Asian Pacific Journal of Reproduction

453–458

Authors: D.A. El-Badry, Gamal A. El Sisy, Amal M. Abo El-Maaty

Objective To relate seminal plasma reproductive and metabolic hormone profile to both semen freezability and post-thaw semen characteristics. Methods Semen was collected from each Arabian stallion (N = 16) for five consecutive weeks and evaluated before conducting this study. Seminal plasma was collected during semen processing for measuring leptin, insulin, Insulin like growth factor-I (IGF-1), cortisol, testosterone, estradiol and thyroid hormones. Stallions were classified into good (N = 10) and poor freezer (N = 6). Semen post-thaw motility (%), viability index, membrane and acrosome integrity (%) were also evaluated. Results Good freezer stallions had significantly (P = 0.0001) high cortisol, estradiol, insulin, IGF-1, T3 and T4, but significantly low leptin (P = 0.003). Post-thaw sperm viability index (P = 0.0001), membrane integrity (%) (P = 0.002) and motility (%) (P = 0.0001) at 0, 1, 2 and 3 h of good freezer stallions was significantly high compared to poor freezers. Testosterone, insulin and IGF-1 in seminal plasma of good freezer stallions had significant (P < 0.01) positive correlation with sperm post-thaw viability index but estradiol and insulin in seminal plasma of poor freezers correlated with sperm post-thaw viability index. Within good freezer stallions, testosterone in seminal plasma correlated positively with both viability and acrosome integrity but within poor freezer stallions it had a negative correlation with both of them. Individual variation existed between animals and significantly affected seminal plasma hormones and semen post-thaw characteristics. Conclusion Good freezer stallions could be selected depending on assaying seminal plasma hormones. Both estradiol and IGF-1 could be used as semen extender supplementation to improve sperm post-thaw motility, viability and acrosome integrity.

462–466

Authors: Zahra Rezaeian, Hossein Yazdekhasti, Sima Nasri, Zahra Rajabi, Parvin Fallahi, Fardin Amidi

Objective To evaluate the effects of pre-freezing treatment of human semen samples with selenium on semen parameters after thawing procedure. Methods Each sample was divided into four groups: two groups were washed and other two remained unwashed. 5 μg/mL of selenium was added to one of the washed and unwashed group. Results The results showed that sperm motility in unwashed selenium-treated samples was higher compared to washed untreated samples (P < 0.05). DNA damage percentage in unwashed treated samples compared to both untreated samples showed no difference (P > 0.05) but in washed sperm samples, DNA damaged percentage decreased in treated samples compared to untreated ones (P < 0.05). Moreover, the effect of selenium on morphology of washed treated sperms was higher than unwashed untreated samples after thawing procedure. Normal morphology percentage in unwashed treated samples was significantly higher than both unwashed samples after thawing (P < 0.001). In the washed treated samples a higher number of sperms with normal morphology compared to washed untreated samples (P < 0.001) were observed. Conclusions Based on the results, we recommend 5 μg/mL of selenium should be used in infertility clinics to increase sperm quality after freezing and thawing procedures.

471–475

Authors: J.B. Minari, O.Y. Salau

Objective To investigate the impact of local brewed gin (ogogoro) on the integrity of sperm DNA through the evaluation of the protamine 1 (PRM1) gene. Methods Twenty four 9-week-old male albino rats were divided into five groups; the first group which served as the control were fed with normal fish feed and water ad libitum while the rest were fed with different volumes of the local gin (1, 2, 3 and 4 mL respectively) in conjunction with the normal fish feed and water for 24 d. The caudal epididymis was macerated in saline water and the sperm solution collected in an Eppendorf tube. The extraction of DNA was done using Jena Bioscience DNA preparation kit and the protocol was based on the spin column based genomic DNA purification from blood, animal and plant cells. The primer was designed to detect mutation in Protamine 1 gene associated with male infertility. BseR I, a restriction endonuclease was used to digest the amplicon at 37 °C for 10 min and then inactivated at 80 °C for 20 min. The digest was separated on 2% agarose gel. Results There was variation in the thickness of bands and the thinnest band was observed in the group that consumed the highest volume of local gin. There was also a close similarity between the amplicons from the group fed with 1 mL of ogogoro and the control group that were given distilled water. There was no visible digestion by BseR I. There was a significant increase in the body weight across the entire experimental group and the highest weight was recorded in the 4 mL group. Multiple tumors were also seen in the livers of all the experimental groups. Conclusion This study has shown that local gin can affect the sperm DNA by making it susceptible to damage because of the reduced viability of the PRM1 gene whose product i.e., PRM1 protein is needed for the proper compaction of the sperm DNA.

481–489

Authors: Thirumala Rao Talluri, A. Arangasamy, J. Singh, S.K. Ravi, Yash Pal, R.A. Legha, M. Alpha Raj, Ashish Chopra, R.K. Singh, B.N. Tripathi

Objective To study the factors affecting length of gestation in foals from artificially inseminated Marwari mares for a period of ten consecutive breeding seasons. Methods Gestation length was measured in 126 pregnancies from which viable foals have born. Time of ovulation was confirmed through ovarian ultrasonography in order to determine true gestation length (ovulation to till foaling) as different to previous studies, in which either the mating to foaling interval or from fertilization to till parturition was used for calculating the gestational length. All the mares were inseminated artificially with frozen thawed stallion semen at appropriate time points by monitoring the point near to ovulation. Pregnancy was diagnosed approximately 11–15 d after ovulation initially, and re-affirmed at 30–60 d of pregnancy. Results The observed mean gestation length for all the foals was recorded to be 342.20 ± 1.91 (with standard deviation 21.4) d, and ranged from 259 to 388 d. Various factors like parity of the mare, fetal gender, fetal birth weight, season of breeding (Artificial Insemination) and season of foaling were considered for their effect in causing variation on the gestation length in Marwari mares. According to the present study point of view and after compilation of the results, season of breeding and foal birth weight were found to show significant effect in causing true variation of the gestation length in Marwari horses. Other factors like sex of the foal and parity of the mare also had an effect on length of gestation, but whose effect was observed to be non-significant. Over the range of gestation lengths observed, season of foaling was not significantly associated either with gestational length of mare or foal birth weight. Conclusion From the current study it can be concluded that the true average gestation length for Marwari foals born is 344.1 ± 0.49 d. The range of gestation lengths was found to be 315–388 d all resulting in viable foals. The length of gestation is not significantly affected by age and parity of the mare. The only factors significantly affecting gestation length are foal gender and month of foaling (P < 0.05).

495–499

Authors: Yayuk Dwirahayu, Sugeng Mashudi

Objective To discover the effect of some nicotine's doses to the maturity of rat's oocyte in reproductive age. Methods This study was using an experimental design with 3 treatment groups (subcutaneous injection of nicotine 35, 52.5 and 70 mg/kgBW/d for 7 d) and 1 control group. After nicotine exposure treatment, the oocytes of rats were taken for the histological purpose to observe the oocyte's maturity grade in germinal vesicle (GV), Germinal Vesicle Breakdown (GVBD), Metaphase I (M I), or Metaphase II (M II). All of the oocytes were stained using Aceto orcein 1%. The data was analyzed based on One-Way Analysis of Variance (α = 0.05). Results The significant result got further analyzed using LSD test. The result obtained that there were no significant differences of GV and GVBD oocytes in all groups. Otherwise, the result of One-Way ANOVA showed that there was significant differences of M−II oocytes in all groups. LSD test obtained that there was significant differences between control group and 3 treatment groups, and nicotine's treatment 52.5 mg/kg body weight (BW) with 35 mg/kg BW. This research obtained that the number of GV and GVBD oocytes were lower than the control group. Conclusion There was no M I and M II oocytes, because nicotine treatment were less than the control group.

