Effects of supplemental dietary vitamin C on quality of semen from Nile tilapia (Oreochromis niloticus) breeders

The objective of this study was to evaluate the effects of vitamin C on growth and quality of semen from Oreochromis niloticus breeders. One hundred and sixty males were fed with different levels of vitamin C (0, 261, 599 and 942 mg/kg diet). The higher weight values were recorded for 599 (166 g) and 942 (175 g) mg of vitamin C/kg diet. Sperm motility, vigour and concentration were higher with 599 and 942 mg of vitamin C/kg diet. The semen volume, gonadosomatic index and plasma protein data from the last week showed a direct relationship with increasing levels of vitamin C. No changes were observed in the hepatosomatic index and blood glucose. The haematocrit and erythrocyte showed higher values estimated by equations derived at 850 and 638 mg vitamin C/kg diet, respectively. The leucocytes were inversely proportional to the increasing levels of vitamin C. After 100 days of feeding, animals fed the diet containing 942 mg vitamin C/kg diet had higher sperm motility, linearity, curvilinear velocity, straight line velocity and average path velocity (p < .05). Higher values of beat cross‐frequency were observed in broodfish fed diets containing 942 and 599 mg vitamin C/kg. The different vitamin C levels did not cause differences in straightness, lateral head displacement and sperm morphology. For Nile tilapia males on intensive rearing and handling conditions, vitamin C levels between 599 and 942 mg/kg may be used for a better performance and quality of semen.     

Effects of Cryopreservation on Bull Spermatozoa Distribution in Morphometrically Distinct Subpopulations

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.     

Effect of β-hydroxy-β-methylbutyrate (HMB) administration on volumetric bone mineral density, and morphometric and mechanical properties of tibia in male turkeys

This study was performed to investigate the effects of β-hydroxy-β-methylbutyrate (HMB) administration on skeletal system properties in turkeys. Thirty-two males were randomly divided into two groups at the age of 35 days of life. The first group included control turkeys (n = 16) treated with placebo, while the second group of birds (HMB group; n = 16) was administered orally with calcium salt of HMB during the last 15 weeks of life. The turkeys were sacrificed at the age of 20 weeks and tibia was isolated for analysis of bone geometrical parameters, volumetric bone mineral density (vBMD) and mechanical properties. Furthermore, assessment of free amino acid concentrations in plasma was performed. The results showed a 6.3% increase of vBMD of tibia in response to HMB treatment (p < 0.01). Cross-sectional area, second moment of inertia, maximum elastic strength and ultimate strength of tibia were significantly increased in HMB-treated turkeys by 21.3%, 49.0%, 27.2% and 28.3%, respectively (p ≤ 0.01). β-hydroxy-β-methylbutyrate administration increased plasma concentrations of proline,glutamate, leucine, isoleucine, valine, alanine, aspartate, phenylalanine and cysteic acid (p < 0.05). These results indicate that long-term administration of HMB improves vBMD, and geometrical and mechanical properties of skeletal system in turkeys, and that these effects are associated with improved plasma amino acid concentrations.     

Late Pleistocene Desiccation of Lake Victoria and Rapid Evolution of Cichlid Fishes

Lake Victoria is the largest lake in Africa and harbors more than 300 endemic species of haplochromine cichlid fish. Seismic reflection profiles and piston cores show that the lake not only was at a low stand but dried up completely during the Late Pleistocene, before 12,400 carbon-14 years before the present. These results imply that the rate of speciation of cichlid fish in this tropical lake has been extremely rapid.     

Assessment of Extent of Apoptosis and DNA Defragmentation in Chilled Semen of Stallions During the Breeding Season

The objective of the study was to assess apoptosis and DNA defragmentation in equine semen diluted and chilled to +4°C. Semen was collected from nine fertile stallions, including four Arabian thoroughbreds and five coldbloods. Examinations were carried out immediately after semen collection (0) and at five storage times (24, 48, 72, 96 and 120 h). The basic semen evaluation was performed in terms of volume, sperm concentration, viable sperm percentage, progressive motility and morphology. Using flow cytometry, DNA defragmentation and cell membrane integrity of spermatozoa were determined. The results of basic tests did not demonstrate significant differences amongst stallions, except for progressive sperm motility, which was significantly higher (p < 0.05) in the semen of Arabian stallions. In the semen of the same stallions, a significant decrease in the percentage of alive spermatozoa was observed at 72, 96 and 120 h of storage, whereas a significant increase in the number of spermatozoa with DNA defragmentation was found after 24 h storage. In the semen of coldblood stallions, significantly reduced live spermatozoa percentage was observed at 96 and 120 h, while increased DNA defragmentation was observed at 48 h. These findings demonstrated that the semen of Arabian stallions chilled to +4°C retained original characteristics until 24 h of storage, whereas in coldbloods, these were preserved up to 48 h of storage.     

Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Effects of Hydrogen Peroxide (H2O2) on Fresh and Cryopreserved Buffalo Sperm Functions During Incubation at 37°C In Vitro

The magnitude of damage to buffalo spermatozoa during incubation with different levels of H₂O₂ was assessed. A total number of 24 ejaculates from four Murrah buffalo bulls were analysed in the study. Each ejaculate was split into two parts (part I and II). Part I was extended in Tris–egg yolk–citrate extender (20% egg yolk:7% glycerol), equilibrated (4 h at 5°C) and cryopreserved in 0.5‐ml French straws and stored in liquid nitrogen. The other part was utilized for fresh semen studies. The sperm in fresh, equilibrated and frozen–thawed semen was separated by centrifugation (1500 g ; 15 min) and were washed with sperm TALP. The sperm cells were re‐suspended in incubation TALP at the rate of 10⁸ sperm cells per millilitre and incubated with 0, 10, 25, and 50 μm H₂O₂ per ml at 37°C. Sperm motility, viability and intact acrosome percentages were assessed at 15‐min intervals up to 60 min of incubation. Lipid peroxidation levels of sperm were assessed at 0 and 60 min of incubation. The results of the experiment revealed that sperm motility decreased drastically during incubation with H₂O₂. Among the different levels of H₂O₂, the 50‐μm H₂O₂‐incorporated group had significantly (p < 0.05) higher malonaldehyde (MDA) level than the other groups. In the 50‐μm H₂O₂‐incorporated group, the MDA levels in fresh, equilibrated and frozen–thawed semen after incubation for 60 min were 961.6 ± 12.7, 991.8 ± 10.3 and 1234.9 ± 9.6 nm per 10⁹ spermatozoa respectively. An inverse relationship was observed between sperm motility, viability, intact acrosome percentages and concentration of H₂O₂ and duration of incubation. The decrease in sperm functions with duration of incubation and concentration of H₂O₂ was significantly (p < 0.05) higher in frozen–thawed than fresh and equilibrated spermatozoa.