Effects of the cryopreservation process on dog sperm integrity.

Sperm cryopreservation has become an indispensable tool in reproductive biology. However, frozen/thawed semen has a short lifespan due to loss of sperm cell integrity. To better understand which sperm cell structures are compromised by the cryopreservation process and apoptosis markers, the sperm of five healthy mature dogs was analyzed in this study. Analysis was performed after collection, cooling, and thawing via computer assisted sperm analyzer (CASA) and evaluation of membrane fluidity and permeability, phosphatidylserine translocation (Annexin V), membrane integrity, mitochondrial membrane potential, membrane lipid peroxidation (LPO) and activity of the apoptotic markers caspases 3 and 7 by flow cytometry. Cryopreservation decreased total and progressive motility and the percentage of rapid sperm (P < 0.01). Damage to sperm cells was confirmed by Annexin V (P < 0.01), indicating that capacitation-like changes were induced by the cryopreservation procedures. An increase in sperm membrane fluidity was also noted in frozen/thawed samples (P < 0.01). Plasma and acrosomal cell membranes were affected (P < 0.01), with decreases in the subpopulation displaying high membrane potential (P < 0.01). Membrane LPO was increased in thawed sperm compared to cooled sperm (P < 0.05) but was not different from that in fresh sperm. No differences were observed in caspase 3 and 7 activity after cooling, freezing, or thawing. In conclusion, total and progressive motility, plasma membrane integrity and mitochondrial membrane potential suffered from the deleterious effects caused by cryopreservation, unlike the activity of caspases that remained stable during the freezing process.

Sperm sexing with density gradient centrifugation in dogs.

Sexed sperm in dogs is of interest because of being polytocous, and as a result, the greatest number of offspring of the same sex can improve the market, although few studies assessing sperm sexing have been performed in this species. The present study, therefore, was conducted to evaluate the effects on sperm quality and the effectiveness of three discontinuous density gradients to separate dog sperm containing X and Y chromosomes. Thirty ejaculates from ten adult dogs were collected by digital manipulation of the penis. Cells were separated using gradients of Percoll® and Percoll® associated with Nycodenz® or Ficoll. The cells were evaluated for motility by the CASA system (Computer-Aided Semen Analyzer) and for concentration and recovered sperm concentration (after centrifugation), sperm morphology, plasma and acrosomal membrane integrity, and mitochondrial function pre- and post-centrifugation. The percentage of sperm containing X and Y chromosomes was also evaluated pre- and post-centrifugation by quantitative real-time PCR (qPCR). The use of the Ficoll gradient resulted in the greatest sperm quality after centrifugation; however, no sperm enhancement containing X or Y chromosome occurred with use of any of the methods (Percoll® 54.8 ± 1.9 compared with 45.2 ± 1.9; Percoll® associated with Nycodenz® 53.2 ± 2.0 compared with 46.8 ± 2.0; and Percoll® associated with Ficoll 55.0 ± 1.5 compared with 45.0 ± 1.5 for the percentages of cells containing the X and Y chromosomes, respectively). Thus, it was concluded that the technique of sexing dog sperm using density gradients was not effective for commercial application.

Lipid peroxidation and generation of hydrogen peroxide in frozen-thawed ram semen cryopreserved in extenders with antioxidants.

The objective of this study was to evaluate the effect of addition of the antioxidants Trolox and catalase to a ram semen cryopreservation extender on lipid peroxidation and hydrogen peroxide generation on the extender and in the thawed semen. Semen was collected from 23 Santa Inês rams (one ejaculate per ram) and diluted at 32°C to a concentration of 400×106 cells/ml in one of the following solution: Tris-egg yolk extender (control), or the same extender supplemented with either 50µM Trolox/108 sperm (Trolox), 50µgcatalase/ml (Catalase) or a combination of Trolox and catalase (Tro+cat, 50µM Trolox/108 sperm and 50µg catalase/ml). The semen was loaded into 0.25ml straws, cooled and frozen in a programmable freezer and subsequently stored in liquid nitrogen. Prior to evaluation, frozen straws were thawed in a water bath (42°C for 20s). Lipid peroxidation (LPO), both spontaneous and catalyzed, on the semen and the extender were measured using the thiobarbituric acid (TBA) assay in accordance with the method described by Buege and Aust (1978). Hydrogen peroxide (H2O2) generation was measured using the horseradish peroxidase-dependent oxidation of phenol red to a derivative with absorbance at 610nm, according to the method described by Pick and Keisari (1980). Spontaneous LPO resulted in the least production of thiobarbituric acid-reactive substances (TBARS) in the Tro+cat (1.37±0.02nMol/108 sperm), compared to amounts in the other treatments groups. In the catalyzed LPO experiments, the least (P<0.05) amounts of TBARS were observed in Trolox (2.52±0.02nMol/108 sperm) and Tro+cat (2.54±0.02nMol/108 sperm) groups, compared to the control (3.81±0.02nMol/108 sperm) and catalase (3.83±0.02nMol/108 sperm) groups. Hydrogen peroxide generation was less (P<0.05) in the Trolox (6.00±0.18nMol/40×10(6)sperm/±40min) and Tro+cat (6.08±0.18nMol/40×106sperm/±40min) groups than in the control (6.97±0.18nMol/40×106 sperm/±40min) and catalase (6.53±0.18nMol/40×106 sperm/±40min) groups. Compared to the control group, Trolox and catalase treatment significantly reduced TBARS in catalyzed LPO and hydrogen peroxide concentrations in the samples (P<0.05). ROS (reactive oxygen species) generation occurred in all extenders, without sperm cells. The data presented provide evidence that ROS are produced in ram semen, both in the extender and during the freezing and thawing process. In addition, the data suggest that the antioxidants Trolox and catalase may be used to control the oxidative stress imposed on ram spermatozoa by the cryopreservation process.