Reprod Dom Anim 50, 221–226 (2015); doi: 10.1111/rda.12473 ISSN 0936–6768
Breed of Boar Influences the Optimal Concentration of Gamma-oryzanol Needed for Semen Cryopreservation P Chanapiwat and K Kaeoket Semen Laboratory, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon-pathom, Thailand
Contents The aim of this study was to investigate the influence of boar breed on the optimal concentration of gamma-oryzanol on the qualities of cryopreserved boar semen. Semen was collected from 20 boars (10 Duroc, 5 Large white and 5 Landrace boars). The semen sample was divided into five groups (A–E) according to the concentration of gamma-oryzanol in extender II, that is 0, 0.08, 0.16, 0.24 and 0.32 mM, respectively. The semen was cryopreserved by nitrogen vapour and storage in nitrogen tank ( 196°C). After storage for a week, samples were thawed at 50°C for 12 s and evaluated for progressive motility, sperm viability and acrosome integrity. The results demonstrated that gamma-oryzanol significantly improved progressive motility, viability and acrosome integrity of frozen–thawed boar semen. Considering the influence of breeds on the optimal concentration of gamma-oryzanol, for Duroc boar, gamma-oryzanol at 0.16 mM (group C) yielded the highest percentage of progressive motility, sperm viability and acrosome integrity. For Large white and Landrace boars, gamma-oryzanol at 0.24 mM (group D) showed a significantly higher percentage of progressive motility, viability (not significant in Landrace) and acrosome integrity than other concentrations. In conclusion, the optimal concentration of gamma-oryzanol needed for boar semen cryopreservation in lactose–egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mM for Duroc and 0.24 mM for Large white and Landrace.
Introduction During cryopreservation, there are many factors that influenced the qualities of frozen–thawed boar semen such as cold shock, osmotic stress and oxidative stress (Aitken and Clarkson 1988; Watson 2000). The oxidative stress caused by reactive oxygen species (ROS) is the most important factor as ROS is a major cause of lipid peroxidation of the sperm plasma membrane that lead to a decreased membrane fluidity and plasma membrane damage (Aitken and Clarkson 1988; Johnson et al. 2000; Saleh and Agarwal 2002; Chanapiwat et al. 2009; Kaeoket et al. 2010a). Generally, small amount of free radicals can be found in semen, which is counterbalanced by resident antioxidants, and the residue amount is effective for stimulating the sperm capacitation, acrosome reaction and sperm–oocyte fusion (Gadea et al. 2008). During boar semen cryopreservation, seminal plasma which contained many antioxidants is discarded to minimize the detrimental effect of ice formation on spermatozoa; however, this approach resulted in a © 2015 Blackwell Verlag GmbH
decreased amount of resident antioxidant, and its antioxidant activity against free radicals (Aitken and Clarkson 1988; Watson 2000; Chatterjee et al. 2001). During the past decade, there are many studies reported about the positive effect of supplementation of antioxidants during boar semen cryopreservation on the qualities of frozen–thawed semen, such as vitamin E, glutathione, DHA and L-cysteine (Breininger et al. 2005; Gadea et al. 2005; Kaeoket et al. 2008, 2010a,b). Rice bran oil (RBO) composes of gamma-oryzanol, alphatocopherol and phytosterol. Gamma-oryzanol is the major antioxidant in RBO; its oxidation protection property has been reported to be ten times higher than vitamin E (Shin et al. 1997). It has also been reported that the supplementary feeding of stallions with commercial RBO increased the total antioxidant potential of semen, sperm concentration and motility (Arlas et al. 2008). Recently, Kaeoket et al. (2012) showed that supplementation of gamma-oryzanol-enriched rice bran oil in lactose–egg yolk (LEY) freezing extender resulted in an improvement of frozen–thawed Duroc boar semen qualities (i.e. progressive motility, viability and acrosome integrity). Nevertheless, in this study, the optimal concentration of gamma-oryzanol for each breed of boar for semen cryopreservation has not been investigated. With regard to the effect of boar, Waterhouse et al. (2006) reported that the differences between boars (i.e. Duroc and Landrace) in cryotolerance after freezing and thawing may be related to the amount of long-chain polyunsaturated fatty acids (PUFA) in the sperm plasma membrane. In addition, Chanapiwat et al. (2009) described that the differences in the freezing ability (i.e. cryotolerant) in Pietrain boars can be classified into poor and good freezability according to their post-thawed progressive motility (i.e. < 30% or > 30%, respectively) and may associated with the differences in lipid composition, fatty acid composition and sterol levels in the sperm plasma membrane of individual boar (Parks and Lynch 1992; Waterhouse et al. 2006; Kaeoket et al. 2008). However, no study has been reported about the influence of boar breeds on the concentration of gamma-oryzanol required during cryopreservation in different breeds. Therefore, the aim of this study was to investigate the effect of boar breeds (i.e. Duroc, Large white and Landrace) on the optimal concentration of gamma-oryzanol needed for semen cryopreservation.
