Polyvinyl alcohol addition to freezing extender can improve the post-thaw quality, longevity and in vitro fertility of ram epididymal spermatozoa.

Recovering and cryopreserving epididymal spermatozoa are suitable methods for preserving the genetic potential of livestock and endangered species. Regarding encouraging reports on the use of polyvinyl alcohol (PVA) in cryopreserving various cell types, we conducted this study to examine the impact of PVA on the post-thaw quality, longevity, and in vitro fertility of ram epididymal sperm. In the first experiment, ram epididymal spermatozoa were frozen in extenders containing 6 % glycerol and 0, 0.5, 1, 2, 5, 10, or 15 mg/ml of PVA. Polyvinyl alcohol at concentrations of 0.5, 1, and 2 mg/ml improved the motility and functional membrane integrity (FMI) of the sperm compared with the control group (P < 0.05). In the second experiment, we investigated whether PVA could partially substitute glycerol in the freezing extender. PVA was added at 0, 0.5, 1, and 2 mg/ml to the extenders containing 1 % or 2 % glycerol. After thawing, the sperm motility parameters of the group containing 1 mg/ml PVA and 2 % glycerol were significantly higher than those of the un-supplemented groups (P < 0.05). In the third experiment, the effect of PVA on the post-thaw sperm longevity were examined. Sperm were frozen in 3 extenders: one containing 6 % glycerol and 1 mg/ml PVA (Gly6P1), another containing 2 % glycerol and 1 mg/ml PVA (Gly2P1), and a control extender with 6 % glycerol. After thawing, the quality of the sperm was evaluated. Sperm were then diluted in human tubal fluid (HTF) and incubated at 37 °C for 3 h. Afterwards, the quality of the sperm was evaluated once more. The presence of PVA in the freezing extender improved motility parameters and FMI. Additionally, PVA-containing groups had lower proportions of capacitated and acrosome reacted sperm compared with the control group (P < 0.05). The Gly6P1 group performed better than the other two groups (P < 0.05). In the fourth experiment, sperm from the Gly6P1 and Control groups were used in the IVF process immediately after thawing (T0) and after a 3-h incubation at 37 °C in HTF (T3). Cleavage, blastocyst and hatching rates in both groups were similar at T0, but they were lower in the Control group at T3 (P < 0.05). In conclusion, PVA as an additive to the freezing extender significantly improves post-thaw motility, viability, acrosome integrity, longevity, and fertile lifespan of ram epididymal spermatozoa.

Maternal quercetin supplementation improved lipopolysaccharide-induced cognitive deficits and inflammatory response in a rat model of maternal immune activation.

BACKGROUND: There is strong evidence that prenatal infection during a specific period of brain development increases the risk of neurodevelopmental disorders, partly through immune-inflammatory pathways. This suggests that anti-inflammatory agents could prevent these disorders by targeting the maternal inflammatory response. In the present study, we used a rat model of maternal immune activation (MIA) to examine whether maternal quercetin (QE) supplementation can alleviate behavioral deficits and inflammatory mediators in the prefrontal cortex (PFC) and hippocampus of adult male offspring. METHODS: Pregnant rats were supplemented with QE (50 mg/kg) or vehicle throughout pregnancy and injected with either lipopolysaccharide (0.5 mg/kg) or saline on gestational days 15/16. At postnatal day 60, we evaluated the offspring's behavior, hippocampal and prefrontal cortex glial density, pro-inflammatory gene expression, and neuronal survival. RESULTS: Our data showed that maternal QE supplementation can prevent working and recognition memory impairments in adult MIA offspring. This behavioral improvement correlates with the decrease in MIA-induced expression of pro-inflammatory genes, microglia, and astrocyte densities, without affecting neuronal survival, in both PFC and CA1 hippocampus areas. CONCLUSION: Therefore, our study supports the potential preventive effect of QE on MIA-induced behavioral dysfunctions, at least in part, by suppressing the glial-mediated inflammatory response.

