Bent Tail Research Articles

Morphological and morphometric sperm analysis of dwarf sperm whale (Kogia sima)

Nowadays, an increase in the number of marine species at risk of extinction has been observed. Therefore, studies on the reproductive characteristics of these animals are essential. This animal is considered relatively rare, and there is scarce information regarding its reproductive biology and physiology. Thus, this study aims to describe the morphology and morphometry of sperm obtained from dwarf sperm whale. The material used in this work was collected during the necropsy of a dwarf sperm whale specimen. Thus, seminal samples were fixed and stained with panoptic stain. In morphometric analysis, the results obtained were: acrosome length of 0.83±0.01 μm, head length of 1.5±0.02 μm, intermediate part of 0.4±0.00 μm and total length of 27.3±0.51 μm. In terms of morphology, the defects observed were double head, heavily curled tail, abnormal small head, simply bent tail, piriform head, heavily bent tail and specimens within the normal range. In this context, the morphometric and morphological sperm analysis of Kogia sima described in this study can assist future studies regarding the reproductive physiology of these animals.

Seminal Morphology and Organ Morphometrics of Rabbit Bucks Fed Piliostigma thonningii Essential Oil Supplemented Diet

This study aimed to investigate whether the inclusive supplementation of Piliostigma thonningii essential oil in rabbit diet was beneficial to their seminal morphometrics. Forty-five clinically healthy weaned male Dutch rabbits of about five weeks of age were used for the experiment. The rabbits were divided into three treatment groups, with fifteen rabbits per group and balanced for their body weight such that rabbits in each group had similar average initial BW of 0.27 kg. The experimental rabbits were administered P. thonningii essential oil at 0, 2 and 4 ml/kg for treatments T1, T2 and T3, respectively for 12 weeks. The results showed that left testis weight increased with increasing levels of P. thonningii essential oil supplementation. The normal cells, right testis weight and right and left testis volume were higher in T2 and T3 treatments than in T1. Right and left testis lengths were higher in T3 treatment than T1, with T2 being intermediate between T1 and T3. Mid-piece droplet, coiled tail, detached head, free tail, bent tail, right and left epididymis volume, head weight and length, body weight and length, weight and tail length and weight were not affected by treatments. It is concluded that P. thonningii essential oil supplementation improved some organ morphometrics without necessarily affecting sperm morphology in the experimental rabbit bucks.

ASKAP reveals the radio tail structure of the Corkscrew Galaxy shaped by its passage through the Abell 3627 cluster

ABSTRACT Among the bent-tail radio galaxies common in galaxy clusters are some with long, collimated tails (so-called head-tail galaxies) shaped by their interactions with the intracluster medium (ICM). Here, we report the discovery of intricate filamentary structure in and beyond the ∼28 arcmin (570 kpc) long, helical radio tail of the Corkscrew Galaxy (1610–60.5, ESO 137-G007), which resides in the X-ray bright cluster Abell 3627 (D = 70 Mpc). Deep radio continuum data were obtained with wide-field phased array feeds on the Australian Square Kilometer Array Pathfinder (ASKAP) at 944 MHz and 1.4 GHz. While the bright (young) part of the Corkscrew Galaxy tail is highly collimated, the faint (old) part shows increasing oscillation amplitudes, break-ups, and filaments. We find a stunning set of arc-shaped radio filaments beyond and mostly orthogonal to the collimated Corkscrew tail end, forming a partial bubble. This may be the first detection of a ‘proto-lobe’ previously seen in 3D MHD simulations, formed by the face-on impact of the Corkscrew Galaxy with a shock front in the cluster outskirts. Interactions of the radio galaxy tail with the ICM are likely responsible for the tail collimation and shear forces within the ICM for its increasingly filamentary structure. We also report the discovery of small (∼20–30 kpc) ram-pressure stripped radio tails in four Abell 3627 cluster galaxies, located between the Corkscrew Galaxy and its prominent neighbour, the wide-angle tail (WAT) radio galaxy 1610–60.8 (ESO 137-G006).

Exposure to thiourea during the early stages of development impedes the formation of the swim bladder in zebrafish larvae.