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Materials and Methods This research project was approved by the Faculty of Veterinary Science-Animal Care and Use Committee (FVS-ACUC-Protocol No. MUVS-2012-43). Animals Twenty boars (10 Durocs, 5 Large whites and 5 Landraces) aged between 1 and 3 years old having proven fertility and used for routine artificial insemination (AI) in a commercial herd were included in the experiment. The boars were kept in individual pens in the same evaporative housing system. Each boar was fed with commercial diet twice daily, and water was provided ad libitum via water nipple. Semen collection and preparation Boar semen was collected once a week using glovedhand technique (Kaeoket et al. 2002, 2005). A total of 20 ejaculates of semen were obtained (one ejaculate each). During collection, semen was filtered through gauze and only sperm rich fraction was collected. Within 30 min after collection, sperm parameters including semen volume, pH, concentration and progressive motility were evaluated. Aliquot of 1 ml semen was collected into Eppendorf tube for sperm viability, sperm acrosome integrity and sperm morphology (Chanapiwat et al. 2009). The only ejaculates with motility of ≥70% and ≥80% morphologically normal were selected for cryopreservation. The remaining fresh semen was diluted with (1 : 1 v/v) ModenaTM extender (extender I) (Swine Genetics International, Ltd., Cambridge, IA, USA) and transported by cell incubator (Micom control system 20Q; Continental plastic CORP, Delavan, WI, USA) in 15°C to semen laboratory, Faculty of Veterinary Science, Mahidol University. Freezing and thawing process All semen samples were frozen using traditional nitrogen method. After collection and evaluation, the semen sample was diluted with (1 : 1 v/v) ModenaTM extender (Extender I) (Swine Genetics International Ltd). Diluted semen was transferred into 50 ml centrifuge tubes, cooled at 15°C for 120 min and later centrifuged at 800 9 g 15°C for 10 min (Hettich Rotanta 460R, Tuttlingen, Germany). After centrifugation, the supernatant was discarded, and the sperm pellets were resuspended to a concentration of 1.5 9 109 sperm/ml with approximately 1–2 ml of extender II (20% of egg yolk in 11% lactose solution). In this step, the semen sample was divided into five groups (Groups A–E) according to the concentration of gamma-oryzanol (KingTM; containing gamma-oryzanol 4 mg/ml of rice bran oil) in extender II, that is 0, 2, 4, 6 and 8 mg (0, 0.08, 0.16, 0.24, 0.32 mM) of gamma-oryzanol, respectively.
P Chanapiwat and K Kaeoket
The diluted semen was cooled at 5°C for 90 min. Each group of sample was mixed with Extender III [89.5% extender II with 9% (v/v) glycerol and 1.5% (v/v) Equex-STMâ; Nova Chemical Sales Inc., Scituate, MA, USA] to final concentration of 1.0 9 109 sperm/ml and 3% (v/v) glycerol (Gadea et al. 2005; Kaeoket et al. 2010a). The sperm suspensions were loaded into 0.5 ml straws (Bio-Vet, Z.I. Le Berdoulet, France). All straws were placed in contact with nitrogen vapour at 3 cm above the liquid nitrogen level for 20 min in an expandable polystyrene box and plunged into the nitrogen tank ( 196°C) for storage (Selles et al. 2003). After storage for a week, samples were thawed by immersing the straw in a thermos at 50°C for 12 s (Gadea et al. 2005). After thawing, the thawed sample was diluted (1 : 4) with ModenaTM extender in a test tube and kept in an incubator at 37°C until evaluation. Evaluation of sperm Sperm progressive motility Progressive sperm motility was subjectively evaluated by visual estimation by the same person throughout the study, at 37°C under phase contrast microscope (Olympus CX31, New York, USA) at 1009 and 4009 magnification (Berger et al. 1985). Progressive motility was expressed as the percentage of motile sperm cells. Sperm concentration Sperm concentration was assessed by Neubauer hemocytometer after dilution (1 : 100 v/v). One drop of this dilution was placed into the chamber, and the number of sperms was evaluated by phase contrast microscope 4009 magnification. Sperm concentration was expressed as sperms 9106 sperm/ml. Sperm viability The sperm viability was performed as described earlier by Chanapiwat et al. (2009). Briefly, 10 ll of diluted semen was mixed with 2.7 ll of SYBR-14 (10 lM in DMSO, final concentration of 0.54 lM) and 10 ll of 1.17 lM Ethidiumhomodimer-1(EthD-1). The mixture was incubated at 37°C for 15 min. At least 200 sperms were assessed using fluorescence microscope at 4009 magnification. The nuclei of live sperms with intact plasma membrane stained green with SYBR-14, while those with damaged membrane or dead sperm stained with red. The results were scored as the percentage of live sperm (viable) and dead sperm (non-viable). Sperm acrosome integrity The integrity of sperm acrosome was evaluated with fluorescein isothiocyanate labelled peanut (Arachis hypogaea) agglutinin (FITC-PNA) staining (Carvajal et al. 2004), and 10 ll of diluted semen was mixed with 10 ll © 2015 Blackwell Verlag GmbH
Gamma-Oryzanol Improved Frozen Boar Semen Qualities
of 1.17 lM EthD-1 and incubated at 37°C for 15 min. Then, smear 5 ll of mixture on glass slide and air dry. Fix with 95% ethanol for 30 s and air dry. Fifty ll of FITC-PNA (100 lg/ml in PBS) was spread over the slides and incubated in a moist chamber at 4°C for 30 min. After that slides were rinsed with cold PBS and air dried. Live sperms, stained with green, at least 100 cells were assessed under fluorescence microscope at 10009 magnification (Carl Zeiss, Inc., Axioskop 40, Germany) and classified to intact acrosome and nonintact acrosome. The results were scored as the percentage of intact acrosome of live sperm and non-intact. Statistical analysis Sperm quality was evaluated from parameters, that is sperm progressive motility, sperm viability and sperm acrosome integrity. Prior to analysis, all data were checked for normal distribution with Shapiro–Wilk test. The effect of treatments on parameters was analysed with two-ways ANOVA according to randomized complete block design (RCBD), in which treatments as fixed factor, parameters as dependent variables and boars as random block factor; and the interaction between treatments, breeds and boar (breed) were test by factorial in RCBD. Duncan’s new multiple range test was used for the mean of parameters comparison. All statistical analysis was performed as a general linear model using the univariate procedure of SPSS version 19.0 (SPSS 19.0; SPSS Inc., Chicago, IL, USA). The parameters were expressed as mean of percentage SD, and the difference between treatment means was considered significant when p < 0.05.
