Objective: To establish reference values for pixel intensity of testicular ultrasonogram of rams in breeding and non-breeding seasons, and to investigate if the seasonal changes in testicular echogenicity and heterogeneity are associated with semen characteristics. Methods: Five Awassi rams aged 3-5 years old and weighing 45-60 kg were subjected to ultrasonographic scanning of the testes twice monthly for one year (from January 2018 to December 2018), together with semen collection and evaluation of ejaculate volume, sperm motility, sperm concentration, sperm morphology and viability. The ejaculate volume was determined in a graduated collection tube (scale of 0.1 mL). Mass and individual sperm motility was expressed in percentage of motile spermatozoa under optical microscope equipped with a warm stage. Sperm concentration was determined by using a Neubauer chamber. Semen smears stained with eosin-nigrosin were used to determine the percentage of live spermatozoa and sperm cell morphology by using a light microscope. Results: The mean testicular pixel intensity was the lowest in winter and increased gradually from breeding season to non- breeding season, reaching its maximum value in summer (P<0.05). Pixel intensity was found to have a significant negative correlation with progressive motility (r=-0.605, P<0.05), and sperm concentration (r=-0.619, P<0.05). It is also positively correlated with the percentage of sperm morphological abnormalities (r=0.666, P<0.05). Conclusions: Pixel intensity values of testicular ultrasonogram in rams undergo marked seasonal changes that are associated with fluctuations in photoperiod and ambient temperature. The resulting values of testicular echogenicity (pixel intensity) throughout the year provide useful reference values for predicting the testicular function in Awassi rams.

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Objective: To examine sex reversal both by oral and by immersion using 17α-methyltestosterone on the methyltestosterone residual concentration and the organ histopathology of tilapia fish. Methods: This study used oral and immersion treatment methods for sex reversal of tilapia fish and used normal fish as the control and each treatment was repeated 4 times. 17α-methyltestosterone at dosages of 60 mg/kg feed and 0.5 mg/L were used for oral and immersion methods, respectively. In the first step, tilapia fry were reared at 100 L aquaria, with a density of 1 fish/L for 2 months. In the next step, male tilapias were reared at happa (net cage) of (2×1×1) m³ size in the controlled pond, with a density of 30 fish/happa for 3 months. The methyltestosterone residual concentrations were analyzed by one-way analysis of variance and Duncan’s multiple range tests, while organ histopathology was analyzed by descriptive method. Results: Residual concentrations in the serum of methyltestosterone- treated fish were significantly lower than that in normal fish, especially in 4- and 5-month-old tilapias with averages of less than 5 μg/L, while in normal fish was more than 5 μg/L. In the flesh, methyltestosterone residual concentrations showed relatively no significant differences between the oral and immersion treatment groups and methyltestosterone-treated fish remained lower compared to normal fish, except in 5-month-old tilapia. Methyltestosterone-treated tilapia exhibited histopathological changes on gill, liver, kidneys, and intestine organs. Conclusions: Sex reversal either by oral or by immersion has methyltestosterone residual concentration, but does not exceed the limits (5 μg/L or 5 μg/kg) of synthetic steroid on the fish body, although methyltestosterone causes histopathological changes on gill, liver, kidneys, and intestine.

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Objective: To find the association between testosterone serum levels and severity of late onset pre-eclampsia. Methods: This case-control study involved 34 patients with preeclampsia and 24 healthy control pregnant women matched for gestational and maternal age. The recruited pregnant women were divided into two groups: the pre-eclampsia group (34 pregnant women with pre-eclampsia) and the control group (24 healthy pregnant women). Lipid profile, blood urea, serum creatinine, proteinuria, total serum testosterone, sex hormone binding globulin and free androgen index were evaluated. Moreover, body mass index and blood pressure profile were measured. Results: There were high systolic and diastolic blood pressures in the pregnant women with late-onset pre-eclampsia when compared with healthy pregnant women (P<0.01). Total serum testosterone was higher in women with pre-eclampsia compared with healthy pregnant women (P=0.001). The free androgen index was higher in women with pre-eclampsia compared with healthy pregnant women (P=0.001). Sex hormone binding globulin level was low in women with pre-eclampsia compared with healthy pregnant women (P=0.001). High total serum testosterone was significantly correlated with all measured variables (P=0.001), except for body mass index and pulse pressure (both P=0.070). Smoking habit was low in those patients compared with healthy pregnant women. Meanwhile, total serum testosterone serum level was significantly correlated with number of cesarean sections (r=0.86, P<0.01) and nulliparty (r=0.56, P<0.01). Conclusions: Late onset pre-eclampsia in pregnant women is associated with high serum levels of total serum testosterone, free androgen index, low sex hormone binding globulin, low smoking habit with positive history for nulliparity and caesarean sections that are correlated with high blood pressure profiles. Therefore, high total serum testosterone which is correlated with most of risk factors of late-onset pre-eclampsia is regarded as a proxy biomarker reflecting the severity of late onset pre-eclampsia.