506–509

Authors: Nurdiana Nurdiana, Elly Mayangsari, Bayu Lestari, Bambang Setiawan

Objective To analyze the effect of administering soybean extracts during pregnancy and breastfeeding on hormonal disturbances and impaired spermatogenesis in male rats born to mothers receiving soybean extract. Methods Twenty-eight male Wistar rat puppies were divided into four groups: the control group (no treatment) and male rat puppies whose mothers have been given soy extracts of various doses (68.88 μg/ml, 137.76 μg/ml and 275.52 μg/ml). Female rats were given soybean extracts during pregnancy and lactation until the male puppies were one month. Analysis of the levels of LH, FSH and testosterone was performed by ELISA technique. Testicular spermatogenesis was analyzed by histopathology. Results FSH levels were significantly lower for all three doses of soy extract than those of the control group (P < 0.05). FSH levels increased significantly in the group treated with the highest dose of soybean extract relative to those of the lower doses (P < 0.05). LH levels were significantly lower for all three doses of soy extract than those of the control group (P < 0.05). Testosterone levels were significantly lower for the highest dose of soybean extract relative to those of the control group (P < 0.05). Histology of the seminiferous tubules revealed that increasing the soy bean extract dose correlated with increasing constraints to spermatogenesis. Conclusion Administration of soybean extract from the intrauterine period, during lactation and at the age of two months, to male rats leads to hormonal changes and impaired spermatogenesis.

514–518

Authors: Gamal A. El-Sisy, Walid S. El-Nattat, Reda I. El-Sheshtawy, Amal M. Abo El-Maaty

Objective To investigate the effect of various concentrations of soybean lecithin as an alternative for egg yolk in bull semen extender on post-chilling and post-thaw sperm quality characteristics. Methods Semen ejaculates were collected from three mature bulls' once/week for 5 weeks. After initial evaluation the approved ejaculates were pooled and extended gradually 1:7 with tris-citrate-fructose egg yolk extender (control) and tris-citrate-fructose (TCF) + different concentration of soybean lecithin (0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5% and 4.0%) to ensure 60 million motile spermatozoa/mL, and then proceeded an adopted international cryopreservation protocol. Frozen straws were thawed at 37 °C for 30 s. The parameters studied were sperm motility, viability, abnormality, membrane integrity and normal intact acrosome percentages in chilled and frozen-thawed semen. Results The substitution of egg yolk into TCF with 1% soya lecithin significantly (P < 0.000 1) ameliorated the maintenance of semen characters motility (86.67% ± 0.80%), life (87.60% ± 0.88%), and membrane integrity (82.47% ± 0.94%), meanwhile it had significantly (P < 0.000 1) reduced the abnormality (13.20% ± 0.60%) of spermatozoa to its least value compared to some other concentrations in use. Moreover, the addition of 1.5% of soya lecithin to TCF had maintained the semen characteristics compared to the control tris-citrate-fructose egg yolk. The significantly higher mean values of post-thawing sperm motility % were observed in 1% soybean lecithin (56.00% ± 1.00%) as compared to the control (41.00% ± 1.00%). Conclusion 1–1.5% soya lecithin can effectively alternate egg yolk as cryoprotective additives for cryopreservation extender, without any detrimental effects on post-chilling and post-thaw semen quality in cattle bull.

524–535

Authors: Mohamed I. Shahba, Reda I. El-Sheshtawy, Abdel-Salam I. El-Azab, Alaa E. Abdel-Ghaffar, Maha S. Ziada, Adel A. Zaky

Objective The present study intended for displaying the effect of different freeze-drying media and temperature of storage on the ultrastructure and DNA of freeze-dried buffalo bull spermatozoa. The semen samples undergone freeze-drying were raw semen and frozen-thawed semen extended in Tris-Fructose-Egg yolk-Glycerol. Methods Semen samples were processed in two portions: First portion was cryopreserved with Tris-Fructose-Egg yolk-Glycerol extender to be freeze-dried with the different media used in this study. Second portion was freeze-dried immediately with the different media used in this study. Semen samples were centrifuged in a percoll gradient (45–90%) for 20 min at 700 × g to remove seminal plasma. Subsequently, sperms were washed twice in Tyrode's albumen lactate pyruvate (TALP) to remove percoll remains, and allocated into the four freeze-drying media (media 1, 2, 3 and 4) respectively. The media tested were: medium 1 (EGTA solution), medium 2 (EDTA solution), medium 3 (TCM199 with Hanks salts and 10% FCS) and medium 4 (TCM199 with Hanks salts and 10% FCS and trehalose). For all the media used, samples were diluted, placed in tubes of 1.5 ml and kept at room temperature for 30 min. Then sperm cell suspensions were cooled in liquid nitrogen vapor (approximately −80 °C for 1 h), by keeping the tubes at a distance of 5 cm from liquid nitrogen surface before plunged into it. Frozen samples were immediately inserted into the freeze-drying machine, previously stabilized at (−40 °C) and 350 × 10−3 Mbar pressure. After 12–16 h of freeze-drying, the tubes containing the samples were covered with aluminum foil and stored for 3 months at different temperatures; 4 °C, −20 °C and −80 °C. Freeze-dried sperm samples were re-hydrated by adding 100 μL of milli-Q water at room temperature. To evaluate sperm ultrastructure, transmission electron microscopy was done. For detection of DNA fragmentation, commet assay was performed. Results Electron microscopy showed that the sperm cell component most affected by freeze-drying was the plasma membrane, which was destroyed in all media either in raw or frozen thawed sperm. Microtubules organization was also disorganized in the majority of the sperm from freeze-drying medium 2 and 3, diverging from freeze-drying media 1 and 4, in which microtubules were intact. Conversely, the acrosome and mitochondria were well protected in all media. However, the storage temperature has no effect. The freeze-drying medium with EDTA solution exhibited and the lowest percent of DNA damage (9.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (13.6) at temperature of storage (4 °C). In contrast, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (15.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (18) at temperature of storage (−20 °C). On the other hand, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (11.3) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (20) at temperature of storage (−80 °C). Consequently, the freeze-drying medium (TCM without trehalose) exhibited the lowest percent of DNA damage (6.5) while the freeze-drying medium with EDTA solution exhibited the highest percent of DNA damage (13.9) at temperature of storage (4 °C). On the contrary, the freeze-drying medium with EGTA solution exhibited the lowest percent of DNA damage (13.2) while the freeze-drying medium (TCM without trehalose) exhibited the highest percent of DNA damage (20.8) at temperature of storage (−20 °C). Conversely, the freeze-drying medium with EDTA solution exhibited the lowest percent of DNA damage (6) while the freeze-drying medium (TCM with trehalose) exhibited the highest percent of DNA damage (12) at temperature of storage (−80 °C). Conclusion From the present study, we demonstrated that the freeze-drying medium containing EGTA and EDTA solution were more efficient in avoiding damage to components of buffalo bull sperm, especially the nuclei. Therefore, the medium used for freeze-drying process directly affected sperm nuclear integrity. Also, the storage temperature of freeze-dried sperm affects sperm nuclear integrity.