Thiourea, a widely used agrochemical, is known to inhibit the activity of thyroid peroxidase, a key enzyme in the biosynthetic pathway of thyroid hormones. Thyroid insufficiency compromises the basal metabolic rate in warm-blooded organisms and embryonic development in vertebrates. In this study, we looked for developmental defects by exposing the zebrafish embryos to an environmentally relevant dose of thiourea (3mg/mL). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to validate thiourea's presence in the treated zebrafish embryos. Structural anomalies like bent tail and pericardial edema were noticed in 96-h post-fertilization (hpf) larvae. On histological examination, underdeveloped swim bladder was noticed in 96 hpf larvae exposed to 3mg/mL thiourea. The treated larvae also failed to follow the characteristic swimming behavior in response to stimuli due to defective swim bladder. Swim bladder being homologous to the lung of tetrapod, the role of Bmp4, a major regulator of lung development, was studied along with the associated regulatory genes. Gene expression analysis revealed that thiourea administration led to the downregulation of bmp4, shh, pcna, anxa5, acta2, and the downstream effector snail3 but the upregulation of caspase3. The protein expression showed a similar trend, wherein Bmp4, Shh, and Pcna were downregulated, but Cleaved Caspase3 showed an increased expression in the treated group. Therefore, it is prudent to presume that exposure to thiourea significantly reduces the expression of Bmp4 and other key regulators; hence, the larvae fail to develop a swim bladder, a vital organ that regulates buoyancy.

Unsaturation effects on lipid transmembrane asymmetry.

Within cell plasma membranes, unsaturated lipids are asymmetrically distributed over the inner and outer leaflets, offering an attractive local structural feature. However, the mechanism to keep lipid transmembrane asymmetry and the closely related transmembrane movement (flip-flop) for unsaturated lipids remain poorly understood. Here, we applied sum frequency generation vibrational spectroscopy to investigate this lipid transmembrane asymmetry upon mimicking the cell membrane homeostatic processes. On the one hand, unsaturated lipids were found to hinder the flip-flop process and preserve lipid transmembrane asymmetry in model cell membranes, owing to the steric hindrance caused by their bent tails. On the other hand, local unsaturated lipids in the mixed unsaturated/saturated lipid bilayer were conducive to the formation of the local asymmetry. Therefore, lipid unsaturation can be recognized as an intrinsic key factor to form and maintain lipid transmembrane asymmetry in cell membranes.

Impact of food additives on neurodevelopmental processes in zebrafish (Danio rerio): Exploring circadian clock genes and dopamine system.

Assessing the impact of food additives on neurodevelopmental processes extends beyond traditional acute toxicity evaluations to address subtler, long-term effects. This study investigates the impact of common food additives (tartrazine, sunset yellow, sodium benzoate, and aspartame) on neurodevelopment in zebrafish embryos, observed from 18hours postfertilization (hpf) to 91 days postfertilization (dpf). Results show reduced 96hpf locomotor activity after aspartame exposure, with elevated additives correlating with decreased heart rates and induced neurodegenerative phenotypes, including bent tails and abnormal pigmentation. Although locomotor activity decreases at 7 days postexposure, a gradual recovery is observed. Transcriptome analysis indicates alterations in clock genes (Cry2 and Per2) and dopamine-related genes (NURR1 and tyrosine hydroxylase) in zebrafish larvae. Dietary additive exposure during embryonic development impacts clock genes, influencing dopamine activity and resulting in neurobehavioral changes. This study underscores potential risks associated with dietary additive exposure during critical developmental stages, warranting reconsideration of consumption guidelines, especially for expectant mothers. Observed neurodevelopmental toxicity, even below recommended levels, emphasizes the importance of safeguarding neurodevelopmental health in early life. Our findings contribute to understanding the neurotoxic effects of dietary additives, emphasizing the necessity of protecting neurodevelopment during vulnerable periods. This study is the first to demonstrate a direct correlation between food additives and the dysregulation of key circadian rhythm and dopaminergic genes in zebrafish, providing new insights into the neurodevelopmental impacts of dietary additives. These findings pave the way for further research into the molecular mechanisms and potential implications for human health.

