Lactoferrin increases sperm membrane functionality of frozen equine semen.

During cryopreservation, sperm was submitted to an increase in reactive oxygen species generation. This work aimed to improve the quality of frozen equine sperm after the addition of antioxidants lactoferrin (Lf) and catalase (Cat) to a freezing extender. Semen from six stallions was frozen with the extenders: F1) control, INRA 82 freezing extender, F2) F1 + 500 µg/ml Lf and F3) F1 + 200 IU/ml Cat. After thawing, sperm motility parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome-reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite, hydroperoxide and iron concentrations of frozen semen were measured with spectrophotometry. The percentage of functional membrane sperm treated with Lf was higher (50.7% ± 11.6%) compared to that of the control (37.6% ± 15.6%), while the iron (61.4 ± 11.6 vs 73.3 ± 13.8 mg/dl) and nitrite concentrations (16.3 ± 7.1 vs 25.9 ± 4.2 µM/µg protein) were lower, respectively (p < .05). Thus, it can be suggested that Lf protect stallion spermatozoon during freezing as it has increased the percentage of sperm with functional membrane and decreased the lipid oxidant agents.

Evaluation of Fertilizing Ability of Frozen Equine Sperm Using a Bovine Zona Pellucida Binding Assay.

BACKGROUND: Frozen equine semen has lower fertility compared to cooled semen. Due to the difficulty to obtain equine oocytes, a heterologous zona pellucida binding assay (ZBA) is an alternative method to predict the fertilizing capability of equine frozen sperm. The rate of capacitated and hyperactivated sperm according to their motility characteristics were analyzed with a Computer Assisted Sperm Analyzer. We believe this report describes for the first time the in vitro hyperactivation induction and the heterologous ZBA to predict the fertilizing ability of frozen equine sperm. OBJECTIVE: This work aimed at developing an assay to evaluate the fertilizing ability of frozen equine sperm using a bovine ZBA with the use of an in vitro capacitation and hyperactivation media with procaine and calcium ionophore A23187, respectively. MATERIALS AND METHODS: The sperm motility characteristics, intact and acrosome reacted sperm rates, and number of stallion sperm bound to the bovine ZP were calculated. RESULTS: The procaine group showed a hyperactivation motility pattern, although it improved ZP sperm binding similarly to the capacitation group. CONCLUSION: The capacitation medium improved the IVF capability of frozen equine sperm, allowing the highest possibility of sperm-oocyte interaction.

Milk, caseinate and lactoferrin addition to equine semen cooling extenders.

Cooled semen has been used routinely to prolong sperm viability until artificial insemination time. However, spermatozoa are subjected to oxidative stress. The aim of the present work was to investigate the protective and antioxidant effect of the milk proteins lactoferrin (Lf) and caseinate added to equine semen cooling extenders. Semen from six stallions was cooled at 5 °C after resuspension with C1) milk- and glucose-based, C2) 0.6% caseinate, C3) C2 + Lf 200 µg ml-1 , C4) C2 + Lf 500 µg ml-1 and C5) C2 + Lf 1000 µg ml-1 extenders, and kept at 5 °C for 24 h. Sperm motility characteristics and intact membrane rates were not different among the treatments (P > 0.05). As a result of the cooling process, the nitrite concentration increased significantly in the cooled semen (69.6 ± 78.9 µm per ×106 spermatozoa) compared with the fresh semen (8.6 ± 1.9 µm per ×106 spermatozoa). In contrast, the H2 O2 concentrations were lower in the 0.6% caseinate extender (265.9 ± 221.3 µm per ×106 spermatozoa) than in the milk extender (430.9 ± 199.8 µm per ×106 spermatozoa, P < 0.05), showing an antioxidative effect of the caseinate compared with the milk. However, in all groups, hydrogen peroxide concentrations were similar to the undiluted fresh semen (332.8 ± 151.3 µm per ×106 spermatozoa). Caseinate showed to be as efficient as milk to protect equine-cooled spermatozoon.

Caseinate protects stallion sperm during semen cooling and freezing.

Extenders with a defined composition containing only components with clearly protective effects on sperm during storage would be an advantage. The aims of the present work were to assess whether caseinate, improves cooled and frozen equine semen quality. Semen from six stallions were suspended with four different cooling extenders C1) Kenney extender; C2) 0.6 % caseinate; C3) 2.7 % caseinate ; and C4) C1 + 2.1 % caseinate, and frozen extenders: F1) INRA 82 extender; F2) 1.35 % caseinate; and F3) 2.7 % caseinate. Although there was no significant difference between the motility rate among the cooled (C1:45.0, C2:36.7, C3:38.3 and C4:48.3) and frozen extenders (F1:16.9, F2:21.1 and F3:18.6), significant higher values of sperm velocity variables were observed with the 1.35 % caseinate extender compared to the control (VSL: 40.8 x 18.9 and VAP: 46.8 x 25.0 µm/s), respectively. Caseinate seemed to be responsible for sperm protection during preservation and showed to be as efficient as milk.

Ribavirin, but not interferon alpha-2b, is associated with impaired osteoblast proliferation and differentiation in vitro.

Hepatitis C treatment with interferon alpha-2b (IFN-alpha) and ribavirin has been related to decreased bone mineral density. The aim of this study was to investigate the in vitro effects of different concentrations of ribavirin and IFN-alpha on osteoblast-like cells. Human osteoblast-like cells obtained by the outgrowth of cells from bone chips were exposed to ribavirin (0.1-10 microg/mL) or IFN-alpha (0.1-1000 UI/mL). At regular time-points, cultures were harvested for posterior analysis. Alkaline phosphatase (ALP) activity was determined on days 7 and 14, and cell growth was accessed by C3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell count on days 1, 3, 5, and 7. Flow cytometry analysis was used for investigating cell death on days 1, 3, 5, and 7. IFN-alpha affected ALP expression only at the higher concentration (1000 UI/mL) after 7 days (P < 0.05). No effects were detected in cell growth. In ribavirin treated cultures, concentrations higher than 2.5 microg/mL were associated with a decrease in ALP activity within 7 and 14 days (P < 0.01 and P < 0.001, respectively). Furthermore, the reduction in cell growth was dose-dependent and was detected after the fifth day. This decrease can be explained by an increase in the number of dead cells and a decrease in cell proliferation. In conclusion, our experiments demonstrated that ribavirin reduced, in a time- and dose-dependent manner, the number of metabolically active cells through a decrease in proliferation and an increase in cell death, and induced an impairment in osteoblast differentiation. These negative effects of ribavirin on osteblast-like cells might contribute to the bone loss reported in vivo.

Prilocaine: an old anesthetic agent and a new ophthalmic procedure.

We present a new local anesthetic technique for ophthalmic surgery that reduces the risks associated with retrobulbar and peribulbar anesthesia. This method uses topical proparacaine plus 1.5 mL of prilocaine (3%) with felypressin injected into the subconjunctival (sub-Tenon's) space. Of 5210 consecutive adult patients in whom the technique was used, all demonstrated adequate analgesia. Sixty-three (1.2%) of the eyes required supplemental analgesia, provided by a single injection of prilocaine (0.5 mL). Ecchymosis and subconjunctival hemorrhage developed in 63 (1.2%) of the eyes. There were no instances of ptosis.