Elizabeth Breininger

Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm

The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques.

Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm.

The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27-29%) and acrosome-reacted sperm (23-26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.

Protein tyrosine phosphorylation under capacitating conditions in porcine fresh spermatozoa and sperm cryopreserved with and without alpha tocopherol

The aim of this study was to evaluate the capacitation behaviour of fresh and α‐tocopherol frozen spermatozoa. Spermatozoa frozen with or without α‐tocopherol and fresh semen were incubated under capacitating conditions. Aliquots were collected at 0, 15, 30, 45, 60, 90, 120 and 180 min of incubation time. Parameters of semen quality were evaluated by optical microscopy and capacitation was determined by the epifluorescence chlortetracycline technique. Protein tyrosine phosphorylation was examined by Western immunoblotting. Motility, viability and intact spermatozoa were higher (P < 0.05) in fresh semen compared with frozen samples. These parameters significantly decreased, in every treatment, throughout the incubation time. Fresh semen showed a progressive increase in capacitated spermatozoa, reaching 25 ± 3% at 180 min. Cryopreserved semen had a fast increase at the beginning of incubation time (28 ± 5% at 45 min and 28 ± 3% at 30 min for samples with or without α‐tocopherol, respectively). The amount of an MW 32 kDa tyrosine‐phosphorilated protein, associated with capacitation, increased throughout incubation for fresh semen and spermatozoa cryopreserved with α‐tocopherol. The supplementation with α‐tocopherol preserved sperm plasma membrane, reflected not only in the acrosome integrity but also in a greater efficiency of energy production.

Boar sperm functionality is related to α-tocopherol content after freezing–thawing

Boar spermatozoa are sensitive to oxidative damage produced during cryopreservation. Our aim was to evaluate the participation of different antioxidants in the improvement of cryopreserved boar sperm functionality. Spermatozoa frozen with 200 μg ml−1α‐tocopherol, 0.5 mm 17β‐oestradiol or seminal plasma were used to evaluate sperm parameters and capacitation‐like changes. The 17β‐oestradiol and α‐tocopherol concentrations were assessed by RIA and HPLC respectively. Motility was improved but lipid peroxidation and capacitation‐like changes were diminished (P < 0.05) in antioxidant samples. A significant increase in 17β‐oestradiol concentration was detected in 17β‐oestradiol or seminal plasma samples. Alpha‐tocopherol content increased in α‐tocopherol, 17β‐oestradiol or seminal plasma samples, obtaining the lowest level in the α‐tocopherol ones. The 17β‐oestradiol or seminal plasma components may be acting in the regeneration of the α‐tocopherol antioxidant capacity. The α‐tocopherol concentration may be conditioning the cryopreserved boar sperm functionality. The addition of antioxidants could be useful to reduce oxidative stress, thus improving the functionality of cryopreserved boar spermatozoa.

Participation of membrane adenylyl cyclase in heparin-induced capacitation in cryopreserved bovine spermatozoa

The aim of this work was to study the participation of membrane adenylyl cyclase in heparin‐induced capacitation in cryopreserved bovine spermatozoa. Sperm suspensions were incubated in Tyrodes albumin lactate pyruvate medium in the presence of heparin (10 IU ml−1) or forskolin (1–75 μm), a well‐known membrane adenylyl cyclase activator. The participation of membrane adenylyl cyclase was confirmed using a specific inhibitor, 2′,5′‐dideoxyadenosine (6–25 μm). Spermatozoa capacitated with forskolin (25 μm) were incubated with bovine follicular fluid to evaluate their ability to undergo acrosome reaction. Capacitation percentages were determined by the fluorescence technique with chlortetracycline, and true acrosome reaction was determined by trypan blue and differential interferential contrast. The forskolin concentrations employed had no effect on progressive motility or sperm viability. Capacitation values induced by 25‐μm forskolin treatment (27.80 ± 2.59%) were significantly higher respect to the control (4.80 ± 1.30%). The inhibitor 2′,5′‐dideoxyadenosine prevented forskolin‐induced capacitation and significantly diminished capacitation induced by heparin. Follicular fluid induced physiological acrosome reaction in spermatozoa previously capacitated with 25‐μm forskolin (P < 0.05). Forskolin acts as a capacitation inducer and involves the participation of membrane adenylyl cyclase as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.

Biochemical and Morphological Alterations in Hearts of Copper-Deficient Bovines

Copper deficiency is an important disease of cattle that produces several clinical signs and lesions, due to alterations in copper-dependent enzymes. One of the organs affected by this deficiency is the heart (falling disease), but nevertheless, these cardiac lesions have not been extensively studied in bovines. The aim of this work was to propose a possible pathogenic mechanism for cardiac lesions in cattle affected by copper deficiency. Because of the possible existence of oxidative distress caused by low levels of copper-zinc-superoxide dismutase and cytochrome oxidase, ultrastructural and histological lesions have been evaluated in the heart of bovines in which a Cu deficiency had been induced using high molybdenum and sulfur levels in the diet. Our results indicated that copper deficiency produces significant damage in myocardium with high levels of lipid oxidation and a significant reduction in copper-zinc-superoxide dismutase activity leading to an oxidative distress situation. However, cytochrome oxidase activity was not significantly reduced. Histological observation revealed a significant increase in the amount of connective tissue, enlarged basement membranes of myocytes, and numerous Anichkov cells, in the hearts of deficient animals. Ultrastructural observation showed a significant enhancement in the mitochondrial volume density, with presence of lesions such as swelling and cristae disruption. We conclude that copper deficiency in bovines causes morphological lesions in the heart due to an oxidative damage produced by copper-dependent enzyme alterations.

Regulation of key enzymes of glucose metabolism in bovine COCs

The aim of this work was to study the regulation of PFK 1 and G6PDH, two key enzymes involved in glucose metabolism in cumulus oocytecomplexes (COCs), and its relationship with the oocyte maturation process. It was observed that the activity of PFK 1 in the presence of ATP was inhibited whereas the addition of AMP increased the activity (P < 0.05). To verify the effect of the physiological modulators on the COC glycolytic pathway, the lactate production during IVM and the maturation rate were evaluated. In accordance with the enzymatic activity, the glycolytic activity evaluated by lactate production and the maturation rate diminished (P < 0.05) with the addition of ATP. While the AMP had a dose response effect on the lactate production, the maturation rate remained unaltered. It was observed that NADPH inhibited the activity of the G6PDH and the addition of NADP increased the activity of the enzyme (P < 0.05). To verify the effect of the physiological modulators on the COC pentose phosphate pathway (PPP), the proportion of colourless oocytes evaluated by brilliant cresyl blue (BCB) and the maturation rate were carried out. In presence of NADPH an inhibition (P < 0.05) on PPP and maturation rate was observed. On the other hand, NADP had no effect on PPP activity and maturation rate. The present study shows that the regulation of key enzymes of glucose metabolism in bovine COCs are regulated mainly by the energetic charge and the redox status. We also reported a tight relation between the activity of the PFK 1 and G6PDH enzymes, glycolytic and PPP activities and the oocyte maturation process.