Assessment of participants’ perception on the use of mass drug administration in communities endemic to <i>Wuchereria bancrofti</i> Cobbold, 1877 in Northern Nigeria

Lymphatic filariasis (LF) is one of the neglected tropical diseases that cause permanent long term disability inform of hydrocoele, lymphoedema and elephantiasis that develop as a result of inflammation of the lymphatic system. This study aimed to assess the prevalence of lymphatic filariasis and perception of participants towards Mass Drug Administration (MDA) programme. The study was a community-based cross-sectional study for screening microfilariae of Wuchereria bancrofti in consenting participants. A total of 1010 participants’ from selected communities on the MDA programme in 5 Local Government Areas (LGAs) of Bauchi, Plateau and Kaduna states were enlisted into to the study. The communities were chosen based on the presence of the people living with LF according to the signs and symptoms. A well-structured adapted questionnaire was administered to collect participants’ information and their perception of the MDA programme. Microflaria of Wuchereria bancrofti were identified based on the sheathed nuclear, caudal nuclei and bent tail in Parasitology and Entomology Research Laboratory, Ahmadu Bello University, Zaria. Data was analysed using descriptive statistics, chi-squared and odds ratio. Participants with knowledge (294/1010; 29.11%) of the MDA programme were less than those without knowledge (716/1010; 70.89%) of the MDA. Similar, the prevalence of LF was higher in participants with knowledge (26.87%) of the MDA than in those without knowledge (25.14%). The status of participation showed that, those that participated (380/1010; 37.62%) in the MDA were lower than those did not participants (630/1010; 62.38%); the infection in participants that did not partook in the MDA programme had higher prevalence (29.68%) of LF than those that participated. Participants gave various reasons for not being part of the MDA programme ranging from absent (177/1010; 17.53%) at the time of the campaign to side effect (2/1010; 0.20%) of the drugs after taking it. However, those that gave reasons for not participating because Community Drug Distributors (CDD) did not come, had highest prevalence (40.74%) of LF compared to those that gave reasons as side effect (0.00%) for not taking the drugs. Overall LF prevalence of 25.64% was found in this study. There is poor knowledge of MDA programme among the participants in the communities examined. Similarly, microfilaria was present in some participants despite the MDA campaign in the selected states. Intensive awareness campaign and drug distribution need to be carried out in most of the remote communities in the affected states. Furthermore, members of the communities must be part of the CDDs for acceptability and effectiveness of the campaign.

Seasonal variation of semen quality among the three genotypes of native chicken

The study, conducted at the Bangladesh Livestock Research Institute, aimed to assess semen quality and characteristics in three types of indigenous cocks and explore seasonal variations. Ninety bird samples from three genotypes, namely non-descript Deshi (ND), Hilly (HI), and Naked Neck (NN), were used in this research. A total of 548 semen samples were collected to evaluate the color, volume, consistency, and transparency of semen in different seasons. Semen collection was performed using an abdominal massage technique, and volume was measured with a tuberculin syringe. For determining microscopic physical and morphological characteristics, computer-assisted sperm analysis (CASA) software was used. Results showed that among three types of indigenous cocks, semen quality differed (p < 0.001) from season to season. Semen quality was comparatively good for ND in the autumn season, for HI in the summer season, and for NN in the spring season. The highest semen volume (0.318 ± 0.010 ml) was recorded in the summer season, and the lowest semen volume (0.218 ± 0.008 ml) was recorded in the spring season. Genotype had a significant (p < 0.01) effect on semen volume, and the effect of semen and genotype interaction on semen volume was non-significant (p > 0.05). The sperm concentration was higher in NN compared to ND and HI. The percentages of static, bent tail, coiled tail, DMR, distal droplet, and proximal droplet were comparatively higher in the HI and NN genotypes. The color, consistency, and volume of semen were affected by genotype and season.