357-360

Authors: Muslim Akmal, Aulanni'am Aulanni'am, M. Aris Widodo, Sutiman B. Sumitro, Basuki B. Purnomo, Widodo

Objective To prove that the decrease in the expression of protamine 2 affects the fertilization ability and the number of child production. Methods During the development stage of elongating spermatids, human spermatozoa chromatin undergoes a complex transition where histone is extensively replaced by protamine. Histone substitution by protamine 1 and 2 plays a significant role in condensing chromatin required to induce the quality of potent spermatozoa. The substitution also has a key role in protecting spermatozoa from effects of free radicals which can degrade the spermatozoa quality. Results The results revealed that protamine deficiency instigated a severe disruption of spermatogenesis affecting male infertility. In addition, the protamine expression disorder caused a decrease in number, motility, and morphology of spermatozoa. Conclusion Our results of a study in mice confirmed that inhibin B injection caused a reduction in the expression of protamine 2 in cauda epididymis. It has implications in the decrease of motility, concentration, and spermatozoa viability so that it affects the fertilization ability and the number of child production.

365-370

Authors: Theresia B. Temu, Gilead Masenga, Joseph Obure, Dominic Mosha, Michael J. Mahande

Objective Preterm delivery is the second most leading cause of under-five deaths in the world and has been associated with poor neonatal outcomes especially in developing countries where management of severe and extreme preterm new-born is a challenge. This study aimed to determine maternal and obstetric factors associated with preterm delivery among women who delivered at Kilimanjaro Christian Medical Centre. Methods This was unmatched case-control study conducted at the Kilimanjaro Christian Medical Centre between April and May, 2014. A total of 1 143 women were recorded to have delivered during the study period. Of these, 162 had preterm delivery which comprised the case group while controls were selected from women who had term birth (n = 209) making a final sample size of 371 women. All participants were interviewed using a standard questionnaire to determine factors associated with preterm delivery. Additional data were extracted from maternal clinic cards and hospital records. Data analysis was performed using statistical package for social science version 20.0. Odds ratios with 95% CI for factors associated with preterm delivery were estimated in a multivariate logistic regression models. A P-value of <0.05 was considered statistically significant. Results The prevalence of preterm birth was 14.2%. Numerous factors were associated with preterm delivery including living alone (AOR 5.26, 95% CI: 1.11–25.14), no formal education (AOR 1.2, 95% CI: 3.55–4.06), heavy physical works during pregnancy (AOR 3.13, 95% CI: 1.44–6.81), being a peasant (AOR 2.24, 95% CI: 1.16–4.33), business women (OR 2.88, 95% CI: 1.44–5.74), and history of still birth (OR 4.93; 95% CI: 1.59–15.35). Furthermore, history of miscarriage (OR 1.84, 95% CI: 1.02–3.31), preeclampsia (OR 6.83, 95% CI: 2.92–15.96), placenta previa (OR 7.54, 95% CI: 1.65–34.51), abruption placenta (OR 4.04, 95% CI: 1.08–15.17), Caesarean section delivery (OR 1.60, 95% CI: 1.06–2.43), inadequate ANC visits <4 (OR 3.25, 95% CI: 2.04–5.19), multiple pregnancy (OR 2.75, 95% CI: 1.15–6.61), low birth weight (OR 34.27, 95% CI: 15.93–73.7) and UTIs during pregnancy (OR 1.678, 95% CI: 1.064–2.649) were also independently associated with preterm delivery. Conclusion The risk factors for preterm delivery identified in this study are consistent with previous studies. Clinicians and other health care providers should routinely assess women at high risk of preterm delivery during prenatal care to prevent the occurrence of preterm delivery and associated adverse perinatal outcomes.

376-380

Authors: Adil Salim Elsheikh, Thoyba Faroug Fadul, Eiman Mohamed-Elkheir Aboagla, Ahmed Abdel Rahim Gameel

Objective To investigate the effects of supplementation of potassium bromate (KBrO3) to drinking water on the growth rate, pubertal weight, testis & epididymal weights and testicular histology of growing male rats. Methods Thirty male Wizard rat [age = 21 d, mean BW = (40.0 ± 5.3) g] were used. The rats were grouped into 5 treatment groups each group consist of 6 rats. T1 was offered drinking water supplemented with 100 mg, T2 200 mg, T3 300 mg, T4 400 mg/L KBrO3 for the duration of the experiment, while the control group was offered KBrO3 free water. The BW weights were taken weekly. Eight weeks after treatment the rats were sacrificed, testes & epididymae were excised and weighted. The testes were fixed and histopathological sections of 5–6 μm were made, stained with H & E and examined under light microscope. Results The results showed that the growth rate, pubertal weight, testes & epididymal weights and testicular histology of growing male rats were significantly (P < 0.001) affected with KBrO3 supplementation to drinking water. The growth rate of the control group and T1 (100 mg/L KBrO3) fit well a sigmoid pattern growth curve and no differences in their growth rates were recorded, while the sigmoid pattern of the growth curves of treatment T2, T3 and T4 was disrupted. No difference (P > 0.05) in BW between the control and T1 was recorded. However, it is clear that T2, T3 and T4 significantly (P < 0.05) reduced the BW at puberty. Furthermore treatment groups recorded significantly (P < 0.001) low testicular and epididymal weights compared to the control. Supplementation of KBrO3 to drinking water caused serious changes in testicular tissue of the rats. The treatment rats' testes had distorted or even collapsed seminiferous tubules and narrow interstitial spaces. Upon magnification the seminiferous tubules appeared very narrow, mostly devoid of spermatogenesis and with no sperms. Conclusion Exposure of prepubertal rats to KBrO3 retards their growth, causes testicular hypoplasia and impairs spermatogenesis, which is a predictor of infertility or even sterility in the future.

387-390

Authors: Abdul Gofur, Sri Rahayu Lestari

Objective To analyze the effect of black soybean natto treatment on the recovery of the reproduction system in hypercholesterolemic male mice model. Methods Thirty male mice were divided into five groups, they were normal mice (K−), a hypercholesterolemic mice model (K+), a hypercholesterolemic mice model with 200 mg/kg BW of natto extract (P1), a hypercholesterolemic mice model treated with 400 mg/kg BB of natto extract (P2), and a hypercholesterolemic mice model treated with 800 mg/kg BB of natto extract (P3). Natto extract was given orally for 30 d. Results Black soybean natto extract influenced on reproduction system of male hypercholesterolemic mice by increasing the testosterone level and increasing the density and motility of sperm. Conclusion Black soybean natto extract is promising as hypercholesterolemic therapy for treating the male reproduction system.