Results Fresh semen analysis The mean percentages of progressive motility, viability and acrosome integrity were 76.3 4.3, 67.1 17.1 and 66.8 11.7, respectively. The volume and concentration of fresh semen were ranged between 168 and 436 ml and 395-460 9 106 sperm/ml (n = 16), respectively.
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Post-thaw semen analysis The effect of breed and boar (breed) According to the statistical analyses of the present results, the effect of breed and boar within the same breed on post-thawed semen qualities (i.e. progressive motility, viability and acrosome integrity) was found (p < 0.001). Progressive motility The results of progressive motility in Duroc, Large white and Landrace boars are presented in Tables 1–3, respectively. In Duroc, the percentage of progressive motility in group C was higher than other groups except group D (p < 0.01) (Table 1). In Large white, the percentage of progressive motility in group D was significantly higher than other groups (p < 0.01) (Table 2). In Landrace, a higher percentage of progressive motility was found in groups C and D, compared with control group (p < 0.05) (Table 3). Sperm freezability with regard to progressive motility (effect of breed) Comparing among control groups (i.e. group A in all breeds, n = 20), a significantly higher percentage of progressive motility was found in Duroc (35.0 9.1) > Landrace (33.0 14.8) > Larger white (26.0 6.5) (p < 0.05). Sperm viability The highest percentage of sperm viability in Duroc and Large white boars were found in groups C and group D, respectively (Tables 1 and 2) (p < 0.05). However, in Landrace, no significant difference in sperm viability was observed among the treatment groups (Table 3). Acrosome integrity In Duroc, the highest percentage of acrosome integrity was found in group C (p < 0.01) (Table 1). In Large
Table 1. The mean (SD) percentages of progressive motility, viability and acrosome integrity of frozen–thawed semen in the different groups (A= control, B to E added with 0.08, 0.16, 0.24 and 0.32 mM of gamma-oryzanol, respectively) (Duroc boars, n = 10 ejaculates in each group) Viability (%) Groups
Progressive motility (%)
Viable
A B C D E Significant differences
35.0 9.1c 44.0 6.1b 50.0 8.2a 45.5 7.2ab 44.5 8.6b p < 0.01
29.2 5.6c 35.0 4.7b 40.0 6.4a 38.1 5.2ab 36.1 5.9ab p < 0.01
Values in each column marked with different superscript letters differ significantly (p < 0.05). NA, not analysed.
© 2015 Blackwell Verlag GmbH
Acrosome integrity (%) Non-viable
Intact
35.9 9.1c 41.5 10.4bc 52.2 9.6a 47.4 9.5ab 44.5 13.1b p < 0.01
66.9 65.1 60.5 61.9 64.0 NA
14.1 4.7 6.4 5.2 6.0
Non-intact 63.1 58.5 47.8 52.6 55.5 NA
8.9 10.4 9.6 9.5 13.1
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Table 2. The mean (SD) percentages of progressive motility, viability and acrosome integrity of frozen–thawed semen in the different (A= control, B to E added with 0.08, 0.16, 0.24 and 0.32 mM of gamma-oryzanol, respectively) (Large white boars, n = 5 ejaculates in each group) Viability (%) Groups
Progressive motility (%)
Viable
A B C D E Significant differences
26.0 6.5c 37.0 12.0b 39.0 8.2b 49.0 6.5a 36.0 8.9b p < 0.01
31.8 5.5b 37.1 4.9ab 36.0 6.3ab 42.2 5.4a 34.2 5.2b p < 0.05
Acrosome integrity (%) Non-viable
Intact
43.9 13.2c 53.0 17.6b 51.5 18.6b 60.3 11.8a 49.9 13.8b p < 0.01
62.8 62.9 64.0 57.8 65.8 NA
5.5 4.9 6.3 5.4 5.2
Non-intact 56.1 47.0 48.5 39.7 50.1 NA
13.2 17.6 18.6 11.8 13.8
Values in each column marked with different superscript letters differ significantly (p < 0.05). NA, not analysed.
