Jian-Hong Hu

The cryoprotective effect of trehalose supplementation on boar spermatozoa quality.

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200 mm), and try to determine the optimum concentration of trehalose. We chose sperm motility, mitochondrial activity, acrosome integrity and membrane integrity as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100 mm trehalose-supplemented extenders, with values of 49.89% for motility, 44.69% for mitochondrial activity, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance diminished significantly for 200 . The synergic effect of trehalose and glycerol resulted in better cryosurvival of boar spermatozoa than that of a single cryoprotectant. In conclusion, when trehalose-supplementation was added up to 100 mm, trehalose confers a greater cryoprotective capacity to the extender, and the sperm motility, mitochondrial activity, membrane integrity and acrosome integrity parameters were significantly improved during frozen-thawed process.

Effects of different extenders on DNA integrity of boar spermatozoa following freezing-thawing.

The sperm-rich fraction, collected from eight mature Yorkshire boars, was frozen in an extender containing 9% LDL (w/v), 100mM trehalose, or 20% yolk (v/v), respectively. Sperm DNA integrity was assessed using the single-cell gel electrophoresis (SCGE). Other sperm quality characteristics such as motility, acrosome and membrane integrity were also monitored. The results showed that freezing-thawing caused an increase in sperm DNA fragmentation, and extender containing 9% LDL could significantly protect sperm DNA integrity (P<0.05) from the damage caused by cryopreservation and decrease DNA damages compared with extender containing 100mM trehalose and 20% yolk (v/v). No significant difference in damaged DNA was detected between frozen and unfrozen semen samples for extender of 9% LDL and 100mM trehalose, but cryopreservation could increase the degree of DNA damage (P<0.05), the percentage of damaged DNA degree of grade 2 and 3 was significantly increased. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. The data here demonstrated that the cryoprotectant played a fundamental role in reducing boar sperm DNA damage and protecting DNA integrity. It can be suggested that evaluation of sperm DNA integrity, coupled with correlative and basic characteristics such as motility, acrosome integrity and membrane integrity, may aid in determining the quality of frozen boar semen.

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing-thawing

Egg low-density lipoprotein (LDL) was added at concentrations of 7-10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen-thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 microm/s; VCL, curvilinear velocity: 50.2 microm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 microm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P<0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P<0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen-thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.

RETRACTED: The cryoprotective effect of trehalose supplementation on boar spermatozoa quality

In order to improve boar sperm quality during frozen-thawed process, the influence of the presence of trehalose on success of cryopreservation of boar sperm were investigated. We evaluated freeze-thawing tolerance of boar spermatozoa in a base cooling extender with the addition of different trehalose concentrations (0, 25, 50, 100 and 200mmol/l), and tried to determine the optimum concentration of trehalose. We chose sperm motility, acrosome integrity, membrane integrity and cryocapacitation as parameters to evaluate cryopreservation capacity of boar spermatozoa. We obtained the best results for 100mmol/l trehalose-supplemented extenders, with values of 49.89% for motility, 66.52% for acrosome integrity and 44.61% for membrane integrity, while freeze-thawing tolerance was diminished significantly for 200mmol/l of trehalose. Before and after capacitation, the CTC score for semen diluted by extender containing 100mmol/l trehalose was 3.68% and 43.82%, respectively. In conclusion, trehalose could confer a greater cryoprotective capacity to boar spermatozoa. Trehalose-supplementation with 100mmol/l concentration in basic extender could significantly improve sperm motility, membrane integrity and acrosome integrity parameters, and reduce boar spermatozoa cryocapacitation during the cryopreservation process.

The advantages of low-density lipoproteins in the cryopreservation of bull semen

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen-thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk (P<0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups (P>0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) (P<0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.

The cryoprotective effects of soybean lecithin on boar spermatozoa quality

the extender supplemented with 6% soybean lecithin, with values of 59.7% for the percentage of total motile sperm (TM%), 44.3% for motility, 45.3% for plasma membrane integrity and 61.9% for acrosome integrity. TM%, motility, acrosome integrity and plasma membrane integrity in the extender containing 6% soybean lecithin were significantly higher than that of other concentrations of soybean lecithin and 20% egg yolk (P < 0.05). However, the percentages of TM, acrosome integrity and plasma membrane integrity decreased with the increasing concentration of soybean lecithin in extender. In summary, the effect of soybean lecithin on spermatozoa quality was superior and the effective concentration of soybean lecithin in extender was 6% (w/v). Soybean lecithin might replace egg yolk in extender in the cryopreservation of boar semen.