Ameliorating Effect of Sodium Selenite on Developmental and Molecular Response of Bovine Cumulus-Oocyte Complexes Matured in Vitro Under Heat Stress Condition.

Selenium (Se), an essential trace element, plays an important role in the antioxidative defense mechanism, and it has been proven to improve fertility and reproductive efficiency in dairy cattle. The present study evaluated the potential protective action of Se supplement of in vitro maturation (IVM) media on the maturation and subsequent development of bovine cumulus-oocyte complexes (COCs) exposed to heat stress (HS). The treatment with Se improved the viability of cumulus cells (CCs) and oocytes (P < 0.05). The proportion of oocytes reached metaphase II (MII) and those arrested at metaphase I (MI) was greater and lower in treatment than control respectively (P < 0.05). Supplementation with Se increased the percentage of cleaved embryos, total blastocysts, and blastocyst/cleavage ratio (P < 0.05). Moreover, the upregulation of CCND1, SEPP1, GPX-4, SOD, CAT, and downregulation of GRP78, CHOP, and BAX in both Se-treated CCs and oocytes were recorded. The upregulation of NRF2 was detected in Se-treated CCs other than in oocytes, which showed upregulation of IGF2R and SOX-2 as the markers of quality as well. Se supplement in IVM media improved the viability, maturation, and the level of transcripts related to antioxidant defense and quality of heat-treated oocytes, which coincided with greater subsequent development outcomes. Se ameliorated the viability of CCs along with upregulation of antioxidative candidate gene expression and downregulation of apoptosis-related ones to support their protective role on restoring the quality of oocytes against compromising effects of HS.

Supplementation of media with gamma-oryzanol as a novel antioxidant to overcome redox imbalance during bovine oocyte maturation in vitro.

This study evaluated the effect of supplementing IVM media with γ-oryzanol (ORY), a nutraceutical derived from rice bran oil, on the development of bovine oocytes and hindering the compromising effect of redox imbalance. An in vitro model of the bovine cumulus-oocyte complex was used for the evaluation of nuclear maturation and development. Antioxidant activity was investigated by assessing the level of ROS (Reactive Oxygen Species) and GSH (glutathione) in oocytes and quantitative changes in gene expression in matured oocytes and their respective cumulus cells. ORY supplementation increased the proportion of MII oocytes, cleaved embryos, and total blastocysts (p < .05) and was linked to higher and lower levels of intracellular GSH and ROS, respectively (p < .05). The treated oocytes and their respective cumulus-granulosa cells showed a modulation in the expression of genes related to apoptosis (downregulation of BAX and CHOP) and oxidative stress (upregulation of NRF2, CAT, and SOD). Also, relative upregulation of OCT-4 and IGF2R in treated oocytes was concomitant with higher subsequent development in terms of cleavage and total blastocyst rates (p < .05). Based on our findings, it appears that ORY supplementation can improve the nuclear maturation and development of bovine oocytes into blastocysts and augment their enzymatic and non-enzymatic antioxidant systems, maintaining the Redox balance and high enzymatic activity against ROS generation.

Development and evaluation of an injectable slow-release progesterone formulation for estrus synchronization in ewes out of the breeding season.

This study was aimed at developing a type of slow-release progesterone micro-particles useable in a single intramuscular injection for estrus synchronization in non-breeding season ewes. A total of 66 ewes were randomly assigned into four groups: CIDR (n = 16): exposed to intravaginal CIDR for 12 days, and three experimental groups, i.e., T100 (n = 16), T150 (n = 17) and T200 (n = 17), receiving a single intramuscular injection of 100, 150 and 200 mg slow-release progesterone, respectively. Blood sampling was performed on all ewes at five different times, and the ELISA method measured progesterone levels. No significant differences were observed in progesterone levels among the groups in each sampling time. More than 90% of ewes in the CIDR, T100 and T150 groups and all those in T200 showed estrus behaviour, and the rate was not significantly different between groups. The difference in the mean interval from progesterone treatment to estrus was also insignificant. The parturition rate declined by increasing the dose of injected progesterone; although it was similar in CIDR and T100 groups, it decreased significantly in T150 and T200 . Since our injectable progesterone formulation was successful in the induction and synchronization of estrus in ewes out of the breeding season, it can be applied as an alternative to the conventional progesterone containing intravaginal devices.

