Cypermethrin triggers apoptosis, depletes granulosa cells, and induces endometrium thinning in female rats Novita Eka Kusuma Wardani, Respatiningrum Respatiningrum, Anis Nur Laili, Dwi Yuni Nur Hidayati, Setyawati Soeharto, Hidayat Sujuti Asian Pacific Journal of Reproduction 2019 8(4):141-147 Objective: To analyze the effects of subchronic Cypermethrin on the ovary and endometrium as well as the involvement of apoptosis in the toxicity of cypermethrin. Methods: A total of 32 female Wistar rats were randomly divided into four groups, with 8 rats in each group. The control group received no treatment, and the other three groups received oral cypermethrin at 10, 15 or 20 mg/kg body weight for 28 days (sub-chronic). The granulosa cells were calculated histopathologically. The apoptotic index was determined by in situ technique. Histopathological examination was performed on the uterus and ovary. Results: There was no significant difference in the number of primary follicular granulosa cells between the treatment groups and the control group (P>0.05). However, the number of secondary and tertiary follicle granulosa cells in the treatment groups was significantly decreased compared to that of the control group (P all<0.05). The apoptotic index of primary follicular granulosa cells increased significantly in the groups treated with cypermethrin compared with the control group (P<0.05). The secondary, tertiary, and endometrial granulosa cell apoptosis index was significantly higher in all treatment groups compared to the control group (P<0.05). The higher the dose of cypermethrin was, the higher the apoptotic index of secondary, tertiary and endometrial granulosa cells was. There was a significant decrease in endometrial thickness in the three treatment groups compared to the control group (P<0.05). Thinning of the endometrial layer was seen in the cypermethrin exposure groups. Conclusions: Exposure to cypermethrin can suppress the number of secondary and tertiary follicular granulosa cells, and trigger thinning of the endometrium through induction of apoptosis.

Effect of hydro–alcoholic extract of Olea europaea on apoptosis–related genes and oxidative stress in a rat model of torsion/detorsion–induced ovarian damage Majid Shokoohi, Malihe Soltani, Seyed-Hosein Abtahi-Eivary, Vahid Niazi, Mohammad Javad Rafeei Poor, Hooman Ravaei, Ramin Salimnejad, Maryam Moghimian, Hamed Shoorei Asian Pacific Journal of Reproduction 2019 8(4):148-156 Objective: To evaluate the impact of Olea (O.) europaea extract on markers of oxidative stress and apoptosis of ovarian tissues in a rat model of torsion/detorsion-induced ovarian damage. Methods: A total of 28 Wistar female rats were randomly assigned into 4 groups, with 7 rats in each group. The sham group received a 2.5 cm longitudinal incision in the midline part of the abdomen which was then sutured with 5-0 nylon thread; the torsion/detorsion group underwent torsion induction for 3 h followed by reperfusion for 10 days; the torsion/detorsion+O. europaea group received 300 mg/kg hydro-alcoholic extract of O. europaea 30 min before detorsion, followed by reperfusion for 10 days; and the O. europaea group only received 300 mg/kg hydro-alcoholic extract of O. europaea for 10 days. After the treatment period, blood samples were taken; the levels of estrogen, glutathione peroxidase, superoxide dismutase, and malondialdehyde were assayed. The histological changes, as well as the rate of apoptosis in ovarian tissues, were also carried out by histomorphometric analysis at day 10 post-procedure. Results: Histological comparisons demonstrated a significant detrimental change in the torsion/ detorsion group as compared with other groups. The number of pre-antral and antral follicles and corpus luteum was significantly decreased in the torsion/detorsion group compared with the sham group, while treatment with O. europaea could enhance their numbers (P<0.05). The index of apoptosis and the number of atretic body in the ovarian tissue were significantly higher in the torsion/detorsion group compared with the sham group (P<0.05). The concentrations of glutathione peroxidase, estrogen, and superoxide dismutase as well as the mRNA expression of Bcl-2 were considerably diminished in the torsion/detorsion group while they were elevated in the torsion/detorsion+O. europaea group (P<0.05) compared with the torsion/detorsion group. The serum malondialdehyde level and the mRNA expression of Bax were markedly increased during ischemia, while treatment with O. europaea significantly diminished the increased concentrations of malondialdehyde and Bax level in the torsion/detorsion+O. europaea group (P<0.05). Conclusions: O. europaea extract can reduce the degree of tissue damage induced by oxidative stress and apoptosis in the ovary following ovarian ischemia/reperfusion.