395-399

Authors: Seyyed Jalil Taheri, Abbas Parham

Objective To investigate the expression of leptin and its functional receptor mRNA by reverse transcription-polymerase chain reaction analysis in sheep oocytes. Methods Sheep ovaries were collected in reproductive season from abattoir and immediately were transported to the lab. Cumulus–oocyte complexes were aspirated from antral follicles and were denuded to obtain denuded oocytes. Total RNA of denuded oocytes was extracted, cDNA synthesis was carried out subsequently and cDNA was subjected to PCR amplification using specific designed primers. Results Gel electrophoresis of PCR products showed the amplification of 162 and 121-bp amplicons for leptin and functional leptin receptor respectively. Sequencing the amplified fragments also confirmed the results. Conclusions The result of present study reveals that leptin and its functional receptor (Ob-Rb) mRNA are expressed in sheep oocyte and further studies should investigate the role(s) of leptin on sheep oocyte physiology and embryo development.

406-410

Authors: K. Kaneko

Objective To investigate the conception rate when cows showing clear signs of estrus and having both normal sized follicles and cystic follicles were artificially inseminated. Methods Thirty parous Holstein cows showing clear signs of estrus and having both normal sized follicles (diameter <25 mm) and cystic follicles (diameter ≥25 mm) simultaneously in their ovaries were artificially inseminated. Changes in the ovaries were observed using real-time ultrasonography for 25 d after artificial insemination. The concentration of plasma progesterone (P4) was measured simultaneously to monitor the function of the corpus luteum. Results The normal sized follicles ovulated in 21 out of 30 cows (ovulation rate: 70.0%), and 11 of these cows successfully conceived (conception rate: 36.7%) (Group C) while 10 cows did not conceive (Group NC). The maining 9 cows, normal sized follicles did not ovulate (Group NO). The change in P4 in cows that ovulated was similar to that in normal cows. Although there were no significant differences in median number and size of cystic follicles among the groups, all cows in Group C only had one cystic follicle. Conclusion These results support artificially inseminated of cows that show clear signs of estrus even if they have both cystic follicles and normal sized follicles, and especially when the cows have only one cystic follicle.

416-418

Authors: Gamal A. El-Sisy, Mohamed I. Shahba, Reda I. El-Sheshtawy

Objective To display the effect of different concentrations of glycerol, as a cryopreservative, on the quality of the frozen-thawed buffalo semen extended in TRIS extender enriched with disaccharides (trehalose or sucrose). Methods Semen samples were extended in Tris-Citric acid-Fructose-Egg yolk without addition of trehalose/sucrose and with 6.4% glycerol as a control (TFEG-C) and with the addition of Trehalose/Sucrose and different concentrations of glycerol to ensure 60 million motile spermatozoa mL−1. Semen cooled slowly up to 5 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 min 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 best sperm motility, sperm liveability, sperm abnormality, sperm cell membrane and DNA integrities appeared with TFES-G 5.5% (41.00 ± 2.08%, 70.40 ± 2.27%, 7.80 ± 1.19%, 68.10 ± 1.55%, 98.90 ± 0.50%, respectively) and TFES-G 7.3% (41.50 ± 1.98%, 70.70 ± 2.03%, 10.80 ± 0.88%, 69.30 ± 1.85% and 96.40 ± 0.88%, respectively). Conclusion From the present study, it can be concluded that addition of glycerol (5.5% or 7.3%) to Tris-Fructose-Egg yolk-Sucrose extender might help in improvement of the post-thawed characteristics of buffalo frozen semen.

428-433

Authors: Gamal A. El Sisy, Amal M. Abo El-Maaty, Zaher M. Rawash

Objective To investigate the antioxidant/oxidant levels in blood serum and seminal plasma of Arab stallion with different ages and their relation to semen quality. Methods Healthy Arabian stallions (n = 57), were divided into three groups. Young (5–10years), Moderate (11–16 years) and Old stallions (>16 years) were subjected to semen evaluation. Seminal plasma and blood samples were collected and stored at −20 °C for measuring glutathione reduced, nitric oxide, Malondialdehyde, ascorbic acid, copper and zinc. Results Old stallions had significantly greater (P < 0.05) ejaculate volume, % live sperm, and total sperm number compared to young and moderate aged groups. The moderate age horses had significantly the lowest (P < 0.05) sperm concentration. Compared to young horses, serum zinc concentrations of moderate and old horses were significantly high (P < 0.0 001), but NO concentrations were significantly (P < 0.05) low. Seminal plasma zinc, ascorbic acid and nitric oxide concentrations were significantly (P < 0.05 and 0.01) high in young stallion group. No significant correlations were observed between seminal zinc, copper, MDA and semen variables. Meanwhile, significant negative correlations were observed between seminal plasma ascorbic acid concentration and all semen variables except total sperm number and sperm abnormities %. Significant correlations were observed between reduced glutathione and both of sperm motility and % of live sperm. Nitric oxide concentrations correlated directly with individual sperm motility but adversely with total sperm number. Conclusion Stallion age has significant effect on some semen variables, antioxidant/oxidant status of either blood serum or seminal plasma.

279-286

Authors: Norihiro Tada, Fumio Kanai, Eri Nakamura, Hongmei Lu, Masahiro Sato

To examine whether functional spermatozoa can be obtained when a whole male gonadal tissue (testes, epididymides, and fat) isolated from neonatal mice is grafted underneath adult mouse testes. Methods Neonatal (1-day-old) male gonadal tissue, isolated from enhanced green fluorescent protein (EGFP)-transgenic (Tg) mice (C57BL/6-Tg(ACTB-EGFP)1Osb/J), was inserted deep in the testis of a non-Tg recipient mouse through a tunica albuginea incision. Two months after transplantation, the fluorescent grafted tissues were retrieved from recipient mice. Results Histological analysis demonstrated that epididymal architecture was well developed and that spermatogenesis in the testis occurred in 30–60% of each seminiferous tubule of all the grafted tissues examined. Interestingly, motile spermatozoa could be successfully retrieved from the portion corresponding to the cauda epididymis in 1 of the 7 transplants obtained. These obtained spermatozoa had transgenes and could support embryonic development when intracytoplasmic sperm injection was performed using frozen-thawed spermatozoa. Conclusion This present technique will be useful for study in various biological fields including the rescue of Tg lines with lethal postnatal phenotypes and cloned animals that die immediately after birth.

295-300

Authors: Mark Anthony C. Mamon, Cheska May M. Menodiado, Glenn L. Siasu, Gliceria B. Ramos

Abstract Objective To assess the histoarchitecture of the maternal liver and kidney as influenced by selenium supplementation within the periconception period and to determine which of the periconception stages of selenium intake is most favorable of the liver and kidney. Methods Thirty-six 7-week-old female mice were divided into four groups where all groups were given 6.0 g of food pellets per day. Un-supplemented group (U) received no selenium in the food pellet, Pre-gestation supplemented group (P) was given 3.0 μg selenium/d for 21 d, Pre-gestation to Gestation supplemented group (PG) was given 3.0 μg selenium/d for 37 d, and Gestation supplemented group (G) was given 3.0 μg selenium/d for 16 d only. Results The occurrences of vacuolated hepatocytes and congested central veins in the liver were observed. The P and the G groups incurred the lowest and highest percent occurrence, respectively, of murine mothers that exhibited presence of vacuolated hepatocytes. The mean percent occurrence of congested central veins did not significantly vary among groups. The occurrences of shrunken and swollen glomeruli were observed in the kidney. The mean percent occurrence of shrunken glomeruli showed that P and G groups were significantly lower than those of U and PG groups. The mean percent occurrences of swollen glomeruli did not vary significantly among groups. Conclusion The histological analyses of the liver and the kidney obtained from different stages of periconception indicate that selenium supplementation would be best during pregestation stage.