Table 3. The mean (SD) percentages of progressive motility, viability and acrosome integrity of frozen–thawed semen in the different groups (A= control, B to E added with 0.08, 0.16, 0.24 and 0.32 mM of gamma-oryzanol, respectively) (Landrace boars, n = 5 ejaculates in each group) Viability (%) Groups
Progressive motility (%)
A B C D E Significant differences
33.0 14.8b 41.0 13.9ab 47.0 12.0a 48.0 10.4a 42.0 14.0ab p < 0.05
Viable 27.8 31.6 35.9 36.7 34.6 NS
9.3a 7.4a 8.4a 7.9a 5.9a
Acrosome integrity (%) Non-viable
Intact
39.6 13.7b 41.4 9.4b 42.0 12.3b 51.6 15.4a 42.2 12.6b p < 0.05
72.2 68.4 64.1 63.3 65.4 NA
9.3 7.4 8.4 8.1 6.3
Non-intact 60.4 58.6 58.0 48.4 57.8 NA
13.7 9.4 12.3 15.4 12.6
Values in each column marked with different superscript letters differ significantly (p < 0.05). NS, not significant; NA, not analysed.
white and Landrace, the percentage of intact acrosome in group D was significantly higher than other groups (p < 0.01) (Tables 2 and 3).
Discussion The present study clearly demonstrated that gammaoryzanol-enriched RBO significantly influenced the frozen–thawed boar semen qualities including progressive motility, viability and acrosome integrity. To the best of our knowledge, this study was the first report to investigate the effect of varied concentrations of gamma-oryzanol on boar semen qualities following cryopreservation with special focus on the different breeds (i.e. Duroc, Large white and Landrace). In the present results, it is worth noting that breed of boar not only resulted in the differences in freezability but also the differences in the optimal concentration of gamma-oryzanol needed for semen cryopreservation. In Duroc boars, it seems likely that gamma-oryzanol at a concentration of 0.16 mM provided a superior post-thawed semen qualities when compared to other groups. In Large white and Landrace boars, the optimal concentration of gamma-oryzanol seems to be at 0.24 mM in which clearly improved progressive motility and intact acrosome of frozen–thawed semen qualities. In addition to a significant difference among
breeds, we also found an important impact of individual boars on the susceptibility of their sperm to cryoinjury as shown by the differences in the postthaw sperm qualities among boars within the same breed, which is in agreement with the previous findings (Larsson and Einarsson 1976; Waterhouse et al. 2006; Chanapiwat et al. 2009). The reason for individual variation among boars in term of sperm cryotolerance is unknown at present. However, the trigger of genetic difference among individual boars may explain this impact (Holt et al. 2005) and the different physiological characteristic of the sperm from each boar may also contribute to the individual variation (White 1993; Watson 1995). Besides the genetic and physiological differences, the explanation of boar and breed effect found in this study may include the differences in lipid composition, fatty acid composition and sterol levels in their sperm plasma membrane (Waterhouse et al. 2006). Recently, in a proteomic investigation of bull (Chen et al. 2012; De Canio et al. 2014) and human (Xu et al. 2012) sperm, it has been reported that each sperm contains different expression of membrane proteins, in which, to some extent, may explained the effect of individual boar and breed found in this study. However, at the moment, there are no data available for the proteomics analysis of boar sperm. © 2015 Blackwell Verlag GmbH
Gamma-Oryzanol Improved Frozen Boar Semen Qualities
In the present results, superior qualities of frozen– thawed boar semen were observed in all treatment groups compare with control group. This can be explained by the chemical properties of gamma-oryzanol in that gamma-oryzanol has hydroxy and phenoxy groups which provide electrons that could bind to free radicals, leading to minimizing the detrimental effect of ROS and thereby stabilizing lipid components in the sperm plasma membrane (Masuda et al. 2006; Srinivasan et al. 2007). Besides gamma-oryzanol, it is worth noting that RBO contains antioxidant, that is alphatocopherol, which has a significant ability to break the covalent links that ROS have formed between fatty acid side chains in sperm membrane lipids, consequently, a decrease in sperm membrane damage (Pen~ a et al. 2003; Breininger et al. 2005; Jeong et al. 2009) and also contains membrane stabilizer, that is phytosterols, which may also stabilized sperm plasma membrane by replacing cholesterol in their membrane and consequently has a protective effect on sperm plasma membrane from oxidative damage (Brufau et al. 2008). Taken together, it can be concluded that the optimal concentration of gamma-oryzanol needed for boar
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semen cryopreservation in lactose–egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mM for Duroc and 0.24 mM for Large white and Landrace. Acknowledgements The research grant was provided by Thailand Research Fund (MRG5680130), Commission of Higher Education of Thailand and Mahidol University. The authors are grateful to Dr. Surasak Jittakhot PhD (Animal Nutrition), Faculty of Veterinary Science, Mahidol University for advice in statistical analysis. Drs. Jiraporn Thamma, Pornpitcha Treenawong, Apitsada Visessripong are thanked for technical assistant.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions P Chanapiwat has contributed to freezing semen, data analysing and manuscript writing. K Kaeoket contributed to data interpretation, mining the data, manuscript writing and supervised the project.