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Objective: To investigate the bacterial infections in repeat breeder dairy cows and to evaluate the treatment effects of hyperimmune serum against two main endometritis bacteria, Escherichia (E.) coli and Trueperella pyogenes. Methods: A total of 29 Holstein multiparous cows with three or more unsuccessful artificial inseminations were carried out and examined to confirm the absence of reproductive tract abnormality and vaginal discharges. Uterine lavage was performed to collect uterine samples for bacterial and fungal cultures. In addition, polymerase chain reaction (PCR) was done to detect Trueperella pyogenes, E. coli, Fusobacterium necrophorum, and Prevotella melaninogenicus. The cytological study was done on cervical mucus. The hyperimmune serums produced against Trueperella pyogenes and E. coli were infused into the uterus of repeat breeder cows and two doses of prostaglandin F2α were administrated intramuscularly within 14 days’ interval. Results: There were 10 positive samples in the bacterial culture with 19 isolations and no growth of the main causative bacteria of endometritis. In the PCR method, no Trueperella pyogenes, Fusobacterium necrophorum, and Prevotella melaninogenicus were found. However, 11 positive samples of E. coli were identified by PCR. Cladosporium was detected in one case of repeat breeder cows. The median (interquartile range) of neutrophils in vaginal discharge was 12 (22.5). Eighteen from twenty-nine (62.1%) repeat breeder and eight from eleven (72%) cows with E. coli infections in PCR were made pregnant in the first artificial insemination after treatment by intrauterine infusion of hyperimmune serum and prostaglandin F2 α. Conclusions: Intrauterine administration of hyperimmune serum could be an alternative to antibiotics for the treatment of repeat breeder cows.

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Objective: To determine the antioxidant status of females with polycystic ovarian syndrome. Methods: Blood samples of 85 females (45 newly diagnosed polycystic ovarian syndrome patients and 40 apparently healthy subjects) between 25 and 45 years of age were obtained from Ekiti State University Teaching Hospital, Ado Ekiti, Ekiti State, Nigeria. Spectrophotometry was used to evaluate the plasma concentrations of malondialdehyde, vitamin A, C and E, reduced glutathione and activities of glutathione peroxidase, catalase and superoxide dismutase. Results: The concentrations of malondialdehyde and glutathione peroxidase activity significantly increased (P<0.05) in patients with polycystic ovarian syndrome compared with the healthy controls, while concentrations of reduced glutathione, vitamins A, C, E and activities of catalase and superoxide dismutase of patients with polycystic ovarian syndrome showed significant decrease (P<0.05) compared to the healthy controls. This study showed that oxidative stress may assume a role in the pathogenesis of polycystic ovarian syndrome. There were significant negative correlations between malondialdehyde and superoxide dismutase, catalase (P<0.01) and vitamin A (P<0.05), while there was a significant positive correlation (P<0.01) between malondialdehyde and glutathione peroxidase. In addition, vitamins A (P<0.05), C (P<0.01) and E (P<0.01) showed significant positive correlations with catalase antioxidant enzyme. However, vitamins C and E showed significant positive correlation (P<0.05) among each other. Conclusions: The involvement of antioxidants in the management of polycystic ovarian syndrome may be helpful as secondary therapy to prevent oxidative damage and may be used as a potential approach to overcome metabolic as well as reproductive disorders associated with infertility in polycystic ovarian syndrome.

