Asian Pacific Journal of Reproduction

104-111

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

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

116-120

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

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

128-132

Authors: El-Sheshtawy RI, El-Nattat WS

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

136-139

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

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

144

Authors: Beuy Joob, Viroj Wiwanitkit

none

54-57

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

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

68-76

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

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

81-84

Authors: El-Sheshtawy RI, El-Nattat WS

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

89-92

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

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

6-12

Authors: Abdulaziz Al-Roujayee

Objective: To evaluate the effect of Quercetin (QT) on erectile dysfunction and oxidative stress in penile tissue of streptozotocin-induced diabetic rats. Methods: Two weeks after diabetes induction, QT was treated to normal and diabetic rats for 5 wk. Sexual behavioral parameters including mount latency, intromission latency, ejaculation latency, post-ejaculatory interval, mount frequency and intromission frequency, were observed against stimulus females. Sperm count and their motility and viability were recorded. Serum glucose and testosterone levels were estimated. In penile tissue levels of cyclic guanosine monophosphate, thiobarbituric acid reactive substances and glutathione, and enzymatic activities of superoxide dismutase and catalase were measured. Histopathological changes were evaluated in a cross-section of penile tissue. Results: Sexual behavioral ejaculation latency, post-ejaculatory interval, mount latency and intromission latency were significantly increased while mount frequency and intromission frequency were decreased in diabetic rats. Treatment with QT corrected the male sexual behavioral levels and also enhanced the inhibited sperm count, motility and viability in diabetic rats. Serum testosterone and penile cyclic guanosine monophosphate levels were significantly increased in QT treated diabetic rats compared to untreated diabetic animals. Penile oxidative stress biomarkers were corrected by the QT treatments in diabetic rats. Histopathological evaluation revealed damaged penile tissues in diabetic rats, which was protected following QT treatment. Conclusions: QT eliminated the diabetic-induced sexual impairment and showed significant antioxidant effects in penile tissue. Further experimental studies are recommended for QT therapeutically usage.