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Submitted: 23 Sep 2014; Accepted: 30 Nov 2014 Author’s address (for correspondence): K Kaeoket, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Phuttamonthon, Nakhon-pathom 73170, Thailand. E-mail: [email protected]
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Breed of Boar Influences the Optimal Concentration of Gamma-oryzanol Needed for Semen Cryopreservation P Chanapiwat and K Kaeoket Semen Laboratory, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Nakhon-pathom, Thailand
Contents The aim of this study was to investigate the influence of boar breed on the optimal concentration of gamma-oryzanol on the qualities of cryopreserved boar semen. Semen was collected from 20 boars (10 Duroc, 5 Large white and 5 Landrace boars). The semen sample was divided into five groups (A–E) according to the concentration of gamma-oryzanol in extender II, that is 0, 0.08, 0.16, 0.24 and 0.32 mM, respectively. The semen was cryopreserved by nitrogen vapour and storage in nitrogen tank ( 196°C). After storage for a week, samples were thawed at 50°C for 12 s and evaluated for progressive motility, sperm viability and acrosome integrity. The results demonstrated that gamma-oryzanol significantly improved progressive motility, viability and acrosome integrity of frozen–thawed boar semen. Considering the influence of breeds on the optimal concentration of gamma-oryzanol, for Duroc boar, gamma-oryzanol at 0.16 mM (group C) yielded the highest percentage of progressive motility, sperm viability and acrosome integrity. For Large white and Landrace boars, gamma-oryzanol at 0.24 mM (group D) showed a significantly higher percentage of progressive motility, viability (not significant in Landrace) and acrosome integrity than other concentrations. In conclusion, the optimal concentration of gamma-oryzanol needed for boar semen cryopreservation in lactose–egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mM for Duroc and 0.24 mM for Large white and Landrace.
Introduction During cryopreservation, there are many factors that influenced the qualities of frozen–thawed boar semen such as cold shock, osmotic stress and oxidative stress (Aitken and Clarkson 1988; Watson 2000). The oxidative stress caused by reactive oxygen species (ROS) is the most important factor as ROS is a major cause of lipid peroxidation of the sperm plasma membrane that lead to a decreased membrane fluidity and plasma membrane damage (Aitken and Clarkson 1988; Johnson et al. 2000; Saleh and Agarwal 2002; Chanapiwat et al. 2009; Kaeoket et al. 2010a). Generally, small amount of free radicals can be found in semen, which is counterbalanced by resident antioxidants, and the residue amount is effective for stimulating the sperm capacitation, acrosome reaction and sperm–oocyte fusion (Gadea et al. 2008). During boar semen cryopreservation, seminal plasma which contained many antioxidants is discarded to minimize the detrimental effect of ice formation on spermatozoa; however, this approach resulted in a © 2015 Blackwell Verlag GmbH
decreased amount of resident antioxidant, and its antioxidant activity against free radicals (Aitken and Clarkson 1988; Watson 2000; Chatterjee et al. 2001). During the past decade, there are many studies reported about the positive effect of supplementation of antioxidants during boar semen cryopreservation on the qualities of frozen–thawed semen, such as vitamin E, glutathione, DHA and L-cysteine (Breininger et al. 2005; Gadea et al. 2005; Kaeoket et al. 2008, 2010a,b). Rice bran oil (RBO) composes of gamma-oryzanol, alphatocopherol and phytosterol. Gamma-oryzanol is the major antioxidant in RBO; its oxidation protection property has been reported to be ten times higher than vitamin E (Shin et al. 1997). It has also been reported that the supplementary feeding of stallions with commercial RBO increased the total antioxidant potential of semen, sperm concentration and motility (Arlas et al. 2008). Recently, Kaeoket et al. (2012) showed that supplementation of gamma-oryzanol-enriched rice bran oil in lactose–egg yolk (LEY) freezing extender resulted in an improvement of frozen–thawed Duroc boar semen qualities (i.e. progressive motility, viability and acrosome integrity). Nevertheless, in this study, the optimal concentration of gamma-oryzanol for each breed of boar for semen cryopreservation has not been investigated. With regard to the effect of boar, Waterhouse et al. (2006) reported that the differences between boars (i.e. Duroc and Landrace) in cryotolerance after freezing and thawing may be related to the amount of long-chain polyunsaturated fatty acids (PUFA) in the sperm plasma membrane. In addition, Chanapiwat et al. (2009) described that the differences in the freezing ability (i.e. cryotolerant) in Pietrain boars can be classified into poor and good freezability according to their post-thawed progressive motility (i.e. < 30% or > 30%, respectively) and may associated with the differences in lipid composition, fatty acid composition and sterol levels in the sperm plasma membrane of individual boar (Parks and Lynch 1992; Waterhouse et al. 2006; Kaeoket et al. 2008). However, no study has been reported about the influence of boar breeds on the concentration of gamma-oryzanol required during cryopreservation in different breeds. Therefore, the aim of this study was to investigate the effect of boar breeds (i.e. Duroc, Large white and Landrace) on the optimal concentration of gamma-oryzanol needed for semen cryopreservation.
