Poor sperm viability post-thaw has resulted in constant research into methods of cryopreservation of canine semen. One factor that may be involved in poor viability is sperm oxidative stress caused by excessive formation of reactive oxygen species. The present study was performed in order to evaluate the effect of different concentrations of ascorbic acid (AA) and glutathione (Glu) added to an extender for the freeze-thawing of dog sperm. Semen from five mature dogs was collected and frozen in two studies. Prior to and after freezing, sperm motility, morphology and membrane status were examined. In addition, sperm motility was examined up to 120 min after thawing to evaluate thermo-resistance. In study I, semen was collected twice from each dog. On both occasions, semen was divided into three aliquots: control, Glu 1 mM and Glu 5 mM. In study II, semen was collected twice and divided into three aliquots; control, AA 50 microM and AA 250 microM. Initial sperm motility was significantly higher in sperm diluted with AA 50 microM; sperm longevity, however, measured by a thermal-resistance test (TRT), was higher for Glu treatments. Higher concentration of Glu produced significant improvement in TRT and membrane status, whereas higher concentration of AA had a negative impact in sperm longevity. Antioxidant supplementation to semen freezing extenders improved semen quality post-thaw. Moreover, Glu had the most beneficial effect when supplemented at 5 mM.

SummaryThe aim of this study was to assess the effect of exogenous DNA and incubation time on the viability of bovine sperm. Sperm were incubated at a concentration of 5 x 106/ml with or without plasmid pEYFP-NUC. Fluorescent probes, propidium iodide/Hoechst 33342, FITC-PSA and JC-1, were used to assess plasma membrane integrity (PMI), acrosome membrane integrity (AMI) and mitochondrial membrane potential (MMP) respectively at 0, 1, 2, 3 and 4 h of incubation. Exogenous DNA addition did not affect sperm viability; however, incubation time was related to sperm deterioration. Simultaneous assessment of PMI, AMI and MMP showed a reduction in the number of sperm with higher viability (integrity of plasma and acrosome membranes and high mitochondrial membrane potential) from 58.7% at 0 h to 7.5% after 4 h of incubation. Lower viability sperm (damaged plasma and acrosome membranes and low mitochondrial membrane potential) increased from 4.6% at 0 h to 25.9% after 4 h of incubation. When PMI, AMI and MMP were assessed separately we noticed a reduction in plasma and acrosome membrane integrity and mitochondrial membrane potential throughout the incubation period. Therefore, exogenous DNA addition does not affect sperm viability, but the viability is reduced by incubation time.

The aim of this research was to analyze oestrogen receptor-alpha (ERalpha), ERbeta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells' total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ERbeta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ERbeta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ERalpha and ERbeta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ERalpha was expressed throughout the oestrous cycle.

The inefficiency of embryo cryopreservation protocols limits the broad use of in vitro production (IVP) of bovine embryos. The aim of this work was to identify the damage caused by cryopreservation and embryo culture of IVP bovine embryos after thawing by in vitro development before and after cryopreservation. Cumulus–oocyte complexes were in vitro-matured, fertilized, and cocultured on granulosa cells in SOF with amino acids (SOFaa) supplemented with FCS. Expanded blastocysts (n = 600) harvested on Days 7 to 9 were submitted to controlled freezing [controlled group: 10% ethylene glycol (EG) for 10 min and 1.2°C min–1 cryopreservation], quick-freezing [quick group: 10% EG for 10 min, 20% EG + 20% glycerol (Gly) for 30 s], or vitrification [vitrification group: 10% EG for 10 min, 25% EG + 25% Gly for 30 s] protocols. The embryos of the control group were not exposed to cryoprotectant or cryopreservation method, and the hatching rate was evaluated on Day 12 post-insemination. The straws (quick and vitrification groups) were first placed on nitrogen vapor (0.8 cm over the liquid nitrogen) for 2 min and then immersed in liquid nitrogen. Embryos were thawed in air for 10 s followed by a 25°C water bath for 20 s. Embryos were rehydrated in PBS + 0.2% BSA + 0.3 m sucrose and PBS + 0.2% BSA for 3 min each. To evaluate development of frozen–thawed embryos, they were cocultured on granulosa cells in TCM-199 or SOFaa both supplemented with FCS for 4 days. Hatching rate of the control group was 46.1%. Data were analyzed by PROC MIXED model of SAS System for Windows®. For TCM-199, the controlled group hatching rate was 44.65 ± 5.94%, quick group did not hatch, and vitrification group showed hatching rates of 9.4 ± 6.8%. For SOFaa, the controlled group hatching rate was 11.6 ± 3.4%, embryos submitted to the quick group did not hatch, and the vitrification group showed hatching rates of 8.7 ± 4.5%. Values were significant at P < 0.05. The controlled group showed a difference compared with the other groups of cryopreservation in both media (TCM-199 and SOFaa). However, TCM 199 showed high rates of re-expansion and hatching. In conclusion, the culture medium influences embryo development after cryopreservation, and TCM-199 is more appropriate than SOFaa. Financial support by FAPESP (04/05335-1).