Association of heat shock protein 90 with motility of post-thawed sperm in bulls

The correlation between the 90 kDa heat-shock protein (HSP90) and sperm quality following the process of freezing-thawing in bulls has not been studied clearly. Therefore, the objective of the present was to clarify the relationship between HSP90 level and semen parameters during the process of cryopreservation in bulls. Semen samples from 5 Holstein bulls were obtained by artificial vagina. Characteristics of these semen at three stages (fresh, after equilibration and frozen-thawed), including motility, plasma membrane integrity and acrosome integrity were evaluated. The mRNA expression level of HSP90 at the three stages was evaluated by using quantitative Real-Time PCR. Meanwhile, the protein level of HSP90 expression at the three stages was detected according to Western blot. The results showed that sperm parameters evaluated in fresh semen was the highest in the three groups. Sperm parameters in semen after equilibration were lower than those in fresh semen (P>0.05) and higher than those in post-thawed semen (P<0.05). Sperm parameters in frozen-thawed semen were the lowest among the three groups (P<0.05). This study indicated that HSP90 expression is proportional to sperm quality. HSP90 expression level in fresh semen was significantly higher than that in frozen-thawed semen (P<0.05). Although no significant differences in HSP90 expression were observed between fresh semen and semen after equilibration (P>0.05). Results in this study suggest that HSP90 level in bull spermatozoa was gradually declined following the process of freezing-thawing, and might be associated with sperm motility, plasma membrane integrity and acrosome integrity.

Effect of Gynostemma Pentaphyllum Polysaccharide on boar spermatozoa quality following freezing–thawing☆

Gynostemma Pentaphyllum Polysaccharide (GPP) was added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0 mg/ml to the extenders used to freeze boar semen and its effects on the quality of frozen-thawed sperm were assessed. The sperm motility was significantly higher in the extenders containing 0.25 and 0.5 mg/ml GPP, as compared to other groups (P<0.05). The extender supplemented with 0.5 mg/ml GPP favored the highest intact membrane and intact acrosome percentages in comparison with other groups (P<0.05), respectively. The mitochondrial activity was significantly higher at the concentrations of 0.25, 0.5 and 1.0 mg/ml GPP than that of other treatments, and the control group (P<0.05). In biochemical assays, the extender supplemented with 0.25 and 0.5 mg/ml GPP significantly improved SOD levels, compared to other groups (P>0.05). However, the extenders supplemented with GPP did not cause significant differences in levels of CAT and GSH-Px, compared to the control (P>0.05). In summary, GPP exhibited a dose-related response and the lower concentration produced greater protective effect. According to the standard semen quality parameters and antioxidant activities measured in this study, the concentration of 0.5 mg/ml GPP caused a beneficial cryoprotective effects on the quality of frozen-thawed boar semen. It is proposed that an extender containing 0.5 mg/ml GPP could be used as cryoprotective medium of better efficiency.

HSP90 expression correlation with the freezing resistance of bull sperm.

To date, there has been little improvement in cryopreservation of bull sperm due to lack of understanding of the freezing mechanisms. Therefore, this study set out to investigate expression levels of fertility-associated proteins in bull sperm, and in particular the relationship between the 90 kDa heat-shock protein (HSP90) and the sperm characteristics after freezing-thawing. Semen was collected from eight Holstein bulls by artificial vagina. Characteristics of these fresh semen, including sperm motility, morphology, viability and concentration, were evaluated. Sperm quality was also assessed after freezing-thawing. Eight ejaculates were divided into two groups based on freezing resistance and sperm motility. Sperm proteins were extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting were performed. SDS-PAGE results showed that there was substantial diversity in 90 kDa proteins in the frozen-thawed sperm and HSP90 was confirmed as one of the 90 kDa proteins by western blot. This study indicated that HSP90 expression correlated positively with sperm quality. The amount of expressed 90 kDa proteins in the high freezing resistance (HFR) group was significantly higher than that in the low freezing resistance (LFR) group (P < 0.05). Thus, higher expression of HSP90 could probably lead to the higher motility and freezing resistance of sperm found after freezing-thawing. Therefore, we concluded that level of HSP90 expression could be used to predict reliably and simply the freezing resistance of bull sperm.

The effect of Laminaria japonic polysaccharide on sperm characteristics and biochemical parameters in cryopreserved boar sperm.

The aim of the present study was to evaluate the cryoprotective effect of Laminaria japonic polysaccharide (LJP) on boar sperm. Semen samples were collected from seven mature Yorkshire boars once a week by the gloved hand technique and frozen-thawed in the extender with LJP added. Extender with LJP added at concentrations of 0.25, 0.5, 1.0, 1.5 and 2.0mg/mL to the extender and its effects on the quality of frozen-thawed boar sperm were assessed. Results showed: (i) sperm motility and plasma membrane integrity were greater in the extender containing 0.5 and 1.0mg/mL LJP, as compared to other groups (P<0.05); (ii) extender added 1.0mg/mL LJP showed the greatest plasma membrane and acrosomal integrity percentages in comparison with other groups (P<0.05); (iii) mitochondrial activity was significantly higher at the concentration of 0.5 and 1.0mg/mL LJP than those of other groups (P<0.05); (iv) in terms of biochemical assessments, 0.5 and 1.0mg/mL LJP improved SOD (superoxide dismutase) and CAT (catalase) concentrations, compared to other groups (P<0.05). However, no significant difference was found in GSH-Px (glutathione peroxidase) concentration when supplemented with LJP. Interestingly, LJP exhibited a dose-related response and the lesser concentration represented greater protective effects. It is also important to note that 1.0mg/mL LJP provides for an enhanced cryoprotective effect in boar semen.