The protective effects of astaxanthin on pre-antral follicle degeneration in ovine vitrified/warmed ovarian tissue.

This study assesses the protective effects of astaxanthin (AST) against vitrification/warming-induced cryoinjuries of ovarian tissue slices in sheep. Cortical slices of slaughterhouse acquired-ovine ovaries were randomly distributed in different groups: fresh, toxicity, and five vitrification groups including vitrification in presence of 0 (control group), 1, 10 and 100 µM astaxanthin or 100 µM vitamin E. After vitrification/warming and 24 h culturing, the samples were subjected to histological studies, antioxidant evaluation by TAC and TBAR assays, and assessment of relative expression of BMP4, BMP15, GDF9 and KITLG genes related to folliculogenesis and follicular growth regulation. According to the results, vitrification reduced the percentage of morphologically intact follicles compared to the fresh and toxicity groups (p < 0.05). In vitrification groups, vitamin E and all three concentrations of AST increased the percentage of intact pre-antral follicles and antioxidant activity relative to the vitrified control (p < 0.05). This enhancement significantly occurred in 10 µM AST group more than vitamin E (p < 0.05). Also, 10 µM concentration of AST enhanced the expression of all the examined genes compared to the control (p < 0.05), while the expression of BMP4, BMP15 and KITLG was higher in the AST than vitamin E (p < 0.05). The latter could increase only the expression of GDF9 compared to the control group (p = 0.011). In conclusion, AST is a highly effective antioxidant for maintaining the survival of pre-antral follicles, retaining cell density, increasing total antioxidant capacity, and increasing the expression of some genes related to follicular development after short-term culture of vitrified/warmed ovarian tissue slices.

Atorvastatin Efficacy in the Management of Mild to Moderate Hospitalized COVID-19: A Pilot Randomized Triple-blind Placebo- controlled Clinical Trial.

BACKGROUND: Statins are first-line lipid-lowering agents with tolerable adverse reactions, low cost, and high availability worldwide. The potent anti-inflammatory, antioxidant, anti-thrombotic and immunomodulatory effects of statins propose them as an option against COVID-19 infection. OBJECTIVE: In this randomized triple-blind placebo-controlled clinical trial, we have investigated the atorvastatin efficacy in the management of mild to moderate hospitalized COVID-19 patients. METHODS: In this study, 52 mild to moderate hospitalized COVID-19 patients who fulfilled the inclusion criteria were allocated to the treatment group to receive 40 mg atorvastatin orally once daily for two weeks (n=26) or the placebo group (n=26). Patients' symptoms and laboratory investigations were assessed at baseline and during the follow-up period. We also evaluated the duration of hospitalization and supplemental oxygen therapy as endpoints. RESULTS: After 14-day of follow-up, the oxygen saturation (SaO2) was significantly higher, and the serum high sensitivity C-reactive protein (hs-CRP) level was lower in the treatment group compared to the placebo group. Moreover, at the end of the followup in the treatment group, the lymphocyte count was higher, and the duration of symptom resolution was shorter but not significant. Additionally, in the treatment group, the length of supplemental oxygen therapy and hospitalization duration were meaningfully shorter. Our results revealed that the mortality rate was almost twice higher in the placebo group compared to the treatment group, without any significant adverse drug reaction. CONCLUSION: Atorvastatin significantly reduces supplemental oxygen need, hospitalization duration, and serum hs-CRP level in mild to moderate hospitalized COVID-19 patients.

The effect of quercetin in the maturation media on cumulus-granulosa cells and the developmental competence of bovine oocytes.