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Objective: To assess the association between genetic variants of transferrin receptor 2 (TFR2) exon 4 and anemia status and to describe the expression levels of several cytokines, hepcidin, soluble transferrin receptor and erythropoietin. Methods: Institutional based comparative study was done randomly to recruit 106 pregnant women who attended antenatal care in three different health centers in Boyolali Regency, Central Java from May 2015 to September 2015. DNA was extracted from peripheral blood samples of selected pregnant women and sequencing was done for TFR2 exon 4. Furthermore, enzyme-linked immunosorbent assay was conducted to measure the expression levels of interleukin 6, interleukin 4, transforming growth factor β and iron-metabolism related proteins such as hepcidin, soluble transferrin receptor, and erythropoietin. Gene alignment was performed by using a CLUSTAL W program. Collected data were analyzed statistically by using parametric and nonparametric tests with Statistical Product and Service Solutions (SPSS) 20.0 for Windows. Results: Three novel genetic variants from TFR2 exon 4 (position 603, 605 and 606) were associated with anemia status. Moreover, the expression levels of interleukin 6, interleukin 4, transforming growth factor β and erythropoietin were higher in anemic pregnant women than those of nonanemic pregnant women but only erythropoietin level reached statistical significance. These results were followed by decreases of hepcidin and soluble transferrin receptor levels. Conclusions: Various factors contribute to anemia prevalence among pregnant women in Boyoali Regency, Central Java, Indonesia. Our novel findings showed that TFR2 exon 4 has 3 mutational sites in position 603, 605 and 606. These novel genetic variants may provide a new insight into the role of TFR2 in anemia.

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Objective: To evaluate changes of feline (Felis catus) oocytes proteins during in vitro maturation by using the proteomic approach. Methods: Immature oocytes (germinal vesicle) isolated from female cats were cultured and collected at 0 h and 24 h. After collection, oocytes were investigated into immature (germinal vesicle) and mature (metaphase II) stages. The qualitative profiles of the proteins at the immature and mature stages were determined by one-dimensional electrophoresis and liquid chromatography-mass spectrometry. Results: Our data revealed that following 24 h in vitro maturation the maturation rate (metaphase II stage) was 58.7%. Eighty-one of the 260 proteins analyzed were differentially expressed between the germinal vesicle stage and the metaphase II -arrest stage. Proteomic analysis of germinal vesicle and metaphase II oocytes showed abundant expression of proteins involved in transportation (10%), indicating that this was a major characteristic of germinal vesicle oocytes. Similarly, analysis of the proteome of metaphase II oocytes indicated that cell cycle proteins were overexpressed. Interestingly, proteins involved in DNA repair and apoptosis were only expressed in germinal vesicle oocytes and proteins involved in fertilization were only expressed in metaphase II oocytes. Conclusions: The overexpression of certain proteins in germinal vesicle and metaphase II is necessary for oocyte development and maturation. Our findings provide a valuable resource for further investigations into protein expression in oocytes at different developmental stages.

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Objective: To assess the estrogenic activity of hydroalcoholic extract of Clitoria ternatea leaves in female Wistar rats. Methods: Hydroalcoholic extract of C. ternatea leaves prepared by using cold maceration method was tested for estrogenic activity. An acute toxicity study was conducted to estimate the safe dose using OECD 423 guidelines. For estrogenic activity, ovariectomized female rats were divided into four groups, with 6 rats in each group. The control and standard groups were administered with 1% carboxymethyl cellulose orally and estradiol valerate at 1 μg/rat/day subcutaneously, respectively. The third group was administered with hydroalcoholic extract of C. ternatea at the dose 500 mg/kg body weight orally and the fourth group was administered with hydroalcoholic extract of C. ternatea at the dose 500 mg/kg body weight orally along with estradiol valerate at dose 1 μg/rat/day subcutaneously. All treatments lasted for 7 consecutive days and estrogenic activity was assessed by observing vaginal cornfication. On day 8, all animals were sacrificed and uterine horns were dissected out. Utrine weight was measured and blood serum was further processed for the estimation of biochemical parameters like cholesterol, total proteins, alkaline phosphatase and estrogen by autoanylser. Histological studies of uterus were also carried out. Results: Acute toxicity studies indicated the hydroalcoholic extract of C. ternatea leave was found to be safe up to the dose level of 2 000 mg/kg. Oral administration of C. ternatea extract at the dose 500 mg/kg body weight and and estradiol valerate (1 μg/rat/day) caused morphological changes i.e. increase in uterine weight, vaginal opening and cornification of cells; biochemical changes i.e. increase in cholesterol, total protein, alkaline phosphatase and estrogen contents; histological changes i.e. increase in uterine diameter, thickness and height of endometrium. Simultaneous administration of C. ternatea extract with estradiol valerate showed a synergistic effect. Histological investigations further confirmed the strong estrogenic nature of C. ternatea extract. Conclusions: C. ternatea extract (500 mg/kg) showed a significant estrogenic activity which is also supported by biochemical and histological studies. So, on the basis of these findings, it can be concluded that C. ternatea can be used as an alternative to synthetic oral contraceptives.

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