BackgroundIn order to improve the efficiency of bovine sperm cryopreservation process, it is important to understand how spermatozoa respond to differences in temperature as well as the ability to recover its own metabolism. The combination between flow cytometry approach and antioxidant enzymes activity allows a more sensible evaluation of sperm cell during cryopreservation. The aim of this study was to evaluate sperm attributes and antioxidant enzymes activity during different stages of cryopreservation process. Semen samples from Holstein bulls (n = 4) were separated in 3 treatments: fresh (37 °C); cooled (5 °C); and thawed. Evaluation occurred at 0 h and 2 h after incubation. Membrane integrity, mitochondrial membrane potential (MMP) and DNA damages were evaluated by flow cytometry; activities of antioxidant enzymes such as catalase, superoxide dismutase and gluthatione peroxidase were measured by spectrofotometry.ResultsThere was an increase in the percentage of sperm with DNA damage in the thawed group, compared to fresh and cooled, and for 2 hs of incubation when compared to 0 h. Considering MMP, there was an increase in the percentage of cells with medium potential in thawed group when compared to fresh and cooled groups. Opposingly, a decrease was observed in the thawed group considering high mitochondrial potential. Also in the thawed group, there was an increase on cells with damaged acrosome and membrane when compared to fresh and cooled groups. Significant correlations were found between antioxidant enzymes activity and membrane or mitochondrial parameters.ConclusionBased on our results, we conclude that cryopreservation affects cellular and DNA integrity and that the critical moment is when sperm cells are exposed to freezing temperature. Also, our study indicates that intracellular antioxidant machinery (SOD and GPX enzymes) is not enough to control cryodamage.

NZB/NZW mice were treated with various immunosuppressive drugs used in human SLE. Cyclophosphamide (5 mg/kg 6 days out of 7), alone or with prednisolone, was better than azathioprine, prednisolone, or azathioprineplus-prednisolone, in prolonging survival and/or reducing proteinuria, Coomb's antibodies, antinuclear antibodies, and glomerular deposits of y-globulin. Intermittent bolus therapy with cyclophosphamide (50 mg/kg/lO days) was as effective as daily therapy. However, 61 % of the mice receiving any cyclophosphamide regimen developed malignant tumors compared to none in the other groups. immune complex glomerulonephritis and numerous circulating autoantibodies, many of which are directed against nuclear constituents. Their illness resembles systemic lupus erythematosus (SLE) in man. Therefore, they provide a useful model for studies of therapeutic agents used in SLE.Numerous reports demonstrate the efficacy of various immunosuppressive agents in New Zealand mice. These include single-drug therapy with corticosteroids ( 1 3 , azathioprine (2-4), and cyclophosphamide (2,5-9), as well as studies of various combinations of these drugs (3,lO). In this investigation, we have compared the effects and toxicities of a) single-drug, high-dose, long-term therapy with azathioprine, prednisolone, and cyclophosphamide, b) double-drug therapy with azathioprine-plus-prednisolone and cyclophosphamide-plus-prednisolone, and c) intermittent and daily cyclophosphamide therapy. Cyclophosphamide proved to be the best agent for suppressing the immune disorder, whether used on a daily basis, intermittently, or in combination with corticosteroid. Unfortunately all cyclophosphamide regimens, in contrast to the other therapies, were associated with a high incidence of malignancy. MATERIALS AND METHODSAnimals. NZB/NZW F, hybrids were bred from NZB/B, and NZW stock colonies maintained a t Washington University. Each therapeutic group contained 20 mice, approximately half of which were female a n d half male.

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Since NZB/NZW mice develop an immune nephritis similar t o that of systemic lupus erythematosus in man, a study was designed in these mice to compare the clinical and immunologic effects of three immunosuppressive drug regimens. For 72 weeks, groups of 20 mice received daily oral therapy with a) no drugs, b) azathioprine, c) prednisolone or d) combined azathioprine-prednisolone. The combined regimen was superior t o either drug used alone in preventing deaths from renal disease. Prednisolone alone also prolonged life significantly, but not as effectively as combination therapy. Azathioprine alone was not effective. All drugs suppressed the antibody response to an exogenous antigen (Vi polysaccharide) equally well. None of the drug regimens prevented the appearance of proteinuria, antinuclear antibodies, Coombs' antibody, or 7-globulin deposits in glomeruli. However, the ability of a therapeutic regimen t o suppress antibodies to native DNA correlated well with its ability t o suppress renal disease. No malignancies were found among 73 animals autopsied, but significant hepatic damage occurred in the groups receiving prednisolone. Thus, combined therapy was superior t o either drug used alone, and the immunosuppressive effect of greatest clinical importance seemed to be the ability to prevent formation of anti-DNA antibodies.New Zealand black and New Zealand white hybrid mice (NZB/NZW F,) spontaneously develop a disease quite similar to systemic lupus erythematosus (SLE) in man. Virtually all mice of this strain develop lethal, immune complex

BackgroundSperm DNA integrity is crucial for transmission of genetic information to future generations and DNA damage can occur during chromatin packaging. Chromatin packaging involves the replacement of somatic nucleosomal histones by nuclear proteins called protamines. Protamine 1 (PRM1) is transcribed and translated in spermatids of all mammals; however, protamine 2 (PRM2) is transcribed in low levels in spermatids and it is not yet described in bull mature spermatozoa.ObjectivesThe aim of this study was to assess gene and protein expression of PRM2 and corroborate gene and protein expression of PRM1 in bull spermatozoa and testis.Materials and methodsFor this purpose, absolute q‐RT‐PCR was performed to calculate the number of copies of PRM1 and PRM2 mRNAs in bovine epididymal spermatozoa and testicular tissue. Western blot and mass spectrometry were performed to identify PRM1 and PRM2 in samples of bovine epididymal spermatozoa. Samples of bovine testicular tissue were collected to identify PRM1 and PRM2 by immunohistochemistry.ResultsWe evaluated that the number of PRM1 mRNA copies was about hundred times higher than PRM2 mRNA copies in sperm and testicular samples (p < 0.0001). In addition, we estimated the PRM1: PRM2 ratio based on mRNA number of copies. In spermatozoa, the ratio was 1: 0.014, and in testicle, the ratio was 1: 0.009. We also evaluated the immunolocalization for PRM1 and PRM2 in bovine testis, and both proteins were detected in spermatids. Western blot and mass spectrometry in bovine epididymal spermatozoa confirmed these results.ConclusionOur work identifies, for the first time, PRM2 in bovine epididymal spermatozoa and in testis. Further studies are still needed to understand the role of PRM2 on the chromatin of the spermatozoa and to verify how possible changes in PRM2 levels may influence the bull fertility.