Inclusion of 1,3-dimethylxanthine improved quality parameters of bull semen

Methylxanthines are a unique class of drug, derived from the purine base xanthine and 1,3-dimethylxanthine (Theophylline) is the most commonly used methyl xanthine. The present study was conducted to study the effect of supplementation of 1,3-dimethylxanthine in semen extender on sperm motility and morphological parameters of semen. A total of 15 semen ejaculates were collected from three adult cattle bulls. Each ejaculate was divided into five groups. First group was kept as control (C) in which samples were diluted with Tris-egg yolk-glycerol extender and samples of other 4 groups (T-5, T-10, T-20 and T-40) were supplemented with 1,3-dimethylxanthine at a dose rate of 5, 10, 20 and 40 mM respectively. The diluted semen samples were incubated at 37°C in water bath and evaluated for various seminal parameters at 15 and 30 min of incubations. Sperm motility, sperm kinetic parameters and sperm abnormalities of semen of all groups were assessed using computer assisted sperm analyser (CASA). Morphological parameters of spermatozoa of control and supplemented groups were also evaluated. The results revealed that 1,3-dimethylxanthine supplementation resulted in significant increase in motile sperm concentration, kinematic parameters, sperm viability, sperms with intact plasma membrane as compared to other groups. However, sperm abnormalities viz., bent tail, coiled tail, distal droplet and distal midpiece reflex varied non-significantly in all groups. Thus, it can be concluded that 1,3-dimethylxanthine (Theophylline) inclusion at 10 mM concentration in semen extender improved the quality of semen.

Effects of mercury chloride on antioxidant and inflammatory cytokines in zebrafish embryos.

In this study, a zebrafish embryo toxicity model was employed, utilizing 24 h postfertilization (hpf) zebrafish embryos. These embryos were treated with varying concentrations of mercuric chloride for 96 h under static conditions. We assessed multiple parameters that reflected developmental abnormalities, behavioral alterations, morphological anomalies, antioxidant enzyme activities, including those of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), and glutathione S-transferase (GST), immune messenger RNA transcription levels of key factors such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and cyclooxygenase 2 (COX-2), as well as protein expression of TNF-α. The results revealed that embryos exposed to higher concentrations of mercury exhibited reduced hatchability and increased rates of morphological abnormalities and mortality at 48, 72, and 96 hpf. In addition, a concentration-dependent increase in developmental abnormalities, including cardiac edema, reduced body length, yolk sac edema, scoliosis, and bent tails, was observed. Larval behaviors, such as touch-induced escape responses, startle reactions, and turning actions, were found to be diminished in a concentration-dependent manner. Additionally, the activities of various antioxidative enzymes, such as SOD, CAT, and GST, exhibited an increase at higher mercury concentrations, with the exception of GPX activity, which decreased significantly in a dose-dependent manner (p < 0.05). Pro-inflammatory cytokine transcription levels, specifically TNF-α, IL-1β, IL-6, and COX-2, were significantly upregulated in a dose-dependent manner in the mercuric (II) chloride (HgCl2 ) treatment group compared with the control group. TNF-α protein expression was notably elevated in the larvae group treated with 300 and 400 nM HgCl2 .

Effects of Using Commercial and Homemade Extenders on Sperm Quality of Liquid Stored Semen of Horro Chicken Breed

This study aimed to evaluate the suitability of homemade tris-egg yolk-based and Commercial Beltsville poultry extenders for short-term storage of semen from the Ethiopian Indigenous Horro chicken breed at refrigeration temperature. A total of 30 Horro roosters with an average age of 40 weeks were used to collect semen. The treatments (T) in the sperm quality experiment were control (semen without extender added), semen extended with homemade extender (E1), and semen extended with commercial Beltsville Poultry Semen Extender (E2). Changes in spermatozoa motility, in vitro viability, and morphology were evaluated in fresh semen and semen diluted as 1:4 (v/v semen to extender) and stored for 4, 8, 12, and 24 hours at 4°C. During semen storage, there was a decrease in mass motility, an increase in morphologically abnormal spermatozoa with a high incidence of the bent tail, and an increase in dead spermatozoa. The commercial Beltsville poultry extender was found to be the most suitable extender regarding mass motility and in vitro viability of stored spermatozoa, but there was no significant difference in sperm abnormalities across all extenders. The results showed locally prepared tris-egg yolk-based extender could be a suitable extender for short-term storage of chicken sperm regarding the sperm quality attributes.

Changes in Sperm Morphology, Morphometry, and Motility from the Epididymis to the Vas Deferens in Rheas (Rhea americana, Linnaeus, 1758).