Influence of Glyphaea brevis twig extract on nucleus, tight junctions and expression of inhibin-β, stem cell factor, and androgen binding protein in TM4 Sertoli cells Janet Olayemi Olugbodi, Oladipupo David, Oluwafemi Adeleke Ojo, Afolabi Clement Akinmoladun Asian Pacific Journal of Reproduction 2019 8(4):157-166 Objective: To examine the influence of Glyphaea (G.) brevis twig extract on the mitochondrial dehydrogenase activity, integrity of the tight junctions between adjacent cells, mitochondria, apoptosis, nucleus and expression of inhibin-ß, stem cell factor, and androgen binding protein in TM4 Sertoli cells. Methods: TM4 cell line was used in this study as it exhibited properties similar to the Sertoli cells. TM4 Sertoli cells were exposed to G. brevis twig extract (0.1, 1.0, 10.0, 100.0, or 1 000.0μg/mL) for 24, 48 and 72 h. Parameters studied included cell viability [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay], mitochondrial membrane potential (tetra methyl rhodamine ethyl ester dye), transepithelial electrical resistance, apoptosis (Annexin V Alexa Fluor®488/propidium iodide assay) and mRNA expression (quantitative reverse transcription polymerase chain reaction). Results: G. brevis twig extract had no cytotoxic impact on cell viability, thus, considerably increasing the activity of mitochondrial dehydrogenase enzyme after 24 and 72 h exposure. Transepithelial electrical resistance values revealed substantial (P<0.05) rise in treated groups, especially after 72 h of treatment. Moreover, there was a significant decrease in mitochondrial depolarization of TM4 Sertoli cells exposed to G. brevis twig extract when compared to controls. In addition, G. brevis twig extract significantly reduced necrosis and apoptosis of TM4 Sertoli cells when compared to control. Nevertheless, fluorescence microscopy revealed that the nuclei were egg-shaped and marked uniformly with consistent cell shape at the middle of the TM4 Sertoli cells. Significant stimulatory effects were observed on mRNA levels of inhibin-β , androgen binding protein and stem cell factor. Conclusions: G. brevis twig extract may increase the secretory roles of TM4 Sertoli cells, cells proliferation, as well as cell-cell tight junction integrity. Thus, G. brevis twig may enhance spermatogenesis.

Effect of lyophilized aqueous leaf extract of Aquilaria subintegra on aphrodisiac properties in mice Nur Hidayat Che Musa, Haniza Hanim Mohd Zain, TA Mohammed Muayad, Husni Ibrahim Asian Pacific Journal of Reproduction 2019 8(4):167-173 Objective: To investigate the effects of Aquilaria subintegra leaf aqueous extracts on the aphrodisiac properties including sexual behaviour, testosterone level, percentage of pregnancy, number of offspring and male to female ratio of offspring in ICR mice. Methods: In this experiment, each male cohabitated with one female in a polysulfone cage. 30 ICR male mice were divided into 6 groups that received normal saline (the control group), 50 mg/kg, 100 mg/kg, 200 mg/kg, 500 mg/kg, and 1 000 mg/kg body weight of Aquilaria subintegra leaf aqueous extracts orally for 21 days consecutively. Sexual behavior, percentage of pregnancy, number of offspring and male to female ratio of offspring in ICR mice were measured according to the established methods. Testosterone level was measured by using enzyme-linked immunosorbent assay. Results: Mice that received Aquilaria subintegra leaf aqueous extracts at 50 mg/kg body weight (day 0) had significantly higher mount frequency as compared to the control group; groups treated with 100, 500, 1 000 mg/kg body weight extracts produced a greater number of offsprings when compared to the control group. All aphrodisiac parameters were similar between the treatment groups and the control group, indicating that Aquilaria subintegra leaf aqueous extract did not significantly alter the aphrodisiac parameters. Conclusions: Aquilaria subintegra leaf aqueous extracts have no effect on the aphrodisiac properties, but could increase the breeding rate in mice.