The present study was designed to investigate the effects of Quercetin on the developmental competence of bovine oocytes and cumulus-granulosa cells (CGs). Two groups of immature cumulus-oocyte complexes (COCs) were subjected to IVM with or without Quercetin. The viability, nuclear status, early and late apoptosis of oocytes and CGs were evaluated using gene expression analysis and staining methods. Embryonic development was assessed morphologically by recording Post-IVF survival, cleavage, and blastocyst rates. The proportion of oocytes reaching the MII stage was greater and the number of early-apoptotic oocytes was lower in the group matured with Quercetin compared to the Control (p < 0.05). Relative upregulation of OCT-4, IGF2R and Bcl-2, and downregulation of CHOP was seen in treated oocytes. Also, downregulation of Bax and upregulation of Glut-4 was detected in treated CGs. The treated oocytes experienced higher post-IVF survival and cleavage rates compared to the untreated group (p < 0.05); more cleaved embryos reached ≥16-cell stage and blastocyst at days 4th and 7th respectively. In addition, total blastocyst rate was significantly improved. It is concluded that supplementing maturation media with Quercetin can enhance the quality of bovine oocytes and endow them with protective potential against early apoptotic damage possibly through CHOP regulation of BCL2 gene family, triggering expression of a gene in CGs to maintain the intactness of oocyte against apoptotic signals and providing oocytes with more energy substrates. It also boosts the subsequent development of oocyte to blastocyst and improves the efficacy of bovine embryo production in vitro.

Personality traits of patients with multiple sclerosis and their correlation with anxiety and depression levels: A cross-sectional case-control study.

OBJECTIVE: Multiple sclerosis is a chronic demyelinating disease of the central nervous system that can cause severe disability and impair the quality of life (QoL). METHODS: In the current cross-sectional, case-control study, we investigated personality traits, anxiety and depression levels, in 101 patients in the case group and 202 individuals as a control group. The personality traits of the participants were collected via the Neuroticism-Extraversion-Openness Five-Factor Inventory (NEO-FFI) questionnaire. We evaluated the level of anxiety and depression based on the Hospital Anxiety and Depression Scale questionnaire. RESULTS: Our study showed in patients with disease duration above 1 year, the rates of agreement (29.78), anxiety (8.83), and depression level (6.39) were significantly higher than the control group (27.19, 6.47, and 4.97, respectively). Although patients with disease duration below 1 year showed a higher level of agreement and conscientiousness (29.65 and 34.35, respectively) than controls (26.6 and 30.86, respectively). The level of anxiety and depression in patients with a disability index above 4.5 was significantly higher than patients with a disability index below 1. Patients with a disability index below 1 showed a higher rate of extraversion and agreement and conscientiousness (31.47, 31.53, and 35.07, respectively) than controls (25.5, 26.23, and 3033, respectively). In addition, patients with a disability index above 4.5 showed a higher level of agreement (35.64), conscientiousness (35.5), anxiety (9.64), and depression (7.5) than controls (25.96, 30.71, 6.96, and 4.71, respectively). CONCLUSIONS: In conclusion, anxiety and depression levels were much higher among MS patients compared with controls and the severity of these conditions correlate with the score of the disability index. Therefore, a complete comprehension of these conditions by the neurologist could be vital in improving patients' QoL and increasing compliance and adherence to pharmacological therapy.

Ram epididymal sperm frozen in an extender containing ethylene glycol have higher post-thaw longevity and in vitro fertility.