311-316

Authors: V.U. Nna, E.O. Ofutet, O.E. Ofem, D.C. Okpokam, E.E. Osim

Objective To determine the prevalence of erectile dysfunction (ED), the use of sex stimulants, the motivation behind the use of sex stimulants and sexual satisfaction with or without the use of sex stimulants among male subjects in Calabar, Nigeria. Methods Male subjects aged 15–74 years were recruited for this study. Exclusion criteria was employed to eliminate responses of subjects who have had no prior engagement in any sexual activity, and were currently having sex less than 4 times a month. After applying the exclusion criteria, 2 010 respondents were obtained. Results Out of the 2 010 subjects, 11 were illiterates (0.5%), 605 (30.1%) had primary education, 1 317 (65.5%) had secondary education, 50 (2.5%) were undergraduates and 27 (1.3%) had post graduate education. Out of the 2 010 respondents, 45 (2.2%) had knowledge of the causes of ED while 1 965 (97.8%) were ignorant of the causes of ED. Fifty one respondents (2.5%) were currently suffering ED, while 1 959 (97.5%) had no history of ED. A significant (P < 0.001) positive correlation (0.531) between age and incidence of ED was observed. Forty one (2%) respondents had never and were currently not using sex stimulants, while 1 969 (98%) were currently using sex stimulants. Out of the 2 010 respondents, 1 745 (86.8%) used sex stimulants to increase their genital size, of which 1 622 (92.9%) were aged 15–34 years. Conclusion The incidence of ED in Calabar, Nigeria, increases with increasing age. Although the respondents between the ages of 15–34 years had the least incidence of ED, this age group used sex stimulants the most following their perceived beneficial effect in increasing genital size.

321-325

Authors: Ali Olfati, Gholamali Moghaddam, Nasroallah Moradi Kor, Behzad Baradaran

Objective This study was conducted to investigate the trace mineral concentrations (Mg, Se, Zn, Cu and Fe) in amniotic fluid (AF) in 40 pregnancy toxemia Ghezel ewes at the time of parturition phase and its association with placental traits. Methods Animals were treated with controlled internal drug release for 14 d and injected 400 IU pregnant mare serum gonadotropin at the time of controlled internal drug release removal. After the detection of estrus by use of teaser rams, ewes were hand-mated. Ewes were classified as having subclinical pregnancy toxemia on the basis of beta-hydroxy butyrate (BHBA) results (BHBA > 0.86 mmol/L). Results The overall mean AF traces of mineral concentrations were 3.13 ng/mL, 22.1 μg/dL, 134.7 μg/dL, 122.5 μg/dL and 166.6 μg/dL, respectively. There was a significant positive correlation between placental efficiency and Zn concentration in AF ewes (r = 0.633, P < 0.01), while the relationship was significantly negative between total volume of amniotic fluid and Fe concentration in AF ewes (r = −0.717, P < 0.01). In this research, no relationship between Se, Mg and Cu trace minerals was observed in AF ewes with placental traits ewes. Results of laboratory analyses demonstrated no relationship between BHBA concentrations and placental traits (P > 0.01), except for placental weight (r = 0.808, P < 0.01). Also, no significant correlation was detected between BHBA with the above trace minerals. Conclusions Overall, determinations of these trace minerals in the AF ewes could have been used to obtain information on nutritional and reproductive status for the diagnosis of pregnancy toxemia in Ghezel ewes.

331-334

Authors: Reda I. El-Sheshtawy, Diya A. El-Badry, Gamal A. El-Sisy, Walid S. El-Nattat, Amal M. Abo Almaaty

Objective To investigate the effect of extender supplementation with different concentrations of a honey bee on post-thawed sperm motility, viability index, membrane and acrosome integrities in Arab stallion. Methods Five ejaculates from each of four Arabian stallions were subjected to cryopreservation with a modified INRA-82, without any supplementation (control) or supplemented with 1%, 2%, 3%, 4% and 5% honey bee. After thawing, all samples were maintained at 37 °C, while analyses were performed at 0, 1, and 2 and 3 h. Sperm motility percentage, viability index percentage, membrane integrity percentage and acrosome integrity percentage of each sample were determined by conventional laboratory methods. Results Relative to the control group, supplementation with honey (2%, 3% and 4% significantly improved (P < 0.01 at least) post-thaw sperm motility, viability index (P < 0.001 at least) and had a positive effect on membrane integrity and intact acrosome percentage (P < 0.001 at least) at 0, 1, 2 and 3 h post-thawing. For all semen parameters, the lower concentration of honey (1%) and higher concentration (5%) did not show significant differences (P > 0.05) compared with the control. Conclusion Honey bee supplementation in Arab stallion semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

340-344

Authors: Mohammad Rahim Ahmadi, Ali Kadivar, Mahmood Vatankhah

Objective This study evaluated the cervical sampling as an easy and safe method for the diagnosis of subclinical endometritis in dairy cattle. Methods One hundred ninety seven lactating Holstein cows were examined at 26–32 d in milk (DIM) for diagnosis of endometritis. Differential cellular counts were also made from stained smears of the cervical mucosa. Using the Receiver/Response Operating Characteristics (ROC) curve, presence of >17.5% polymorphonuclear (PMN) cells was calculated for detection of subclinical endometritis with sensitivity and specificity of 56.5% and 83.3% respectively. Results Cows with subclinical endometritis had significantly more open days and all service conception rate than normal cows. The results of survival analysis showed that normal cows became pregnant at a significantly faster rate than cows with subclinical endometritis. Conclusions The results of the present study introduced the cervical smear sampling as an easy and suitable method for diagnosis of subclinical endometritis in dairy cattle.

351-353

Authors: Ferjaoui Mohamed Aimen, Youssef Atef, Gharrad Majed, Achour Radhouane, Mbarki Manel, Malek Monia, Néji Khaled, Reziga Hedi

The vaginal aplasia is a rare Mullerian duct malformation characterized by the absence of the vagina. The management of this malformation is based on surgery to restore a normal sexual life and eventually to hope having a normal fertility. In this case, we presented a spontaneous pregnancy in a 15 year-old girl who underwent a vaginoplasty with re-implantation of the uterus corpus in the neovagina.

356

Authors: Beuy Joob, Viroj Wiwanitkit

no

179-181

Authors: Nirav R. Soni

Zika virus (ZIKV) is a member of the virus family Flaviviridae and the genus Flavivirus, transmitted by daytime-active Aedes mosquitoes, such as A. aegypti. ZIKV will continue to spread and it will be difficult to determine how the virus will spread over time. Sign and symptoms of ZIKAVD (Zika virus disease) were conjunctivitis (red eyes), back pain, birth defect-abnormal brain development known as microcephaly and it is diagnosed through PCR (polymerase chain reaction) and virus isolation from blood samples.