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Materials and Methods This research project was approved by the Faculty of Veterinary Science-Animal Care and Use Committee (FVS-ACUC-Protocol No. MUVS-2012-43). Animals Twenty boars (10 Durocs, 5 Large whites and 5 Landraces) aged between 1 and 3 years old having proven fertility and used for routine artificial insemination (AI) in a commercial herd were included in the experiment. The boars were kept in individual pens in the same evaporative housing system. Each boar was fed with commercial diet twice daily, and water was provided ad libitum via water nipple. Semen collection and preparation Boar semen was collected once a week using glovedhand technique (Kaeoket et al. 2002, 2005). A total of 20 ejaculates of semen were obtained (one ejaculate each). During collection, semen was filtered through gauze and only sperm rich fraction was collected. Within 30 min after collection, sperm parameters including semen volume, pH, concentration and progressive motility were evaluated. Aliquot of 1 ml semen was collected into Eppendorf tube for sperm viability, sperm acrosome integrity and sperm morphology (Chanapiwat et al. 2009). The only ejaculates with motility of ≥70% and ≥80% morphologically normal were selected for cryopreservation. The remaining fresh semen was diluted with (1 : 1 v/v) ModenaTM extender (extender I) (Swine Genetics International, Ltd., Cambridge, IA, USA) and transported by cell incubator (Micom control system 20Q; Continental plastic CORP, Delavan, WI, USA) in 15°C to semen laboratory, Faculty of Veterinary Science, Mahidol University. Freezing and thawing process All semen samples were frozen using traditional nitrogen method. After collection and evaluation, the semen sample was diluted with (1 : 1 v/v) ModenaTM extender (Extender I) (Swine Genetics International Ltd). Diluted semen was transferred into 50 ml centrifuge tubes, cooled at 15°C for 120 min and later centrifuged at 800 9 g 15°C for 10 min (Hettich Rotanta 460R, Tuttlingen, Germany). After centrifugation, the supernatant was discarded, and the sperm pellets were resuspended to a concentration of 1.5 9 109 sperm/ml with approximately 1–2 ml of extender II (20% of egg yolk in 11% lactose solution). In this step, the semen sample was divided into five groups (Groups A–E) according to the concentration of gamma-oryzanol (KingTM; containing gamma-oryzanol 4 mg/ml of rice bran oil) in extender II, that is 0, 2, 4, 6 and 8 mg (0, 0.08, 0.16, 0.24, 0.32 mM) of gamma-oryzanol, respectively.
P Chanapiwat and K Kaeoket
The diluted semen was cooled at 5°C for 90 min. Each group of sample was mixed with Extender III [89.5% extender II with 9% (v/v) glycerol and 1.5% (v/v) Equex-STMâ; Nova Chemical Sales Inc., Scituate, MA, USA] to final concentration of 1.0 9 109 sperm/ml and 3% (v/v) glycerol (Gadea et al. 2005; Kaeoket et al. 2010a). The sperm suspensions were loaded into 0.5 ml straws (Bio-Vet, Z.I. Le Berdoulet, France). All straws were placed in contact with nitrogen vapour at 3 cm above the liquid nitrogen level for 20 min in an expandable polystyrene box and plunged into the nitrogen tank ( 196°C) for storage (Selles et al. 2003). After storage for a week, samples were thawed by immersing the straw in a thermos at 50°C for 12 s (Gadea et al. 2005). After thawing, the thawed sample was diluted (1 : 4) with ModenaTM extender in a test tube and kept in an incubator at 37°C until evaluation. Evaluation of sperm Sperm progressive motility Progressive sperm motility was subjectively evaluated by visual estimation by the same person throughout the study, at 37°C under phase contrast microscope (Olympus CX31, New York, USA) at 1009 and 4009 magnification (Berger et al. 1985). Progressive motility was expressed as the percentage of motile sperm cells. Sperm concentration Sperm concentration was assessed by Neubauer hemocytometer after dilution (1 : 100 v/v). One drop of this dilution was placed into the chamber, and the number of sperms was evaluated by phase contrast microscope 4009 magnification. Sperm concentration was expressed as sperms 9106 sperm/ml. Sperm viability The sperm viability was performed as described earlier by Chanapiwat et al. (2009). Briefly, 10 ll of diluted semen was mixed with 2.7 ll of SYBR-14 (10 lM in DMSO, final concentration of 0.54 lM) and 10 ll of 1.17 lM Ethidiumhomodimer-1(EthD-1). The mixture was incubated at 37°C for 15 min. At least 200 sperms were assessed using fluorescence microscope at 4009 magnification. The nuclei of live sperms with intact plasma membrane stained green with SYBR-14, while those with damaged membrane or dead sperm stained with red. The results were scored as the percentage of live sperm (viable) and dead sperm (non-viable). Sperm acrosome integrity The integrity of sperm acrosome was evaluated with fluorescein isothiocyanate labelled peanut (Arachis hypogaea) agglutinin (FITC-PNA) staining (Carvajal et al. 2004), and 10 ll of diluted semen was mixed with 10 ll © 2015 Blackwell Verlag GmbH
Gamma-Oryzanol Improved Frozen Boar Semen Qualities
of 1.17 lM EthD-1 and incubated at 37°C for 15 min. Then, smear 5 ll of mixture on glass slide and air dry. Fix with 95% ethanol for 30 s and air dry. Fifty ll of FITC-PNA (100 lg/ml in PBS) was spread over the slides and incubated in a moist chamber at 4°C for 30 min. After that slides were rinsed with cold PBS and air dried. Live sperms, stained with green, at least 100 cells were assessed under fluorescence microscope at 10009 magnification (Carl Zeiss, Inc., Axioskop 40, Germany) and classified to intact acrosome and nonintact acrosome. The results were scored as the percentage of intact acrosome of live sperm and non-intact. Statistical analysis Sperm quality was evaluated from parameters, that is sperm progressive motility, sperm viability and sperm acrosome integrity. Prior to analysis, all data were checked for normal distribution with Shapiro–Wilk test. The effect of treatments on parameters was analysed with two-ways ANOVA according to randomized complete block design (RCBD), in which treatments as fixed factor, parameters as dependent variables and boars as random block factor; and the interaction between treatments, breeds and boar (breed) were test by factorial in RCBD. Duncan’s new multiple range test was used for the mean of parameters comparison. All statistical analysis was performed as a general linear model using the univariate procedure of SPSS version 19.0 (SPSS 19.0; SPSS Inc., Chicago, IL, USA). The parameters were expressed as mean of percentage SD, and the difference between treatment means was considered significant when p < 0.05.