BACKGROUND: Establishing an efficient, simple and inexpensive method for freezing ram epididymal sperm so that the quality and fertility of spermatozoa could be maintained for a longer period after thawing is of great practical value. OBJECTIVES: To optimize freezing and thawing protocol for ram epididymal sperm using either ethylene glycol (EG) or glycerol (GLY) as cryoprotectants (CPAs). Then, to evaluate the post-thaw longevity and in vitro fertility of spermatozoa that were frozen and thawed according to the optimized protocol. MATERIALS AND METHODS: At first, an optimum protocol for freezing and thawing sperm using EG or GLY were investigated, and the next experiments were performed using the spermatozoa that had been frozen and thawed according to the optimized protocol for each CPA. In the next experiments, frozen-thawed and fresh sperm were diluted in an isotonic culture medium and subsequently incubated at 39°C for 4 h. The motility characteristics and functional membrane integrity (FMI) of spermatozoa were evaluated after thawing, after dilution (t0 ), and after incubation (t4 ). The in vitro fertility of the spermatozoa was assessed at t0 and t4 . RESULTS: For both CPAs, the highest motility parameters and FMI was found for spermatozoa frozen at 3 cm above LN2 and thawed at 50 and 65°C (P < 0.05). In comparison to the spermatozoa of GLY group, the spermatozoa of the EG group had higher total and progressive motility at t0 , as well as higher FMI, total and progressive motility, and linearity at t4 (P < 0.05). Fertility of frozen-thawed sperm was higher than that of fresh sperm at t0 (P < 0.05). Incubation treatment increased the fertility of fresh sperm while decreased the fertility of frozen-thawed sperm, and this decline was more severe in GLY than in the EG group. CONCLUSION: Based on the findings, EG can be a more suitable CPA for freezing ram epididymal sperm.

Quercetin effect on the efficiency of ovine oocyte vitrification at GV stage.

The widely adopted method of vitrification is known to induce some negative effects on oocytes. In order to enhance the efficiency of this process performed on ovine oocytes at germinal vesicle stage, vitrification and warming (VW) solutions, and maturation media were supplemented with 5 µM Quercetin (Q). Four groups of vitrified and fresh immature oocytes were subjected to IVM, IVF and IVC, and their survival rate, apoptosis, nuclear status and developmental competence were assessed. Non-vitrified oocytes treated with Quercetin experienced higher cleavage rate relative to those matured without Quercetin (p < 0.05). Supplementation of VW and maturation media with Quercetin resulted in increased survival, cleavage and total blastocyst rates relative to the untreated oocytes. The post-IVM survival rate of non-vitrified oocytes showed no difference among those matured with and without Quercetin, but was higher for oocytes vitrified, warmed and matured with Quercetin relative to VW group lacking Quercetin. The proportion of early-apoptotic (AV+) oocytes was affected by Quercetin supplementation in both control and VW groups (p < 0.05). The number of AV positive oocytes was lower and the proportion of oocytes reaching MII stage was greater in non-vitrified and VW groups matured with Quercetin, in comparison with their untreated respective controls (p < 0.05). There was no difference in the number of late-stage apoptotic oocytes among different groups. It is concluded that supplementing vitrification and warming solutions with Quercetin endows vitrified ovine oocytes with protective potential against early apoptotic damage, and improves viability, maturation rate and developmental competence at GV stage.

Evaluation of the efficiency of TaqMan duplex real-time PCR assay for non-invasive pre-natal assessment of foetal sex in equine.

Accurate diagnosis of foetal sex in pregnant mare is helpful for many breeders, both for private or commercial purposes. In this study, in order to pre-natal foetal sexing in equine, we used TaqMan duplex real-time PCR to detect the specific regions of SRY and TSPY genes on extracted cell-free foetal DNA from maternal blood. Peripheral blood samples from 50 pregnant Arabian mares with singleton foetuses were collected. Cell-free foetal DNA was extracted from maternal plasma, and duplex real-time PCR assays were performed with TaqMan probes and primers. Amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as control of DNA extraction procedure. From the 50 sampled mares, 28 cases had female and 22 mares had male foetuses. The final results for 46 samples were conclusive, and from them, 43 cases were predicted correctly. Sensitivity, specificity and accuracy of the test were 90.48%, 96% and 93.48%, respectively. In conclusion, a TaqMan duplex real-time PCR was set up to pre-natal detection of foetal sex in equine. The method was fast and decreased the false-positive and false-negative results. The technique can be used as a routine procedure in farms by collecting only a blood sample.

Cryopreservation and its effects on motility and gene expression patterns and fertilizing potential of bovine epididymal sperm.