188-192

Authors: T. Auta, A.T. Hassan

Objective The study was designed to investigate the alterations in oestrus cycle and implantation in Mus musculus administered aqueous wood ash extract of Azadirachta indica. Methods Mice were randomly grouped into four groups (5/group) where 0, 5, 50 and 100 mg/kg doses of the extract were orally administered for 20 d and microscopy of vaginal smear carried out daily to determine anti-ovulatory activity. Oestrus cycle, including metestrus, diestrus and oestrus phases and histopathology of uterus were examined. To assess abortifacient activity, pregnant mice were also administered extracts for 7 d and laparotomised on the 10th day of pregnancy to observe implantation in the horns of the uterus. Results Duration of metestrus and diestrus phases varied significantly, while the frequency at which the oestrus phase occurred was lowest at 100 mg/kg, (2.8 ± 0.2) and (1.2 ± 0.4) d, with degeneration of epithelial cells of glands in the uterus. Failure of implantation (0% implantation) was recorded in mice exposed to 50 and 100 mg/kg of the extract. Conclusions Aqueous wood ash extract of A. indica altered the duration of metestrus, diestrus and oestrus phases of the oestrus cycle and compromised implantation in female mice.

198-203

Authors: R. Heru Prasetyo, Erma Safitri

Objective Auto-regeneration of the intestinal and ovarian tissue are experiencing degenerative due to protein energy malnutrition (PEM) through of auto-mobilization, increase of immune response and differentiation of endogenous stem cells. Methods Female rat model of PEM obtained through fasting meals for 5 d and only to drink, causing malnutrition and damage of the intestinal and ovarian tissue. Furthermore, the therapy of honey with a dose of 30% water (T1) and 50% water (T2), respectively for 5 d and compared with the positive control, were fasted without being given honey (T0+) and negative control, not fasted and without being given honey (T0−). Observations on: auto-mobilization, increased immune response, differentiation of stem cells, and regeneration of the intestinal and ovarian tissue with HE staining. Results Auto-mobilization of stem cells based on the expression of CD34+ and CD45+, which is a marker of endogenous stem cells (hematopoietic stem cells/HSCs). Increased immune response is based on Hsp70 expression and PGE2 in intestinal tissue. Differentiation of stem cells into progenitor cells that expected based expression of growth differentiation factor-9 (GDF-9) by immunohistochemistry in ovarian tissue. Conclusions Expression of CD34+ and CD45+, which significantly different in treatment 2 (2). Furthermore, increase of immune response (decrease Hsp70 expression and increased PGE2) in intestinal tissue. Increased immune response causes expression of GDF-9 in ovarian tissue. Decreased of Hsp70 expression, increased PGE2 and increased GDF-9 followed the process of regeneration of the intestinal and ovarian tissue.

210-213

Authors: Raichel Jacob, Chandirasekar Ramachandran, Calistus Jude, Uthayakumar Venkatachalam, Sasikala Keshav Rao

Objective To investigate the frequency of Pro12Ala polymorphism in PPAR gamma gene among PCOS of South Indian Population. Understanding this polymorphism may help us in better diagnosis prevention and therapeutic approaches towards management of PCOS. Method A total of 75 women with the diagnosis of PCOS and 75 healthy controls were included in this study. The Pro12Ala variant in the PPAR gamma gene was analysed by PCR-RFLP. Results Genotypic frequencies for PPAR gamma gene Pro12Ala polymorphism showed the frequency of Pro/Pro genotypes was 77% in PCOS and 81% in controls. The odds ratio was 0.71 (95% CI 0.31–1.63) and 2.66 (95% CI 0.21–32.9) and alleles expressed 0.87 (95% CI 0.42–1.79). Regarding hormone levels, there were significant differences between PCOS and non PCOS. Conclusion PPARγ2 gene Pro12Ala polymorphism was supposed to be susceptible genes in PCOS. The present study demonstrated that there is a statistical difference between the distributions of PPAR gamma Pro12Ala polymorphism in South Indian Population.

221-226

Authors: Amir Hossein Asgari Safdar, Ali Asghar Sadeghi

Objective This study was conducted in order to the administration of 50 and 100 mg of bovine somatotropin (bST) at the beginning of the estrus synchronization and natural mating of the sheep to evaluate the improvement of the ovulation rate, embryonic development and pregnancy rate of the transferred embryos. Methods Forty eight donors were treated with three different types of treatment; Group A: treated with bST-100 (n = 15), received 100 mg of bST at the beginning of the synchronization and natural mating, Group B: treated with 50 mg of bST (n = 15) same as the previous group and control (n = 18) did not receive any type of bST. Each recipient received two embryos, (n = 108): 30 recipients received the embryos from bST-100s, 45 recipients received the embryos from bST-50 and 33 recipients received embryos from the control group. Using SAS related GENMOD method, rate of superovulatory, recovered structure percentage, cleavage rate, transferable embryo percentage, quality of embryos, rates of pregnancy and embryonic development were analyzed. Using GLM procedure, numbers of corpus luteum and blastocyst cells were analyzed. Results The bST administration had no significant effect on rate of superovulatory, number of CL and recovered structures (P ≥ 0.05). Number of transferable embryos and embryos that had access to the blastocyst in bST-50 (P ≤ 0.01) was more than bST-100 and control group. Conclusions The treatment 50 mg bovine somatotropins enhance the ratio and growth of the transferable embryos. Embryos of bST-50 treatment indicated an improved embryonic development but bST did not affect the pregnancy rates of transferred embryos.

232-235

Authors: Syed Ali Abid Bukhari, Shujait Ali, Muhammad Zubair, Ijaz Ahmad, Ubaid Ur Rehman

Objective To evaluate the ovulation inducing effect of clomiphene citrate along with human chorionic gonadotropin (hCG) in prepubertal Sahiwal heifers. Clomiphene citrate (an anti-estrogen) causes gonadotropin secretion from pituitary gland by removing the negative feedback effect of estrogen. This causes more FSH secretion which ultimately stimulates follicular growth and upon hCG administration (luteinizing hormone activity) ovulation occurs. Methods Twelve prepubertal Sahiwal heifers were divided into experimental and control groups, with six animals in each group. The grouping of animals was made on the basis of plane ovary. The status of ovarian puberty was checked through rectal palpation and history of previous estrus signs. Experimental heifers were given clomiphene citrate at 300 mg/heifer for 9 d orally, while heifers in control group were given placebo tablets. The dose of clomiphene citrate was selected on the basis of previous literature. On day 10, hCG (IVF-C 5000 iu (hCG) LG Life Sciences, Korea) was given to all heifers of both groups at 2 500 IU/heifer through intravenous route. Two base line blood samples were collected before start of the treatment at each rectal palpation examination for selection of animals. Subsequently, during and after treatment three blood samples per week (on alternate days) for 4 weeks were collected for measuring the progesterone (P4) in serum. The concentration of progesterone was measured though enzyme linked immunosorbent assay. For the comparison of variables, student t-test was applied. Results The P4 concentrations in five heifers (83.3%) in the experimental group with (P4 > 1 ng/mL) and one heifer (16.7%) in the control group along with corpus luteum on ovary showed ovulation. One heifer in control group ovulated that might be due to hCG. Mean progesterone concentration was significantly higher (P < 0.05) in treated heifers than in control heifers. Conclusion Administration of clomiphene citrate and hCG can induce ovulation induction in prepubertal Sahiwal heifers.