Results Fresh semen analysis The mean percentages of progressive motility, viability and acrosome integrity were 76.3 4.3, 67.1 17.1 and 66.8 11.7, respectively. The volume and concentration of fresh semen were ranged between 168 and 436 ml and 395-460 9 106 sperm/ml (n = 16), respectively.
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Post-thaw semen analysis The effect of breed and boar (breed) According to the statistical analyses of the present results, the effect of breed and boar within the same breed on post-thawed semen qualities (i.e. progressive motility, viability and acrosome integrity) was found (p < 0.001). Progressive motility The results of progressive motility in Duroc, Large white and Landrace boars are presented in Tables 1–3, respectively. In Duroc, the percentage of progressive motility in group C was higher than other groups except group D (p < 0.01) (Table 1). In Large white, the percentage of progressive motility in group D was significantly higher than other groups (p < 0.01) (Table 2). In Landrace, a higher percentage of progressive motility was found in groups C and D, compared with control group (p < 0.05) (Table 3). Sperm freezability with regard to progressive motility (effect of breed) Comparing among control groups (i.e. group A in all breeds, n = 20), a significantly higher percentage of progressive motility was found in Duroc (35.0 9.1) > Landrace (33.0 14.8) > Larger white (26.0 6.5) (p < 0.05). Sperm viability The highest percentage of sperm viability in Duroc and Large white boars were found in groups C and group D, respectively (Tables 1 and 2) (p < 0.05). However, in Landrace, no significant difference in sperm viability was observed among the treatment groups (Table 3). Acrosome integrity In Duroc, the highest percentage of acrosome integrity was found in group C (p < 0.01) (Table 1). In Large
Table 1. The mean (SD) percentages of progressive motility, viability and acrosome integrity of frozen–thawed semen in the different groups (A= control, B to E added with 0.08, 0.16, 0.24 and 0.32 mM of gamma-oryzanol, respectively) (Duroc boars, n = 10 ejaculates in each group) Viability (%) Groups
Progressive motility (%)
Viable
A B C D E Significant differences
35.0 9.1c 44.0 6.1b 50.0 8.2a 45.5 7.2ab 44.5 8.6b p < 0.01
29.2 5.6c 35.0 4.7b 40.0 6.4a 38.1 5.2ab 36.1 5.9ab p < 0.01
Values in each column marked with different superscript letters differ significantly (p < 0.05). NA, not analysed.
© 2015 Blackwell Verlag GmbH
Acrosome integrity (%) Non-viable
Intact
35.9 9.1c 41.5 10.4bc 52.2 9.6a 47.4 9.5ab 44.5 13.1b p < 0.01
66.9 65.1 60.5 61.9 64.0 NA
14.1 4.7 6.4 5.2 6.0
Non-intact 63.1 58.5 47.8 52.6 55.5 NA
8.9 10.4 9.6 9.5 13.1
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Table 2. The mean (SD) percentages of progressive motility, viability and acrosome integrity of frozen–thawed semen in the different (A= control, B to E added with 0.08, 0.16, 0.24 and 0.32 mM of gamma-oryzanol, respectively) (Large white boars, n = 5 ejaculates in each group) Viability (%) Groups
Progressive motility (%)
Viable
A B C D E Significant differences
26.0 6.5c 37.0 12.0b 39.0 8.2b 49.0 6.5a 36.0 8.9b p < 0.01
31.8 5.5b 37.1 4.9ab 36.0 6.3ab 42.2 5.4a 34.2 5.2b p < 0.05
Acrosome integrity (%) Non-viable
Intact
43.9 13.2c 53.0 17.6b 51.5 18.6b 60.3 11.8a 49.9 13.8b p < 0.01
62.8 62.9 64.0 57.8 65.8 NA
5.5 4.9 6.3 5.4 5.2
Non-intact 56.1 47.0 48.5 39.7 50.1 NA
13.2 17.6 18.6 11.8 13.8
Values in each column marked with different superscript letters differ significantly (p < 0.05). NA, not analysed.
