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.