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    Combination of trehalose and low boron in presence of decreased glycerol improves post-thawed ram sperm parameters: A model study in boron research
    (Wiley, 2022) Bucak, Mustafa Numan; Keskin, Nazan; Bodu, Mustafa; Bulbul, Bulent; Kirbas, Mesut; Ozturk, Ali Erdem; Frootan, Fateme
    Background Sperm cryopreservation has been widely used in the field of reproductive biotechnology. It applies to certain males of economic and scientific values, including livestock breeds or endangered animal species. The development of a semen extender with a low cryoprotectant concentration and an appropriate amount of trehalose and boron can prevent the deterioration of sperm parameters. Objective The main goal of this study is to establish a suitable ram extender model, by examining different combinations of high (5%) and low (3%) glycerol concentrations (to reduce its toxic effects on sperm freezing), a fixed amount of trehalose and an increased dose of boron to prevent the deterioration of sperm parameters, and investigate the levels of gene expressions. Materials and methods The Merino ram ejaculates were collected. The collected ejaculates providing the defined criteria were pooled. The pooled ejaculates were divided into eight aliquots and diluted with the Tris extender including different combinations of glycerol (5% and 3%) and boron (0.25, 0.5, and 1 mm) concentrations and a fixed amount of trehalose, then frozen. After freeze-thawing process, sperm motility, mitochondrial membrane activity, plasma membrane integrity, acrosomal membrane integrity, DNA damage (single cell gel electrophoresis (COMET) and TUNEL assays) as well as NAD(P)H quinone oxyreductase (NQO1), glutamate-cycteine ligase (GCLC), and glutathione S-transferase (GSTP1) for molecular mechanisms of sperm cell response to oxidative stress were assessed for different extender groups following freeze-thawing process: 5% glycerol + 0 mm boron (G5B0.00), 5% glycerol + 0.25 mm boron (G5B0.25), 5% glycerol + 0.5 mm boron (G5B0.50), 5% glycerol + 1 mm boron (G5B1.00), 3% glycerol + 0 mm boron (G3B.00), 3% glycerol + 0.25 mm boron (G3B0.25), 3% glycerol + 0.5 mm boron (G3B0.50), and 3% glycerol + 1 mm boron (G3B1.00). Results G3B0.25 presented higher percentages of subjective motility, mitochondrial activity, and viability of spermatozoa comparing with G5B0.00 and groups with boron. Supplementation of 0.25 mm boron with and without trehalose (G3B0.25 and G5B0.25) showed higher acrosome integrity, compared with G5B0.00, G5B1.00, G3B0.50, and G3B1.00. For TUNEL analysis, G3B1.00 showed the highest DNA integrity among the experimental groups which was statistically significant only with G5B0.50 (p < 0.05). The mRNA levels of NQO1 were significantly decreased in G5B1.00, G3B0.50, and G3B1.00, when compared to G5B0.00. In comparison with G5B0.00, supplementation of 1 mm boron with and without trehalose had significantly lower expression of GCLC. The level of GSTP1 gene was significantly lower (approximately threefold) in G3B1.00, compared to G5B0.00 (p < 0.05). Discussion and conclusion It can be assumed that the increase of the boron concentration in the extender may have important adverse effects on sperm parameters and antioxidant gene expression after thawing. The results obtained from this study will help to understand the toxicity limits of boron and eliminate the toxicity of glycerol in studies of gametes and tissue freezing. Therefore, it can be concluded that the use of sufficient boron can decrease cryodamages of cryopreservation of mammalian spermatozoa as well tissue engineering.
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    Relationship of blood and seminal plasma ceruloplasmin, copper, iron and cadmium concentrations with sperm quality in Merino rams
    (Elsevier Science Bv, 2015) Akalin, Pinar Peker; Bulbul, Bulent; Coyan, Kenan; Baspinar, Nuri; Kirbas, Mesut; Bucak, Mustafa Numan; Gungor, Sukru
    The aim of the current study was to investigate the concentrations of ceruloplasmin, copper, iron, zinc and cadmium concentrations in blood serum and seminal plasma obtained from Merino rams. In addition, their relationship with sperm parameters, fertility rate and litter size were also studied. Blood and ejaculate samples (6 replicates) were taken in October from 19 Merino rams, aged between 18 and 24 months. Ceruloplasmin, copper, iron, zinc and cadmium in blood serum and seminal plasma were determined. Sperm parameters including volume, mass motility, motility, concentration, Hos-test, viability, abnormal sperm and acrosome abnormality in semen, fertility rate and litter size were also evaluated. Highly positive correlation was found between blood ceruloplasmin and blood copper concentrations (r=0.812, p<0.001), whereas negative correlation were determined between these parameters in seminal plasma (r=0.195, p<0.05). Seminal plasma copper concentration was positively correlated with seminal plasma cadmium (r=0.206, p<0.05) and seminal plasma iron (r=0,305, p<0.01) concentrations. Negative correlation was determined between blood ceruloplasmin level and acrosomal defect (r=0.443, p<0.05). Seminal plasma ceruloplasmin level was positively correlated with volume (r=0.255, p<0.01) and negatively correlated with abnormal sperm (r=0.186, p=0.058) and acrosome abnormality (r=0.213, p<0.05). Seminal plasma iron concentration was positively correlated with other abnormality (r=0.257, p<0.01). Seminal plasma cadmium concentration was positively correlated with sperm abnormality (r=0.207,p=0.052) and other abnormality (r=0.262,p <0.05) and negatively correlated with fertility rate (r=0.449,p =0.054). Blood cadmium concentration was negatively correlated with litter size (r=0.579, 9<0.01). In conclusion, blood and seminal plasma ceruloplasmin may be suggested to have positive influence regardless of copper with its antioxidant property whereas iron and cadmium have negative influence on sperm parameters and fertility in Merino rams. (C) 2015 Elsevier B.V. All rights reserved.