Duchenne’s muscular dystrophy (DMD) is the most common human muscular affliction with high frequency in male individuals. The condition is characterized by progressive muscle degeneration, weakness, and loss of motion capacity. These individuals exhibit longer lifespans resulting from improvements in technical patient care and now raise new questions ranging from ethical to physiological issues never observed before, such as the possibility of reproduction. Thus, this study used Golden Retriever muscular dystrophy (GRMD)-affected dogs, a natural experimental model of DMD, to evaluate semen quality and oxidative stress by thiobarbituric acid reactive substances (TBARS) test. Thirty-seven ejaculates from 4 non-affected Golden Retriever and 5 GRMD-affected dogs (from 1.5 to 4.5 years old) were collected monthly and evaluated as 5 different replicates. Semen samples were processed, and initial analysis comprised of sperm concentration, percent of straightforward motility and morphology. Samples were then diluted to a final concentration of 106 spermatozoa mL–1 of prior incubation with each fluorescent probe. Acrosome integrity assessment was conducted with PSAFITC (100 g mL–1), and mitochondrial activity was assessed by JC1 (50 g mL–1). Fluorescence-activated cell sorting was performed in 103 spermatozoa, from a pre-selected gate that contained only these cells. Seminal plasma was submitted to TBARS quantification by spectrophotometer under normal and oxidative conditions (stress). Parametric data were compared by Student’s t-test. There were no significant differences in ejaculate volume, sperm concentration, acrosomal integrity, mithocondrial activity, and ejaculate TBARS tests between GRMD and normal dogs. Data are summarized in the table. GRMD does not affect the production and quality of semen of dogs. This animal model suggests that ejaculates from DMD men can be obtained by the proper stimulation and that conventional in vitro fertilization techniques combined with preimplantation genetic diagnosis for single gene disorders may be able to satisfy the desire of paternity of DMD patients with the birth of non-affected children. Table 1.Sperm analysis and TBARS tests of ejaculates from GRMD and normal dogs FAPESP for the financial support (06/50272-3); Surgery Department and Reproduction Department of FMVZ-USP; Canil GRMD Brazil.

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

Heat-stress induced maternal hyperthermia has been shown to compromise the series of events associated with oocyte growth and maturation reducing oocyte competence. Such events are regulated by a variety of growth factors and dynamic communication between the oocyte and its surrounding cumulus cells. The objective of the current study was to evaluate the modulatory effects of COCs quality and IGF-I on mitochondrial membrane potential (MMP) and apoptosis in cumulus cells induced by heat shock. In this study high (≥3 layers of compact cumulus cells and homogeneous cytoplasm) and low-grade COCs (<3 layers of less compact cumulus cells and irregular cytoplasm) derived from slaughterhouse ovaries were exposed to control (CTR: 39°C) or heat shock (HS: 41°C) treatments in the presence of 0 or 100 ng mL-1 IGF-I during the first 12 h of in vitro maturation (12 h-IVM). Immediately after 12 h-IVM COCs were denuded by repeated pipetting and cumulus cells evaluated for MMP (MitoProbe JC-1 assay kit. JC-1 is a cationic dye that exhibits potential-depend accumulation in the mitochondria) and apoptosis (Annexin V-FITC and propidium iodide) by flow cytometry (Guava EasyCyte Mini Flow Cytometry System, Millipore, Billerica, MA, USA). This factorial experiment was replicated 4 times using 75-100 COCs per treatment. Data were subjected to three-way analysis of variance using the General Linear Models procedure of SAS. Results are shown in Table 1. Exposure of high and low-grade COCs to HS reduced (P < 0.01) the percentage of cumulus cells carrying high MMP regardless of IGF-I. Even though HS caused cumulus cells mitochondrial membrane depolarization there was neither temperature nor COCs quality effect on cumulus cells apoptosis as indicated by the lack of phosphatidylserine (PS) translocation from the inner to the outer leaflet of the plasma membrane. On the other hand, addition of IGF-I to maturation medium reduced (P < 0.05) the percentage of cumulus cells labeled with Annexin V + PI regardless of COCs quality or temperature. There was no statistical interaction between COCs quality × IGF-I × temperature. In conclusion, exposure of COCs to HS during 12 h-IVM caused cumulus cells mitochondrial depolarization without inducing apoptosis. It is possible that a period longer than 12 h is required for most PS translocation to occur in cumulus cells. Moreover, IGF-I exerted protective effect reducing cumulus cells late apoptosis/necrosis events. Table 1.Effect of heat-shock and IGF-I on cumulus cells mitochondrial membrane potential and apoptosis. Results are least-squares means ± SEM.