Soybean lecithin–based extender improves Damascus goat sperm cryopreservation and fertilizing potential following artificial insemination Mohamed Fathi, Rawash Zaher, Dohreig Ragab, Inas Gamal, Assi Mohamed, Eman Abu-El Naga, Magdy Badr Asian Pacific Journal of Reproduction 2019 8(4):174-180 Objective: To study the effect of adding different concentrations of soybean lecithin in Tris based extender and to compare the results of Tris soybean lecithin extender with two commercial diluents (egg yolk-based: BullXcell and plant-based: OptiXcell) for Damascus goat sperm cryopreservation. Methods: The ejaculates from 4 mature male Damascus goats were obtained by using an artificial vagina. Semen samples were pooled and diluted in Tris extender supplemented with soybean lecithin at different concentrations of 1.5%, 3.0%, 6.0% and 10.0% to get better concentration to be used for further experiments, with a final concentration of 240x106 spermatozoa/mL. Semen samples were packed in straws (0.25 mL), frozen by using an automated system and stored in liquid nitrogen at -196 °C for 48 h. After thawing (37 °C/30 s), the samples were evaluated for sperm quality parameters, including sperm motility, membrane integrity and acrosome integrity. Malondialdehyde concentration was estimated as a marker for lipid peroxidation. Based on the previous investigations, only Tris extender supplemented with 3.0% soybean lecithin (based on its positive results) was used versus BullXcell and OptiXcell for sperm ultrastructure evaluation and artificial insemination by using electron microscope and artificial insemination of the synchronized does. Results: There was no significant difference between Tris-soybean lecithin at 3.0% and BullXcell/OptiXcell diluents in post-thaw sperm parameters and fertility following artificial insemination; meanwhile, the other concentrations of soybean lecithin (1.5%, 6.0% and 10%) showed lower sperm parameters following cryopreservation. Conclusions: Using of Tris-soybean lecithin based extender at the level of 3.0% can be an appropriate alternative to either BullXcell or OptiXcell for Damascus goat sperm cryopreservation.

Pregnancy outcome of lactating dairy cows assigned for Presynch-Ovsynch synchronization program and inseminated either at detected standing heat or at scheduled fixed time Abdel-Tawab Abdel-Razek Yassyn Khalil, Mahmoud Mohamed Hussein Asian Pacific Journal of Reproduction 2019 8(4):181-186 Objective: To determine if insemination at standing heat results in a similar or higher pregnancy rate compared with fixed time artificial insemination, and to study some factors affecting the pregnancy rate. Methods: A total of 8 944 inseminations were included in this study, from which 6 823 inseminations were done in Holstein cows and 2 121 inseminations were performed in Simmental cows. All cows were subjected to a Presynch-Ovsynch protocol. Cows detected in estrus (n=7 424) were artificially inseminated, whereas cows not observed in estrus (n=1 520) were submitted to fixed time artificial insemination. Results: The overall pregnancy rate of cows inseminated on the basis of the detected standing heat was comparable to that recorded for cows receiving fixed time artificial insemination. A higher pregnancy rate was recorded for cows during cold months than that recorded during hot months (P=0.000). A higher pregnancy rate was recorded for Simmental compared with that recorded for Holstein cows (P=0.001). Regarding parity, significant differences in the pregnancy rate were detected between primiparous and multiparous cows (P=0.040). In addition, artificial insemination technicians had no sigificant effect on pregnancy rate (P>0.05). Meanwhile, the used artificial insemination sires significantly (P=0.000) impacted the pregnancy rate. Conclusions: Insemination of cows detected in standing heat prior to predetermined fixed time results in similar pregnancy outcome and decreases days to the first service compared with insemination at the scheduled fixed time at the end of the Presynch-Ovsynch synchronization program. The overall pregnancy outcome is not affected by the breeding program, but it is highly impacted by cow’s breed, parity, artificial insemination sire and breeding season.