Combination of trehalose and low boron in presence of decreased glycerol improves post-thawed ram sperm parameters: A model study in boron research.

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.

Proteomic fertility markers in ram sperm.

Precise estimation of ram fertility is important for sheep farming to sustain reproduction efficiency and profitability of production. There, however, is no conventional method to accurately predict ram fertility. The objective of this study, therefore, was to ascertain proteomic profiles of ram sperm having contrasting fertility phenotypes. Mature rams (n = 66) having greater pregnancy rates than average (89.4 ± 7.2%) were assigned into relatively-greater fertility (GF; n = 31; 94.5 ± 2.8%) whereas those with less-than-average pregnancy rates were assigned into a lesser-fertility (LF; n = 25; 83.1 ± 5.73%; P = 0.028) group. Sperm samples from the outlier greatest- and least-fertility rams (n = 6, pregnancy rate; 98.4 ± 1.8% and 76.1 ± 3.9%) were used for proteomics assessments utilizing Label-free LC-MS/MS. A total of 997 proteins were identified, and among these, 840 were shared by both groups, and 57 and 93 were unique to GF and LF, respectively. Furthermore, 190 differentially abundant proteins were identified; the abundance of 124 was larger in GF while 66 was larger in LF rams. The GF ram sperm had 79 GO/pathway terms in ten major biological networks while there were 47 GO/pathway terms in six biological networks in sperm of LF rams. Accordingly, differential abundances of sperm proteins between sperm of GF and LF rams were indicative of functional implications of sperm proteome on male fertility. The results of this study emphasize there are potential protein markers for evaluation of semen quality and estimation of ram sperm fertilizing capacity.

Varied Approach of Using MSCs for Bovine Embryo In Vitro Culture.

The objective of the present study was to evaluate the effect of porcine mesenchymal stem cells (MSCs) secreted factors on bovine in vitro embryo development using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer, and in reverse drop and by embryo culture in coculture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in coculture with MSCs (P < 0.0001), and between SOF and SOF conditioned (monolayer and drop) (P < 0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analyzed by TUNEL only in two out of four experimental groups: SOF and SOF in coculture with MSCs. There were no significant differences between any of analyzed blastocysts' groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in coculture with MSC turned out to be effective.

Varied approach of using MSCs for bovine embryo in vitro culture.

The objective of the present study was to evaluate the effect of porcine Mesenchymal Stem Cells (MSCs) secreted factors on bovine in vitro embryo development by using MSCs in different culture systems: SOF medium, SOF medium conditioned by MSCs in monolayer and in reverse drop and by embryo culture in co-culture with MSCs. Statistically highly significant differences were noted between the number of blastocysts derived cultures in all tested culture systems. The in vitro culture in SOF turned out to be the most optimal. Statistically highly significant differences were observed in the number of blastocyst obtained between SOF and SOF in co-culture with MSCs (p < 0.0001), and between SOF and SOF conditioned (monolayer and drop) (p < 0.00001). The trials to produce blastocysts in SOF conditioned by MSCs in reverse drops and monolayer failed. The blastocysts were obtained and analysed by TUNEL only in two out of four experimental groups: SOF and SOF in co-culture with MSCs. There were no significant differences between any of analysed blastocysts' groups neither in the total number of nuclei nor in the apoptotic features. Neither medium conditioning by MSCs in monolayer and in reverse drop nor embryo culture in co-culture with MSC turned out to be effective.