Despite encountering new challenges in using epididymal sperm recovered from cauda epididymides, this accessible and, in some species, worthwhile sample makes inevitable the further development of a suitable cryopreservation protocol. In this study, sperm was recovered from the epididymis of 4°C overnight stored slaughtered bulls' testes and the effects of cryopreservation on the bovine epididymal sperm motility (with CASA) and gene expression patterns (with quantitative Real time-PCR) were evaluated. Moreover the fertilizing potential of cryopreserved epididymal sperm was used in in vitro fertilization (IVF). After freezing and thawing of epididymal sperm, total and slow progressive sperm motility, VCL, VAP, MAD, ALH and BCF were significantly decreased (p < .05), while in the parameters of fast progressive motility, VSL, LIN, WOB and STR there were not any significant variations in the frozen sperm compared to fresh (non-frozen) counterpart. The assessment of abundance of transcripts encoding motility (TSSK6) and fertility (PRM1 and PRM2)-related genes in epididymal sperm, showed that these transcripts were affected by freezing especially in slow progressive motility status (p < .01). Furthermore, cleavage and blastocyst rate did not present any significant differences between bovine embryos produced in vitro by fresh or frozen-thawed epididymal sperm. It can be concluded that epididymal sperm has enough freezability after overnight testes storage, and cryopreservation could not affect the percentage of in vitro produced embryos in spite of the changes of relative abundance of some transcripts and direction progressive motility pattern of sperm.

Peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARß/δ) gene expression profile on ram spermatozoa and their relation to the sperm motility.

Peroxisome proliferator-activated receptors (PPARs) are a member of nuclear receptors superfamily, which mainly regulate the expression of target genes involved in lipid and energy metabolism. These receptors are divided to three isotypes: PPARα, PPARγ and PPARß/δ. Each isotype has a distinct tissue distribution relating to the distinct functions. In this study, the mRNA abundance for PPARα, PPARγ and PPARß/δ was evaluated and compared with high and low motile ram spermatozoa. Semen samples from 6 adult rams were fractionated on a two layer discontinuous Percoll gradient to high and low motile sperm and quantitative parameters of sperm motility were determined by CASA. Total RNA was extracted and the mRNA abundance for each gene was measured by relative quantification technique with Real time PCR. The levels of three isotypes of PPAR transcripts were significantly higher in high motile semen samples using quantitative RT-PCR. Some of sperm motility indices were also significantly correlated with PPARα and PPARγ relative expression. This study revealed the novel association of PPAR gene isotypes with sperm motility. Data from our study suggested PPARs are one of the possible factors that can be studied in male infertility.

Fine Structures of the Oocyte in Relation to Serum, Follicular Fluid Steroid Hormones and IGF-I in the Ovulatory-Sized Follicles in One-Humped Camel (Camelus dromedarius).

BACKGROUND: The following study was carried out to determine the ultrastructural features of the oocyte of the ovulatory-sized follicles in relation to concentrations of steroids and IGF-I in the Follicular Fluid (FF) and serum in the dromedary camel. METHODS: CAMEL FOLLICLES WITH A CLEAR AND HEALTHY APPEARANCE WERE CATEGORIZED INTO THREE CLASSES: follicles 10 to 13.9, 14-17.9 and 18-30 mm diameter. The FF and serum samples were assayed for estradiol-17ß, progesterone and IGF-I. Recovered Cumulus-Oocyte Complexes (COCs) were prepared for transmission electron microscopy. RESULTS: The mean (±SD) FF concentrations of progesterone and IGF-I was significantly (p < 0.05) higher in follicles 18 to 30 mm diameter compared to other groups of follicles. There was no difference in the mean (±SD) serum estradiol-17ß, progesterone and IGF-I concentrations between camels with different ovulatory-sized follicles (p > 0.05). Oocytes from follicles 18 to 30 mm diameter (group 3) showed more advanced signs of maturation including the disappearance of the nuclear envelope, increased number of microvilli in erect position, the increase in number and size of vesicles and more even distribution of the mitochondria throughout the ooplasm. CONCLUSION: The final stages of oocyte maturation in dromedary camel is associated with increasing progesterone and IGF-I concentrations and constant high estradiol concentration in the follicular fluid which are paralleled with well-defined ultrastructural changes in oocytes.