The objective was to characterize morphological, morphometric, and ultrastructural changes in rhea spermatozoa between the epididymis and the vas deferens. Sperm samples were collected from the reproductive tracts of seven adult individuals and evaluated for sperm characteristics using brightfield microscopy as well as ultrastructural features using scanning electron microscopy (SM). Mean sperm count tended to increase in the vas deferens (378.0 ± 135.0 × 106) compared to the epididymis (201.0 ± 77.4 × 106). Percentages of motile sperm grew from 37.0 ± 4.9% in the epididymis to 58.5 ± 7.7% in the vas deferens. The proportion of normal spermatozoa was 75.6 ± 1.8% and most common defects were bent tails (9.7 ± 0.9%). However, these proportions were not different between epididymis and vas deferens. SM analysis revealed further features of rhea spermatozoa. Normal rhea spermatozoa were threadlike with an acrosome (0.95 ± 0.0 µm), head (7.53 ± 0.01 µm), midpiece (2.08 ± 0.01 µm), and tail (30.7 ± 0.06 µm). Lengths of sperm acrosome, head, midpiece, and tail were longer in the vas deferens compared to the epididymis. Our findings suggest that rhea spermatozoa undergo a maturation process during the passage from the epididymis to the vas deferens.

DNALI1 interacts with the MEIG1/PACRG complex within the manchette and is required for proper sperm flagellum assembly in mice.

The manchette is a transient and unique structure present in elongating spermatids and required for proper differentiation of the germ cells during spermatogenesis. Previous work indicated that the MEIG1/PACRG complex locates in the manchette and is involved in the transport of cargos, such as SPAG16L, to build the sperm flagellum. Here, using co-immunoprecipitation and pull-down approaches in various cell systems, we established that DNALI1, an axonemal component originally cloned from Chlamydomonas reinhardtii, recruits and stabilizes PACRG and we confirm in vivo, the co-localization of DNALI1 and PACRG in the manchette by immunofluorescence of elongating murine spermatids. We next generated mice with a specific deficiency of DNALI1 in male germ cells, and observed a dramatic reduction of the sperm cells, which results in male infertility. In addition, we observed that the majority of the sperm cells exhibited abnormal morphology including misshapen heads, bent tails, enlarged midpiece, discontinuous accessory structure, emphasizing the importance of DNALI1 in sperm differentiation. Examination of testis histology confirmed impaired spermiogenesis in the mutant mice. Importantly, while testicular levels of MEIG1, PACRG, and SPAG16L proteins were unchanged in the Dnali1 mutant mice, their localization within the manchette was greatly affected, indicating that DNALI1 is required for the formation of the MEIG1/PACRG complex within the manchette. Interestingly, in contrast to MEIG1 and PACRG-deficient mice, the DNALI1-deficient mice also showed impaired sperm spermiation/individualization, suggesting additional functions beyond its involvement in the manchette structure. Overall, our work identifies DNALI1 as a protein required for sperm development.

Radical scavenging activity of Chlorophytum borivilianum L. root extract and its protective role in cauda epididymal sperm integrity in Mus musculus after gamma irradiation.

Background: Chlorophytum borivilianum L. is a recognized herbal medicine for the management of impotency in South Asian countries. In Ayurveda, it is used for the management of multiple health conditions, including diabetes, infection, and cardiovascular diseases. Parts of the plant have been used as excellent antioxidants and scavengers of free radicals. Since oxidative stress plays an important role in spermatogenesis and fertility in male populations, this study evaluated the role of ethanolic extract of C. borivilianum roots in epididymal sperm maturation against adversities posed by ionizing gamma irradiation. Materials and methods: Antioxidant potential of C. borivilianum root extract (CRE) was evaluated through DPPH (2,2-diphenylpicrylhydrazyl) and NO (nitric oxide) scavenging assays. Four groups of healthy Swiss albino mice were constituted, which were labeled as follows: Group I: sham control, Group II: 7-day pre-treatment with 50mg/kg CRE, Group III: 6Gy irradiation without pre-treatment, and Group IV: 7-day pre-treatment with 50mg/kg CRE and 6Gy irradiation on day 7. Swiss albino mice were observed for 30 days and later sacrificed to evaluate sperm quality parameters. Results: CRE showed a remarkable antioxidant potential with IC50 values of 46.37μg/ml and 98.39μg/ml for DPPH and NO, respectively. A significant decline (p < 0.001) in cauda epididymal sperm count, motility, and viability was observed in Group III animals. Group IV also showed a substantial decline (p < 0.01) in all three parameters compared to Group I; nonetheless, these were significantly higher than Group III. Morphological alterations indicated a coiled and bent tail, with the presence of cytoplasmic droplets in Group III, which declined substantially in Group IV. The ultrastructure of sperm indicated higher curvature of hook in Group III than Group IV, indicating specific interferences in the sperm maturation process. Conclusion: It was concluded that pre-treatment with 50mg/kg body weight of CRE could protect sperm during epididymal maturation against oxidative stress.