18-22

Authors: Gliceria B Ramos, Alfonso Noel, Oyong Glen, Sia Aleli Jillian L

Objective: To investigate if the protective effect of毩-tocopherol against the impact of ethanol on brain morphogenesis involved the activity of the selenoproteins phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) and selenoprotein P (SelPP) that have roles against oxidative stress. Methods: Forty female mice were randomly assigned into natural control (CON), positive control (ETOH), low-, medium-, and high-Objective: To investigate if the protective effect of α-tocopherol against the impact of ethanol on brain morphogenesis involved the activity of the selenoproteins phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) and selenoprotein P (SelPP) that have roles against oxidative stress. Methods: Forty female mice were randomly assigned into natural control (CON), positive control (ETOH), low-, medium-, and high-α-tocopherolsupplemented- ethanol groups (LTOC, MTOC, HTOC, respectively). CON received drinking water without ethanol while 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 day 14 before mating onwards to the day 9 of gestation. At 10.5 ED of gestation (1100 h), the pregnant females were sacrificed by cervical dislocation and the embryos were harvested. Total RNA were extracted, cDNA synthesis and qRT- PCR analyses were carried out. Results: The level of expression of PHGPx in the positive control was significantly lower than that of the natural control. Among the threeα- tocopherol-supplemented groups, only the medium dose- group was significantly higher than the positive control. The level of expression of SelPP in the positive control was significantly lower than those of the natural control, the low- and medium- dose α-tocopherol supplemented groups. In the high dose-α-tocopherol supplemented group, the level of expression was not significantly different from the positive control but significantly lower than the natural control. Conclusions: The activity of the selenoproteins PHGPx and SelPP are involved in the internetwork of antioxidative enzymes with vitamin E when given up to a medium dose only and is one of the possible pathways of shielding embryonic development against the impact of ethanol on brain morphogenesis. This study strengthens the impact of dietaryα-tocopherol and Selenium supplement during the critical period of pregnancy.Objective: To investigate if the protective effect of α-tocopherol against the impact of ethanol on brain morphogenesis involved the activity of the selenoproteins phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) and selenoprotein P (SelPP) that have roles against oxidative stress. Methods: Forty female mice were randomly assigned into natural control (CON), positive control (ETOH), low-, medium-, and high-α-tocopherolsupplemented- ethanol groups (LTOC, MTOC, HTOC, respectively). CON received drinking water without ethanol while 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 day 14 before mating onwards to the day 9 of gestation. At 10.5 ED of gestation (1100 h), the pregnant females were sacrificed by cervical dislocation and the embryos were harvested. Total RNA were extracted, cDNA synthesis and qRT- PCR analyses were carried out. Results: The level of expression of PHGPx in the positive control was significantly lower than that of the natural control. Among the threeα- tocopherol-supplemented groups, only the medium dose- group was significantly higher than the positive control. The level of expression of SelPP in the positive control was significantly lower than those of the natural control, the low- and medium- dose α-tocopherol supplemented groups. In the high dose-α-tocopherol supplemented group, the level of expression was not significantly different from the positive control but significantly lower than the natural control. Conclusions: The activity of the selenoproteins PHGPx and SelPP are involved in the internetwork of antioxidative enzymes with vitamin E when given up to a medium dose only and is one of the possible pathways of shielding embryonic development against the impact of ethanol on brain morphogenesis. This study strengthens the impact of dietaryα-tocopherol and Selenium supplement during the critical period of pregnancy.Objective: To investigate if the protective effect of α-tocopherol against the impact of ethanol on brain morphogenesis involved the activity of the selenoproteins phospholipid hydroperoxide glutathione peroxidase (PHGPx; GPx4) and selenoprotein P (SelPP) that have roles against oxidative stress. Methods: Forty female mice were randomly assigned into natural control (CON), positive control (ETOH), low-, medium-, and high-α-tocopherolsupplemented- ethanol groups (LTOC, MTOC, HTOC, respectively). CON received drinking water without ethanol while 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 day 14 before mating onwards to the day 9 of gestation. At 10.5 ED of gestation (1100 h), the pregnant females were sacrificed by cervical dislocation and the embryos were harvested. Total RNA were extracted, cDNA synthesis and qRT- PCR analyses were carried out. Results: The level of expression of PHGPx in the positive control was significantly lower than that of the natural control. Among the threeα- tocopherol-supplemented groups, only the medium dose- group was significantly higher than the positive control. The level of expression of SelPP in the positive control was significantly lower than those of the natural control, the low- and medium- dose α-tocopherol supplemented groups. In the high dose-α-tocopherol supplemented group, the level of expression was not significantly different from the positive control but significantly lower than the natural control. Conclusions: The activity of the selenoproteins PHGPx and SelPP are involved in the internetwork of antioxidative enzymes with vitamin E when given up to a medium dose only and is one of the possible pathways of shielding embryonic development against the impact of ethanol on brain morphogenesis. This study strengthens the impact of dietaryα-tocopherol and Selenium supplement during the critical period of pregnancy.α-tocopherolsupplemented- ethanol groups (LTOC, MTOC, HTOC, respectively). CON received drinking water without ethanol while ETOH, LTOC, MTOC and HTOC groups received 20% ethanol in drinking water. The supplemented groups were given respective dosages of 毩-tocopherol, 0.410, 0.819, and 1.640 mg/g body weight, at day 14 before mating onwards to the day 9 of gestation. At 10.5 ED of gestation (1100 h), the pregnant females were sacrificed by cervical dislocation and the embryos were harvested. Total RNA were extracted, cDNA synthesis and qRT- PCR analyses were carried out. Results: The level of expression of PHGPx in the positive control was significantly lower than that of the natural control. Among the threeα- tocopherol-supplemented groups, only the medium dose- group was significantly higher than the positive control. The level of expression of SelPP in the positive control was significantly lower than those of the natural control, the low- and medium- dose α-tocopherol supplemented groups. In the high dose-α-tocopherol supplemented group, the level of expression was not significantly different from the positive control but significantly lower than the natural control. Conclusions: The activity of the selenoproteins PHGPx and SelPP are involved in the internetwork of antioxidative enzymes with vitamin E when given up to a medium dose only and is one of the possible pathways of shielding embryonic development against the impact of ethanol on brain morphogenesis. This study strengthens the impact of dietaryα-tocopherol and Selenium supplement during the critical period of pregnancy.