240-246

Authors: Mark Anthony C. Mamon, Julie Andrea P. Añano, Louise C. Abanador, Genesis Julyus T. Agcaoili, Caira B. Sagum, Ria Lorraine H. Pagliawan, Jean Mherck B. Tapere, Jessie Bob M. Agravante, John Harold G. Arevalo, Abdul Jabbar A. Minalang

Objective To determine the effects of the water quality of Manila Bay on allometric parameters and histological biomarkers of selected organs of P. viridis. Methods Green mussels were collected from two coastal sites of Manila Bay, Las Piñas – Parañaque (LPP) and Bacoor, Cavite (BC). Twenty-four green mussels from each site were used for the assessment of allometric parameters, and six green mussels from LPP and eight from BC were used for the assessment of histological structures of gonads, gut, and digestive glands. Gonad development was categorized into five stages, whereas gut and digestive glands were scored into four categories. Results Allometric parameters that include shell height, weight, and total wet and dry soft tissue weight were significantly different between LPP and BC. It was also observed that exposure to the pollutants in Manila Bay resulted to delays in gonadal development, and detrimental changes and lesions in the histostructure of digestive gland and gut. Conclusions Pollutants in Manila Bay have detrimental effects to the growth, reproductive development, and histological structure of digestive organs of P. viridis.

252-255

Authors: Reda I. El-Sheshtawy, Walid S. El-Nattat, Samy I.A. Shalaby, Mohamed I. Shahba, Islam E. Al-Se'dawy

Objective The benefits of dates palm and their pollen grains in the human nourishment system have their consideration and importance in medical and nutritional point of view. The present study aimed to investigate a new perspective for the use of date palm pollen grains (DPPG) aqueous extract or infusion in preservation of chilled and frozen buffalo semen. Methods Pooled buffalo semen were diluted by Tris-Citrate-Fructose egg yolk (TCFY) diluent (considered as control) and diverse concentrations of clarified soaked pollen grains in Tris-Citrate-Fructose (TCF) diluent in concentrations of 50, 100, 150, 200 and 250 mg DPPG/5 mL TCF. Five concentrations were kept without egg yolk (TPG) while other five concentrations contained 20% egg yolk (TPGY). Each of the two dilutions was enumerated according to the concentration of DPPG. Extended semen is then chilled (for 7 d) and cryopreserved. Motility, alive sperm and intact sperm membrane (HOST) % were recorded as characteristic sperm parameters. Results After 7 d of chilling, significant (P < 0.000 1) high percent of motile sperms was recorded for TPG 50, 100, 150, 200 and 250 (66.67, 65.00, 61.67, 61.67 and 63.33, respectively). After freeze thawing, TPGY 150 and 250 showed the significant (P < 0.000 1) high percent of motile sperms (43.75 and 45.00, respectively) while TPGY 100, 200, 250 and TPG 50, 150 revealed the high results concerning HOST (80.61, 83.99, 80.61, 88.33 and 83.33, respectively) whereas the highest alive sperm percentage was recorded with TPGY 150 (96.30) compared to the control. Conclusions The aqueous cold infusion or extract of the DPPG, added to the TCF extender with or without the addition of egg yolk, proved its good preserving and maintaining capacity of chilled and thawed buffalo bull sperms which was expressed mainly by the sperm motility.

263-265

Authors: Achour Radhouane, Neji Khaled

Corpus callosum agenesis (CCA) was evaluated by ultrasound examination and magnetic resonance imaging (MRI) with many studies. Ultrasonography was able to suspect CCA by indirect signs but a definitive diagnosis of CCA was achieved in rare cases. MRI was able to diagnose complete CCA in majority of cases. Additional neurological abnormalities including heterotopia, gyration anomaly, asymmetry of the cerebral hemispheres, and Dandy-Walker variant were documented, as well as an ocular anomaly which was described, by MRI examination. Prenatal counseling for fetal agenesis of the corpus callosum is difficult as the prognosis is uncertain. The association with other cerebral abnormalities increases the likelihood of a poor outcome and ultrasonographic assessment of the fetal brain is limited. We found MRI to be a safe and useful additional procedure to complement ultrasonographic diagnosis or suspicion of CCA.

269

Authors: Beuy Joob, Viroj Wiwanitkit

no

96-101

Authors: B.O. Omontese, P.I. Rekwot, I.U. Ate, J.O. Ayo, M.U. Kawu, J.S. Rwuaan, A.I. Nwannenna, R.A. Mustapha, A.A. Bello

This review is aimed at providing information on current status of oestrus synchronization as a management tool in goats in Nigeria. Oestrus synchronisation is a reproductive tool that enables goat farmers breed their animals within a short pre-determined period. The principle of oestrus synchronisation is chiefly the control of the luteal phase of the oestrous cycle and two basic mechanisms are employed. These include the use of prostaglandins or its analogues to shorten luteal life/induce premature luteolysis or the use of exogenous progesterone to prolong luteal life, thereby simulating the activity of natural progesterone produced by the corpus luteum. The latter is advantageous where the reproductive status of the flock is unknown. However, the former is easy to apply and only effective in cycling animals. Pharmaceutical products that have been employed in ES protocols in Nigeria include Lutalyse®, Estrumate®, EstroPLAN®, Fluorogestone acetate®, Sil-Oestrus®, Medroxyl-progesterone acetate®, Synchromate-B®, PMSG®, and more recently, the Controlled Internal Drug Release® (CIDR) devices. Over the last three decades, many attempts have been made at assessing the response of goats to various oestrus synchronisation agents and protocols in Nigeria. However, the low availability of these pharmaceutical agents and cost preclude the widespread use of oestrus synchronisation technique in goat production. The inclusion of gonadotrophins in oestrus synchronisation protocols have been reported to improve oestrus responses in goats, while season has been reported to influence the efficiency of oestrus synchronisation programmes. Increasing the dose levels of exogenous hormones in oestrus synchronisation protocols has caused variable and sometimes inconsistent effects. The effects of combining the male stimulus with exogenous hormones, seasonal variation, extra-label use of products and short-term nutritional manipulation on oestrus responses in goats require further evaluation. It is concluded adoption of oestrus synchronisation practice portends enhanced goat production in Nigeria.

105-110

Authors: I. Onyeso, K.W. Nkpaa, S. Omenihu

Objective To investigate the effect of Citrillus lanatus (C. lanatus) seeds and caffeine on blood glucose levels and testicular functions of alloxan-induced diabetic male Wistar rats. Methods Alloxan was administered at a single dose of 150 mg/kg BW to induce diabetes. A dose of either 200 mg/kg C. lanatus or 100 mg/kg caffeine or both was administered daily to alloxan-induced diabetic rats for three weeks, after which results were compared with a normal control group and a positive control group that received both alloxan and glybenclamide. Results C. lanatus seeds extract significantly decreases (P < 0.05) blood glucose level and significantly (P < 0.05) increased sperm motility, sperm count, normal sperm morphology, sperm viable cells and testosterone in plasma level of alloxan-induced diabetic rats treated with C. lanatus seed extract and caffeine. Conclusions The present study showed that co-administration of caffeine and hydromethanolic fraction of C. lanatus seed extract have hypoglycemic effect and may consequently ameliorate the impaired testicular general architecture and inhibits sperm death or testicular damage caused by alloxan-induced diabetes.