The Comparative Effects of Azanza garckeana Fruit Pulp and Melatonin on Serum and Testicular Oxidative Stress Changes, Live Sperm Cells and Spermatozoa Abnormalities Evoked by Chronic Administration of Bisphenol A in Rabbit Bucks

The study aimed to elucidate serum and testicular oxidative stress changes induced by bisphenol A (BPA) and their amelioration by Azanza garckeana (AG) pulp extract and melatonin. Adult New Zealand White rabbit bucks (n = 42), with average live weight of 1.2 ± 0.03 kg and aged 10-18 months were fed ad libitum on a commercial diet. They were randomly divided into seven groups of six (6) bucks each. Group A was administered distilled water (1.5 mL); group B, BPA (100 mg/kg); group C, AG (500 mg/kg); group D, melatonin (1.0 mg/kg); group E was pre-dosed for six weeks with BPA (100 mg/kg), then AG (500 mg/kg) for another six weeks; group F was pre-dosed for six weeks with BPA (100 mg/kg ), then melatonin (1.0 mg/kg) for another six weeks; and group G was pre-dosed for six weeks with BPA (100 mg/kg), then AG (500 mg/kg) and melatonin(1.0 mg/kg) for another six weeks. There was a significant increase (p &lt; 0.05) in the activities of both serum and testicular superoxide dismutase and catalase, concentrations of and reduced glutathione concentration as well as a decrease in MDA concentration in treatment groups. Percentage dead sperm, spermatozoa abnormalities such as detached sperm heads, free, coiled and bent tails in the groups exposed to BPA increased significantly (p &lt; 0.05) compared to controls. It is concluded that BPA-induced oxidative stress. The administration of AG only ameliorated this negative effects better than melatonin. However, optimum results was seen when both substances were administered synergistically.

Sulfamethoxazole (SMX) Alters Immune and Apoptotic Endpoints in Developing Zebrafish (Danio rerio)

Sulfamethoxazole (SMX) is a broad-range bacteriostatic antibiotic widely used in animal and fish farming and is also employed in human medicine. These antibiotics can ultimately end up in the aquatic ecosystem and affect non-target organisms such as fish. To discern the effect of SMX on developing zebrafish embryos and larvae, we investigated a broad range of sub-lethal toxicity endpoints. Higher concentrations of SMX affected survivability, caused hatch delay, and induced malformations including edema of the yolk sac, pericardial effusion, bent tail, and curved spine in developing embryos. Lower levels of SMX provoked an inflammatory response in larvae at seven days post fertilization (dpf), as noted by up-regulation of interferon (ifn-γ) and interleukin 1β (il-1β). SMX also increased the expression of genes related to apoptosis, including BCL2-Associated Agonist of Cell Death (bad) and BCL2 Associated X, Apoptosis Regulator (bax) at 50 µg/L and decreased caspase 3 (casp3) expression in a dose-dependent manner. SMX induced hyperactivity in larval fish at 500 and 2500 µg/L based upon the light/dark preference test. Collectively, this study revealed that exposure to SMX can disrupt the immune system by altering host defense mechanisms as well as transcripts related to apoptosis. These data improve understanding of antibiotic chemical toxicity in aquatic organisms and serves as a baseline for in-depth environmental risk assessment of SMX and antibiotics.

Toxicity and teratogenicity investigation of methanolic bark extract of Terminalia arjuna (Roxb.) wight and arn in zebrafish (Danio rerio) embryo model