29-34

Authors: Mamak Rezaeian-Tabrizi, Sadeghi AA

Objective: To evaluate the effects of nano-particle of selenium (nSe) on plasma antioxidant capacity, sexual and thyroid hormones and spermatogenesis in male rats exposed to oxidative stress. Methods: Forty rats were randomly divided into four treatments with ten replicates. Treatment groups were: C, the control group received normal saline as gavage and injection (i.p.); OS, received tert-butyl hydroperoxide (0.2 mmol/kg body weight) for inducing oxidative stress; nSe, received nSe (0.3 mg/kg body weight) as gavage, and OS+nSe, received tertbutyl hydroperoxide and nSe. All groups were treated for 28 d and administrations were done each 48 h. Results: Oxidative stress decreased and gavage of nSe to stressed rats increased the antioxidant capacity and activities (P<0.05). Oxidative stress decreased and nSe increased the levels of thyroid, gonadotropic and testosterone hormones as compared with the control group (P<0.05). Gavage of nSe to stressed rat resulted in increase (P<0.05) of gonadotropin and testosterone hormones compared to oxidative stress group. There were no significant differences (P>0.05) between rats exposed to oxidative stress and those in the control group for sperm quantity and quality. Gavage of nSe to stressed rats had no effect (P>0.05) on the sperm parameters, except increased viability and progressive percentages. Conclusion: Nano-particle of Selenium administration in stressed rats could ameliorate the negative effects of oxidative stress on the antioxidant capacity and activities, but not on the quantity and quality parameters of sperm.

43-48

Authors: Mohamed MM Kandiel, Ahmed RM El-Khawagah, Karima Gh Mahmoud

Objective: To assess the effects of epidermal growth factor (EGF) on the Egyptian buffalo bull frozen semen, EGF was incorporated at 0 (control), 50, 100, 200 and 400 ng/mL of extender (Bioxcell®). Methods: Semen features, spermatozoa biometry, total liberated amounts of enzymes (aspartate transaminase, alanine aminotransferase, lactate dehydrogenase, acid phosphatase and alkaline phosphatase) and lipid peroxidation markers (thiobarbituric acid reactive substances, malondialdehyde, glutathione peroxidase, nitric oxide, catalase (CAT) and superoxide dismutase (SOD)) were determined in the spermatozoa-free extracellular extender. Results: Spermatozoa membrane integrity significantly (P<0.05) increased, but DNA integrity decreased with EGF 200 ng/mL. Spermatozoa head (dimensions, area and perimeter), but not shape, as well as acrosome and midpiece measures substantially differed with regard to EGF. Principle piece length and volume markedly decreased (at 100 and 200 ng/mL), while total tail/flagellum length increased (at 50 ng/mL) after EGF supplementation. EGF 50 ng/mL was associated with the decline of nitric oxide levels and catalase enzyme activity, but EGF 100 ng/mL significantly decreased the total liberated amounts of enzymes (aspartate transaminase, lactate dehydrogenase, acid phosphatase and alkaline phosphatase) as well as lipid peroxidation markers (thiobarbituric acid reactive substances and malondialdehyde). Conclusion: EGF in vitro supplementation would affect the semen characteristics of buffalo bull with 100 ng/mL counteracted the freezing mediated oxidative stress indicated with the lowest enzymes leakage and lipid peroxidation.

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.