During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen-thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen-thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR-14/Ethidiumhomodimer-1 (EthD-1) staining and acrosome integrity by using FITC-PNA/EthD-1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose-dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group-III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post-thaw plasma membrane integrity and progressive motility.

The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50 degrees C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P < 0.05), and the supplementation of L-cysteine in combination with DHA-enriched hen egg yolk (group IV) improved both progressive motility (P < 0.05) and acrosome integrity (P < 0.01). The use of DHA-enriched hen egg yolk alone (group II) did not enhance any of the post-thawed semen qualities (P > 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

Cryopreservation is associated with the production of reactive oxygen species, which leads to lipid peroxidation of the sperm membrane and consequently a reduction in sperm motility and decreased fertility potential. The aim of this study was to determine the optimal concentration of L-cysteine needed for cryopreservation of boar semen. Twelve boars provided semen of proven motility and morphology for this study. The semen was divided into four portions in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various concentrations of L-cysteine to reach 0 mmol L −1 (group I, control), 5 mmol L −1 (group II), 10 mmol L −1 (group III) and 15 mmol L −1 (group IV). Semen suspensions were loaded in straws (0.5 mL) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability using SYBR-14/EthD-1 staining and acrosome integrity using FITC-PNA/EthD-1 staining. There was a significantly higher (P < 0.01) percentage of progressive motility, viability and acrosomal integrity in two L-cysteine-supplemented groups (group II and group III) compared with the control. There was a biphasic effect of L-cysteine, with the highest percentage of progressive motility, viability and acrosomal integrity in group III. In conclusion, 5 or 10 mmol L −1 was the optimum concentration of L-cysteine to be added to the LEY extender for improving the quality of frozen-thawed boar semen.

ABSTRACT. The aim of this study was to determine the effect of gamma-oryzanol-enriched -rice bran oil on the quality of cryopreserved boar semen. Ten boars provided semen of proven motility and morphology for this study. The semen was divided into three portions in which lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with 2 types of rice bran oils, at a gamma-oryzanol concentration of 0 mg/ml of lactose egg yolk (LEY) freezing extender (group A, control), 0.1 mg/ml(0.16 mMol) of freezing extender (group B) and 0.1 mg/ml of freezing extender (group C). Semen suspensions were loaded in medium straws (0.5 ml) and placed in a controlled-rate freezer. After cryopreservation, frozen semen samples were thawed and investigated for progressive motility, viability and acrosomal integrity. There was a significantly higher percentage of progressive motility (34 versus 47.0 and 48.5, P<0.001), viability (35.5 versus 48.1 and 50.1, P<0.001) and acrosomal integrity (39.8 versus 50.8 and 54.9, P<0.001) in the gamma-oryzanolenriched rice bran oil-supplemented groups (groups B, C) than in the control group (group C), respectively. In conclusion, addition of gamma-oryzanol-enriched rice bran oil to LEY freezing extender is appropriated for improving the quality of frozen-thawed boar semen. Cryopreservation of boar spermatozoa offers an effective way to maintain their valuable genetic material and transport it between countries, thereby minimizing boar transportation. It has been demonstrated that certain processes in cryopreservation including elimination of seminal plasma by centrifugation and reduction in the temperature result in cold shock, owing to a decrease in quantity of antioxidants and an increase in reactive oxygen species (ROS) contents in the semen [6,8,43]. The ROS can lead to oxidative stress and lipid peroxidation of the sperm plasma membrane, which have detrimental effects on spermatozoa during cryopreservation, for instance, reduction in progressive motility, acrosomal integrity, sperm-oocyte fusion and fertilizing potential [1-3, 17, 41, 53].During the past decade, several studies have focused on supplementation of freezing extender for frozen boar semen with a variety of antioxidants (e.g., vitamin E, vitamin C, L-cysteine, glutathione, taurine, pyruvate, SOD, catalase) in an attempt to minimize the detrimental effects of ROS, which occur during the freezing process [7,10,16,23,26,38,39].Gamma-oryzanol, a phytosteryl ferulate mixture extracted from rice bran oil, has received a great deal of attention, because of its various significant health-promoting functions such as antioxidant activity, inhibition of lipoperoxidation by its scavenging activity [4], reduction in LDL cholesterol and induction of HDL cholesterol [40,41,48] and inhibition of platelet aggregation [13]. In humans, many studies have been reported on its potential implications as a UV-A filter in sunscreen cosmetics [14] and as a treatment for type 2 diabetes mellitus [12] and allergic reactions [3...