distant and uterine metastasis, while, there was no significant relation between MMP-2 expressions and lymph node metastasis or bilaterality of the EOCs. Conclusions MMP-2 expression was associated with advanced, aggressive EOCs and there was direct relation between expression of MMP-2 and degree of invasiveness and metastasis of EOCs.