Table 3. The mean (SD) percentages of progressive motility, viability and acrosome integrity of frozen–thawed semen in the different groups (A= control, B to E added with 0.08, 0.16, 0.24 and 0.32 mM of gamma-oryzanol, respectively) (Landrace boars, n = 5 ejaculates in each group) Viability (%) Groups
Progressive motility (%)
A B C D E Significant differences
33.0 14.8b 41.0 13.9ab 47.0 12.0a 48.0 10.4a 42.0 14.0ab p < 0.05
Viable 27.8 31.6 35.9 36.7 34.6 NS
9.3a 7.4a 8.4a 7.9a 5.9a
Acrosome integrity (%) Non-viable
Intact
39.6 13.7b 41.4 9.4b 42.0 12.3b 51.6 15.4a 42.2 12.6b p < 0.05
72.2 68.4 64.1 63.3 65.4 NA
9.3 7.4 8.4 8.1 6.3
Non-intact 60.4 58.6 58.0 48.4 57.8 NA
13.7 9.4 12.3 15.4 12.6
Values in each column marked with different superscript letters differ significantly (p < 0.05). NS, not significant; NA, not analysed.
white and Landrace, the percentage of intact acrosome in group D was significantly higher than other groups (p < 0.01) (Tables 2 and 3).
Discussion The present study clearly demonstrated that gammaoryzanol-enriched RBO significantly influenced the frozen–thawed boar semen qualities including progressive motility, viability and acrosome integrity. To the best of our knowledge, this study was the first report to investigate the effect of varied concentrations of gamma-oryzanol on boar semen qualities following cryopreservation with special focus on the different breeds (i.e. Duroc, Large white and Landrace). In the present results, it is worth noting that breed of boar not only resulted in the differences in freezability but also the differences in the optimal concentration of gamma-oryzanol needed for semen cryopreservation. In Duroc boars, it seems likely that gamma-oryzanol at a concentration of 0.16 mM provided a superior post-thawed semen qualities when compared to other groups. In Large white and Landrace boars, the optimal concentration of gamma-oryzanol seems to be at 0.24 mM in which clearly improved progressive motility and intact acrosome of frozen–thawed semen qualities. In addition to a significant difference among
breeds, we also found an important impact of individual boars on the susceptibility of their sperm to cryoinjury as shown by the differences in the postthaw sperm qualities among boars within the same breed, which is in agreement with the previous findings (Larsson and Einarsson 1976; Waterhouse et al. 2006; Chanapiwat et al. 2009). The reason for individual variation among boars in term of sperm cryotolerance is unknown at present. However, the trigger of genetic difference among individual boars may explain this impact (Holt et al. 2005) and the different physiological characteristic of the sperm from each boar may also contribute to the individual variation (White 1993; Watson 1995). Besides the genetic and physiological differences, the explanation of boar and breed effect found in this study may include the differences in lipid composition, fatty acid composition and sterol levels in their sperm plasma membrane (Waterhouse et al. 2006). Recently, in a proteomic investigation of bull (Chen et al. 2012; De Canio et al. 2014) and human (Xu et al. 2012) sperm, it has been reported that each sperm contains different expression of membrane proteins, in which, to some extent, may explained the effect of individual boar and breed found in this study. However, at the moment, there are no data available for the proteomics analysis of boar sperm. © 2015 Blackwell Verlag GmbH
Gamma-Oryzanol Improved Frozen Boar Semen Qualities
In the present results, superior qualities of frozen– thawed boar semen were observed in all treatment groups compare with control group. This can be explained by the chemical properties of gamma-oryzanol in that gamma-oryzanol has hydroxy and phenoxy groups which provide electrons that could bind to free radicals, leading to minimizing the detrimental effect of ROS and thereby stabilizing lipid components in the sperm plasma membrane (Masuda et al. 2006; Srinivasan et al. 2007). Besides gamma-oryzanol, it is worth noting that RBO contains antioxidant, that is alphatocopherol, which has a significant ability to break the covalent links that ROS have formed between fatty acid side chains in sperm membrane lipids, consequently, a decrease in sperm membrane damage (Pen~ a et al. 2003; Breininger et al. 2005; Jeong et al. 2009) and also contains membrane stabilizer, that is phytosterols, which may also stabilized sperm plasma membrane by replacing cholesterol in their membrane and consequently has a protective effect on sperm plasma membrane from oxidative damage (Brufau et al. 2008). Taken together, it can be concluded that the optimal concentration of gamma-oryzanol needed for boar
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semen cryopreservation in lactose–egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mM for Duroc and 0.24 mM for Large white and Landrace. Acknowledgements The research grant was provided by Thailand Research Fund (MRG5680130), Commission of Higher Education of Thailand and Mahidol University. The authors are grateful to Dr. Surasak Jittakhot PhD (Animal Nutrition), Faculty of Veterinary Science, Mahidol University for advice in statistical analysis. Drs. Jiraporn Thamma, Pornpitcha Treenawong, Apitsada Visessripong are thanked for technical assistant.
Conflict of interest None of the authors have any conflict of interest to declare.
Author contributions P Chanapiwat has contributed to freezing semen, data analysing and manuscript writing. K Kaeoket contributed to data interpretation, mining the data, manuscript writing and supervised the project.
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Submitted: 23 Sep 2014; Accepted: 30 Nov 2014 Author’s address (for correspondence): K Kaeoket, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Phuttamonthon, Nakhon-pathom 73170, Thailand. E-mail: [email protected]
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