The present research work aims to establish the teratogenic effect of the Terminalia arjuna (T.arjuna) bark extract using fish embryos through zebrafish embryo model. In the present study, selected zebrafish embryos were exposed to a range of methanolic extract of T.arjuna bark and the changes the embryos undergo were identified by comparing them with a control group. The embryo toxicity was assessed by observing morphological abnormalities after 24, 48, 72 and 96 h exposure and end teratogenicity points were assessed after 48 h exposure. All the embryos were imaged and analyzed for various developmental anomalies such as coagulated embryos, lack of somite formation, non-detachment of the tail, and lack of heartbeat, using imageJ software. From the results obtained, it was found that the embryos exposed with 10 mg/L of T.arjuna showed regular developmental patterns similar to control up to 72 h 5 % mortality was observed post 72 h period. 50 and 100 mg/L showed 100 % mortality after 96 h and 72 h respectively. At 48 h, Embryos treated with 50 mg/L of T.arjuna showed 5.58 % decreased heart beat rate, 7.89 % of decrease in eye diameter decreased head trunk angle of 3.56 % and increased yolk sac width of 0.79 %. After 48 h exposure to 50 and 100 mg/L of T.arjuna extract, 45 % and 85 % of the embryos showed the teratogenic endpoint of coagulation respectively with bent tail. Increased yolk sac width was observed in 100 mg/L treated embryos with 1.01 % after 24 h, also at 48 h decreased heart beat rate of 4.02 %,16.8 % of increased eye diameter, with decreased head trunk angle of 2.35 %, decreased yolk sac width of 5.13 % were observed. After 48 h exposure of T.arjuna bark extract, the morphological, physiological changes and teratogenic end points were eminent in 50 and 100 mg/L treated embryos, thus the results signify that methanolic bark extract of T. arjuna possess teratogenic effects at this concentration in zebrafish embryos.

Semen cryopreservation in Bornean Sun Bear (Helarctos malayanus euryspilus)

Semen cryopreservation is one common tool for extended genome storage and serves as insurance for declining biodiversity. In the endangered Bornean sun bear, semen was collected with electroejaculation and this is the first attempt at semen cryopreservation for this subspecies. Chilled semen with motility of more than 50 % was extended with Caniplus freezing medium, and cryopreserved with slow freezing protocol. A total of 22 cryopreserved semen straws were evaluated with computer-assisted sperm analysis and conventional semen evaluation tests as well as additional functional tests including evaluation of the acrosome (Rose Bengal Fast Green stain), plasma membrane (hypoosmotic swelling test), and chromatin (toluidine blue stain). Post-thaw semen quality was compromised with poor viability (27.6 ± 11.2 %), motility (8.3 ± 7.1 %), and progressive movement (1.3 ± 2.0 %) but maintained high integrity for the acrosome (71.1 ± 10.8 %), plasma membrane (54.7 ± 9.6 %), and chromatin (89.7 ± 7.6 %). The total post-thaw sperm abnormality was 51.7 ± 10.7 %, predominantly bent tail (28.0 ± 7.6 %) and proximal droplets (20.0 ± 11.8 %). Although this current cryopreservation was not a success, the compromised frozen-thawed semen may be a valuable resource in assisted reproductive technologies such as intracytoplasmic sperm injection. The three additional functional tests demonstrated were simple and affordable and hence are recommended to be part of the routine semen evaluation. Further research is required to develop species-specific cryopreservation protocols and to explore other assisted reproductive technologies in the Bornean sun bear.

Sperm factors associated with the production of equine blastocysts by intracytoplasmic sperm injection (ICSI) using frozen/thawed semen

