Effects Of Sodium Alginate, Betaine And Melatonin On Cryopreservation Of Boar Spermatozoa

Boar sperm is susceptible to cold shock due to the high proportion of polyunsaturatedfatty acid (PUFA) of plasma membrane, the lipid peroxidation (LPO) of boar spermmembrane during freezing-thawing process resulted in the damage of sperm membraneintegrity. The relatively low viability and fertility in artificial insemination (AI) offrozen-thawed boar spermatozoa largely restricted the extensive application of cryopreservedboar sperm. In this research, we collected semen samples from Duroc boars, variousconcentrations of sodium alginate, betaine and melatonin (MLT) were firstly added to thefreezing media. The sperm quality was evaluated by analyzing thawed boar permatozoamotility, viability, mitochondrial membrane integrity, plasma membrane integrity andacrosomal integrity. The experimental techniques of eosin staining, hypoosmotic swelling test(HOST), propidium iodide (PI) and rhodamin123(Rh123) staining, and fluoresceinisothiocyanate-peanut agglutinin (FITC-PNA) staining were used to test sperm viability,plasma membrane integrity, mitochondrial membrane integrity and acrosomal integrity. Inaddition, the antioxidase activities (SOD and GSH-Px) and MDA levels were evalueated bySOD, GSH-Px and MDA assay kits. The results of this research were shown below:1. The sperm motility, viability, plasma membrane integrity, acrosomal integrity andmitochondrial membrane integrity of frozen-thawed boar spermatozoa were significantlyimproved with the addition of different concentrations of sodium alginate in freezing media,compared to that of the control (P<0.05), and with0.8and1.0mg/mL sodium alginate addedthawed spermatozoa showed the best motility and viability. The highest plasma membraneintegrity, mitochondrial integrity and acrosomal integrity were achieved when sodium alginatewas added at0.6mg/mL;2. The sperm motility, viability, plasma membrane integrity, acrosomal integrity andmitochondrial membrane integrity of frozen-thawed boar spermatozoa were significantlyincreased with the supplementation of different concentrations of betaine to the freezing media, compared to the control (P<0.05). When betaine concentration was1.0mg/mL, thawedspermatozoa exhibited the highest motility, viability, plasma membrane integrity andacrosomal integrity;3. The sperm motility, viability, plasma membrane integrity, acrosomal integrity andmitochondrial membrane integrity of frozen-thawed boar spermatozoa were significantlyincreased with the supplementation of different doses of MLT to the freezing media,compared to the control (P<0.05). The best sperm quality was achieved when MLT was addedat1mM after thawing, however, when MLT concentration reached to10mM, there was notsignificant difference between10mM group and the control(P>0.05);4. Adding0.4~1.0mg/mL sodium alginate significantly increased frozen-thawed boarsperm SOD and GSH-Px activities, and significantly reduced the MDA levels, compared tothat of the contral (P<0.05), and0.8mg/mL sodium alginate showed the best antioxidantcapacity compared to other treatment groups (P<0.05). SOD and GSH-Px activities weresignificantly increased when various concentrations of betaine were added, the MDA levelwas also reduced accordingly. Antioxidant capacities of5.0and7.5mg/mL betaine werebetter than other two groups of1.0and2.5mg/mL betaine (P<0.05), but no significantdifference was discovered between5.0mg/mL and7.5mg/mL betaine. SOD and GSH-Pxactivities were also significantly increased, and the MDA level was significantly reducedwhen various doses of MLT were added, compared to that of the control(P<0.05), and adding1mM MLT exhibited the best antioxidant capacity compared to those of othergroups(P<0.05).