116-122

Authors: P. Sushma Reddy, Nazia Begum, Sumith Mutha, Vasudha Bakshi

Objective To investigated beneficial effect of Curcumin (a phenolic curcuminoid derivative from Curcuma longa) in Letrozole induced PCOS in female Wistar rats. Methods Letrozole (1 mg/kg) was administered per orally (p.o) for a period of 21 days for the induction of PCOS, followed by dose of Curcumin (100 mg/kg and 200 mg/kg, p.o) for 15 days using 0.5% w/v CMC as vehicle. Results The administration of Letrozole led to abnormalcy in serum sex steroid profile, lipid profile, glucose and glycosylated hemoglobin levels and depletion in antioxidant activity. Curcumin was able to successfully exert its protective effect by restoring all the parameters to normal and disappearance of cysts in ovaries. Conclusion Curcumin showed beneficial effects in Letrozole induced PCOS in female Wistar rats. Its effect was comparable to that of Clomiphene citrate, most widely used treatment for ovulation induction in PCOS condition.

128-131

Authors: Sneha Panchal, Ramtej J. Verma

Objective To determine the toxic effect of diethanolamine on testicular steroidogenesis and its possible amelioration by curcumin in mice. Methods Adult Swiss strain male albino mice were orally administered with diethanolamine (110, 165 and 330 mg/kg body weight) for 45 days. In another set of experiment, male albino mice were orally administered with curcumin (10, 25 and 50 mg/kg body weight) along with diethanolamine (330 mg/kg body weight) for 45 days. On completion of treatment, animals were humanely sacrificed, testis dissected out, blotted free of blood and used for biochemical analysis. The obtained serum samples were analyzed for serum testosterone level. Results Diethanolamine caused significant (P < 0.05), dose-dependent reduction in total lipid and cholesterol contents, activities of 3β- and 17β-hydroxysteroid dehydrogenases in testis as well as significant reduction in serum testosterone level. However, co-treatment of curcumin along with diethanolamine caused significant (P < 0.05) dose-dependent amelioration in diethanolamine-induced changes in total lipid and cholesterol contents, activities of 3β- and 17β-hydroxysteroid dehydrogenases and serum testosterone levels. Conclusions The results of the present study showed that curcumin significantly ameliorates diethanolamine reduced testicular steroidogenesis.

140-143

Authors: Afifi Inayah, Sri Rahayu, Nashi Widodo, Widya Ayu Prasdini

Objective To determine the polymorphism of GDF-9 gene in FH cattle that may affect its interaction with the BMP-15 protein. Method Blood was taken from the jugular vein using 10 mL sterile syringes from ten Friesian Holstein cattle. The DNA were isolated from the whole blood and used as a template to amplify GDF-9 gene. The Amplicon (PCR product) was sequenced to identify the new SNP. The three-dimensional structure of GDF-9 protein was modeled by SWISS-MODEL. The characteristic of the protein structure was analyzed by using projectHOPE. The binding affinity GDF-9 into MBP was examined by PatchDock and FireDock. Results The result indicates a new variant G1358A changed amino acid residue at position 435 from Arginine into Histidine (R453H). In silico analysis using projectHOPE predicted that the variant altered side chain of GDF-9 and changed its interaction with BMP-15. Further study suggested that the polymorphism R453H also reduce the binding affinity of the GDF-9 into BMP-15 from −85.58 kcal/mol (R453) into −80.79 kcal/mol (H453). Conclusion The new variant G1358A at an estrous FH a cattle has potency alter GDF-9 profile that may affect to reproduction performance.

148-155

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

Objective To determine the effect of different concentrations of heparin, caffeine or calcium ionophore and incubation time on motility, hyperactivity (HA) and acrosome reaction (AR) of ram sperm in vitro. Methods Semen samples were collected by artificial vagina from three mature ram. Split fractions (0.1 mL) of the pooled semen were layered under 1 mL of S-TALP medium supplemented with either heparin (10, 25, 50, 75 and 100 μg/mL), caffeine (0.5, 0.75, 1, 2 mg/mL), or calcium ionophore A23187 (0.5, 0.75, 1, 1.55 mM/mL). Individual motility, hyperactivity percentage and acrosomal status were recorded at 0, 1, 2, 3 and 4 h post-incubation for all treatments and control. Moreover, they were examined for ability to fertilize sheep oocyte in vitro. Results Heparin, caffeine and calcium ionophore A23187 at a concentration of 75 μg/mL, 1 mg/mL, and 1.55 mM/mL respectively can be used as a protocol to provide the best results for in vitro cap citation and acrosome reaction in ram. The penetration rates of rate of oocytes inseminated with spermatozoa treated with calcium ionophore A23187, and heparin were higher as compared with caffeine. Moreover, heparin achieved higher fertilization rates but without significant difference with others. Conclusion The best concentration of heparin, caffeine and ionophore A23187 are 75 μg/mL, 1 mg/mL, 1.55 mM/mL for 3, 1, 4 h incubation respectively and can be used for in vitro fertilization in sheep.

161-170

Authors: Loganathan Kousalya, V. Narmatha Bai

Objective An efficient in vitro plant regeneration protocol for Canscora decussata Schult. (Gentianaceae) (C. decussate), a threatened medicinal herb used in Ayurvedic system of medicine was developed. Seed germination was achieved on MS growth regulator free medium. Methods The nodal explants were excised from the in vitro raised seedlings and inoculated on MS medium supplemented with various plant growth regulators such as BAP, KIN, TDZ and Zeatin individually and in combinations with or without GA3. BAP (2 mg/L) was proved to be effective for multiple shoot induction (30.20 ± 6.53) among the cytokinin tested individually. Addition of NAA (1 mg/L) to cytokinin containing medium resulted in callus, KIN (3 mg/L) with NAA (1 mg/L) produced highest percentage of callus (82%) per explant. Results Among the various combination of cytokinin tested, BAP (0.5 mg/L) in combination with KIN (2 mg/L) induced highest number of multiple shoots (72.10 ± 1.05 shoot per explant). Addition of 1 mg/L of GA3 to the above medium induced highest number of shoots (100.80 ± 3.20) with an average shoot length of 6.98 ± 0.66 cm. Rooting was optimized in half-strength MS medium supplemented with IBA at 1.0 mg/L. The plantlets were successfully transferred to hardening medium containing vermiculite with 83% survival rate. Among the antioxidant activity of methanol extract of wild-grown plants and in vitro regenerants tested, half-MS medium supplemented with NAA (0.5 mg/L) derived callus has promising activity for total phenolics, DPPH, ABTS, FRAP and phosphomolymbdenum assays. Total flavonoid content was found to be high in callus derived from MS medium supplemented with KIN (2 mg/L) in combination with NAA (1 mg/L). Conclusion Our present study suggest that in vitro derived callus of C. decussata represent a promising alternative source to meet the pharmaceutical demands for commercial formulations and the protocol could effectively be applied for the conservation of C. decussata Schult.