Intracytoplasmic Sperm Injection (ICSI) using frozen/thawed sperm is a common procedure to obtain embryos from fertile or subfertile mares and stallions. Stallion-associated factors that impact the efficiency of ICSI have been studied less than those associated with the mare. Three experiments were conducted: Experiment 1: the effect of freezing extender composition and cryoprotectant; Experiment 2: the effect of sperm exposure to seminal plasma prior to freezing (ejaculated vs. epididymal sperm; two-freeze/thaw cycles each); and Experiment 3: the effect of sperm morphologic feature used for fertilization (normal vs. cytoplasmic droplet vs. bent tail); on the blastocyst rate after ICSI. In Experiment 1, stallion sperm was cryopreserved using commercially available extenders containing: a) 2% egg-yolk + milk + 4% glycerol (MFR5); b) 2% egg-yolk + milk + 2% glycerol + 3% methyl formamide (CMMFR5); c) 20% egg-yolk + 4.75% glycerol (LE); or d) 20% egg-yolk + 2% glycerol + 3% methyl formamide (CMLE). Sperm from each of the treatment groups were used for Piezo-driven ICSI on in vitro-matured equine oocytes (n = 321). Extender CMLE resulted in a lower cleavage rate (35%) than the other treatment groups (MFR5: 74%, CMMFR5: 62%, LE: 68%; P < 0.05). Extender MFR5 yielded a higher blastocyst rate per injected oocyte (21/82 [26%]) than the Groups LE (8/77 [10%]), CMLE (4/80 [5%]) or CMMFR5 (4/82 [5%]; P < 0.05). Extender MFR5 also yielded a higher blastocyst rate per cleaved oocyte (34%) than Groups LE, CMLE or CMMFR5 (15%, 14%, 8%; respectively P < 0.05). In Experiment 2, ejaculated (EJ) and epididymal (EPD) sperm from a fertile stallion which was initially cryopreserved in the CMLE extender, was thawed and re-cryopreserved in MFR5 extender for use in ICSI. Sperm from both groups (EJ vs. EPD) were used for ICSI on in vitro matured oocytes (n = 127). Differences were not detected for cleavage rate (EJ: 36/63 [57%] vs. EPD: 49/64 [77%]), blastocyst rate per injected oocyte (EJ: 11/63 [17%] vs. EPD: 11/64 [17%]), or blastocyst rate per cleaved oocyte (EJ: 31% vs. EPD: 22%) between treatment groups (P > 0.05). In Experiment 3, morphologically normal sperm (N), or sperm with proximal droplets (PD) or bent tails (BT), were obtained from a single fertile stallion and were used for ICSI on in vitro matured oocytes (n = 75). No differences were detected among treatment groups for cleavage rate (N: 19/25 [77%] vs. PD: 20/25 [88%] vs. BT: 18/25 [72%]), blastocyst rate per injected oocyte (N: 6/25 [24%] vs. PD: 5/25 [20%] vs. BT: 2/25 [8%]), and blastocyst rate per cleaved oocyte (N: 32% vs. PD: 23% vs. BT: 11%; P > 0.05). In conclusion, the current study indicates that freezing extender composition used for stallion sperm cryopreservation has an impact on the developmental competence of in vitro-matured equine oocytes after ICSI and in vitro culture. Furthermore, we were unable to detect differences on cleavage and blastocyst rates when performing ICSI when using: 1) ejaculated or epididymal sperm; or 2) sperm with different morphologic features. The results from the current study provide additional insight regarding stallion-related factors that should be considered when performing ICSI in horses.

Phthalate plasticizer decreases the prion-like protein doppel essential for structural integrity and function of spermatozoa

Di-n-butyl phthalate (DBP), a well-known endocrine disruptor, causes male reproductive dysfunction. To understand the underlying mechanisms, we performed histological, endocrinological, and biochemical analyses and assessed the expression of genes involved in spermatogenesis and sperm function according to OECD test guideline 407. Following 28 days of administration of the lowest observed adverse effect level dose of DBP to mice, no significant changes in body weight, testis and epididymis weights and histology, serum testosterone level, or testicular daily sperm production were found. Nonetheless, the motility of the epididymal sperm of the DBP group was significantly decreased together with an increase in the incidence of bent tails and abnormal heads. In the testes of the DBP group, lipid peroxidation (LPO) level was significantly increased and testicular Bcl-2 mRNA level was significantly decreased together with an increase in the Bax/Bcl-2 mRNA ratio. In the testes of the DBP group, levels of Prnd mRNA and protein and Pou4f1 mRNA, an activator of the Prnd promotor, were significantly decreased. Of note, prion-like protein doppel (PRND) was significantly decreased together with decreased PRND immunoreactivity in the head, midpiece, and tail of sperm. In the testes of the DBP group, levels of Sox9, Sgp1, and Sgp2 mRNA, which are functional Sertoli cell markers, were significantly decreased. Level of Amh mRNA, a Sertoli cell immaturity marker, was significantly increased together with that of Inha mRNA, suggesting deregulation of the brain-gonadal axis. Together, our findings suggest that DBP at present dosage may potentiate LPO generation and Sertoli cell immaturity via downregulation of Sox9 and disruption of the Pou4f1-Prnd gene network in post-meiotic germ cells without visible changes in spermatogenesis or testosterone level. This may result in structural and functional abnormalities in spermatozoa. Additionally, our findings suggest that assessment of the male reproductive toxicity of phthalate ester plasticizers based on conventional OECD test guidelines should be reconsidered.