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Animal Science Journal (2015) 86, 863–868
doi: 10.1111/asj.12395
R A P I D C O M M U N I C AT I O N The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen Panida CHANAPIWAT and Kampon KAEOKET Semen Laboratory, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Phuttamonthon, Nakhon-Pathom, Thailand
ABSTRACT The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A-F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at −196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P < 0.001). A significantly higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (P < 0.01). However, there was no significant difference in percentage of viability among groups. In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen.
Key words: antioxidant, boar semen, cryopreservation, curcumin.
INTRODUCTION Cryopreservation is an effective technique for preservation and distribution of top-quality genetic materials, targeting improvement of pig herd productivity. However, during boar semen cryopreservation processes, detrimental effects such as oxidative stress, cold shock, osmotic changes and lipid–protein reorganisations within the cell membranes occur and are able to damage spermatozoa, consequently causing low fertilization rate, low farrowing rate and small litter size (Eriksson et al. 2002). Nevertheless, in the last decade, many studies have been reported on field fertility trials in which different techniques were applied, for example, using seminal plasma during the thawing process and/or seminal plasma during artificial insemination (AI) (Kaeoket et al. 2010a, 2011; Okazaki & Shimada 2012; Chanapiwat et al. 2014), adding oxytocin during AI (Okazaki et al. 2014), with compromised results. Sperm plasma membrane consists of phospholipids, sterols, saturated fatty acids and polyunsaturated fatty acids (PUFA). Viability of sperm during cryopreser© 2015 Japanese Society of Animal Science
vation is related to alteration of amounts of PUFA in membrane lipids (Waterhouse et al. 2006; Kaeoket et al. 2010b). It is well documented that oxidative stress (i.e. the detrimental effect during cryopreservation) is an imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of the antioxidants in a particular milieu (Baumber et al. 2000). When the production of ROS exceeds the antioxidant defense system, this substantial ROS results in the oxidative damage of membrane lipids, proteins and DNA in the sperm which leads to an impairment of sperm plasma membrane (loss of function), decreased motility and viability (Watson 1995; Baumber et al. 2000; Hernandez et al. 2007; Jeong & Kim 2009). It has
Correspondence: Kampon Kaeoket, Semen Laboratory, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Phuttamonthon, Nakhon-pathom 73170, Thailand. (Email: vskkk@mahidol .ac.th) Received 9 October 2014; accepted for publication 24 December 2014.
864 P. CHANAPIWAT and K. KAEOKET
also been shown that addition of antioxidants such as vitamin E and glutathione (Kaeoket et al. 2008), L-cysteine (Chanapiwat et al. 2009; Kaeoket et al. 2010a), Gamma-oryzanol (Kaeoket et al. 2012) and Docosahexaenoic acid (DHA) from fish oil (Kaeoket et al. 2010c) to the freezing extender improved frozenthawed boar semen qualities (i.e. motility, viability and acrosome integrity) by minimizing the harmful effects of ROS (Maldjian et al. 2005). Currently, natural antioxidants are gaining popularity for medical and cosmetic purposes because they are commercially available and have the least side effects (Khopde et al. 1999). Curcumin, a yellow pigment from turmeric (Curcuma longa) extract, a polyphenolic compound, is dietary spice turmeric that has been used as a food ingredient, cosmetic and medical treatment for many years (Cousins et al. 2007). Curcumin is one of the natural antioxidants which act as an antiinflammatory, antitoxic and anticancer agent for medical treatment (Motterlini et al. 2000; Jang et al. 2009). Reddy and Lokesh (1994) and Jayaprakasha et al. (2006) demonstrated, in the experimental study of inhibition of ROS formation, that curcumin showed eight times more potent than vitamin E as a free radical scavenger. In addition, Sreejayan and Rao (1994) proved that curcumin exhibits protective effects against cold shock and oxidative damage by inhibiting lipid peroxidation, as a result of its powerful scavenging activity against free radicals, such as superoxide anion, hydroxyl radicals and nitric oxide in in vitro assays (Ak & GÜlcin 2008). With regard to its biological, pharmacological and antioxidant activities, curcumin has been shown to improve frozen-thawed semen qualities when supplemented in freezing extender for cryopreservation of goat (Bucak et al. 2010) and bovine (Bucak et al. 2012) semen. No previous study has been performed for investigating the significant roles of curcumin on the qualities (i.e. progressive motility, viability and acrosome integrity) of cryopreserved boar semen. The aim of this study was, therefore, to investigate the optimal concentration of curcumin needed for cryopreservation of boar semen.
Semen collection and preparation The semen samples (n = 9) from boars were collected by using the glove-hand technique (Kaeoket et al. 2005) with a vinyl glove. Only sperm rich fraction was collected and evaluated within 20 min following collection. Fresh semen parameters including semen volume, concentration, pH and progressive motility were evaluated. Only ejaculates with progressive motility and normal morphology, more than 80%, were used for cryopreservation. After evaluation, fresh semen was diluted with Modena™ extender to 1:1 (v/v) (Swine Genetics International, Ltd, Cambridge, IA, USA) and transported by cell incubator (Micom control system 20Q, Continental plastic CORP, Delavan, WI, USA) at 15°C to the semen laboratory, Faculty of Veterinary Science, Mahidol University.
Semen freezing and thawing process The freezing protocol was performed according to Chanapiwat et al. (2009). Briefly, after incubation at 15°C for 120 min, diluted semen was transferred into 50 mL centrifuge tubes and centrifuged at 800 × g, 15°C (Hettich Universal 32R, Tuttlingen, Germany) for 10 min. After centrifugation, the supernatant was discarded and the sperm pellet was re-suspended with freezing extender I (20% of egg yolk in 11% lactose solution) to a concentration of 1.5 × 109 sperm/mL. Freezing extender I was divided into six groups (A-F) according to the different concentrations of curcumin in 0.5% dimethyl sulfoxide (DMSO) (Curcumin c1386; Sigma-Aldrich Chemical, Buchs, Switzerland) as follows: 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively. After cooling at 5°C for 90 min, diluted semen was mixed with freezing extender II (89.5% freezing extender I with 9% (v/v) glycerol and 1.5% (v/v) Equex-STM® (Nova Chemical Sales Inc., Scituate, MA, USA) to a final concentration of 1.0 × 109 sperm/mL (Gadea et al. 2005; Kaeoket et al. 2010b). The processed semen were loaded into 0.50 mL polyvinyl chloride medium straws (Bio-Vet, Z.I. Le Berdoulet, France), and were placed on a rack in an expandable polystyrene box (20.5 cm × 31 cm × 18.5 cm) which contained liquid nitrogen and all the straw was left in contact with nitrogen vapor at 4 cm above the liquid nitrogen level for 20 min and was plunged into liquid nitrogen tank (at −196°C) for storage (Chanapiwat et al. 2009). After storage for a week, thawing was done at 50°C for 12 s (Kaeoket et al. 2010b). The thawed sample was diluted with ModenaTM extender (1:4 v/v) and kept in an incubator at 37°C until semen evaluation.
Evaluation of sperm MATERIALS AND METHODS
Progressive motility
This research project was approved by the Faculty of Veterinary Science – Animal Care and Use Committee, Mahidol University, Phuttamonthon, Thailand (FVS-ACUC-Protocol No. MUVS-2013-03).
Progressive sperm motility was evaluated at 37°C under a phase contrast microscope at 100× and 400× magnification and progressive motility was expressed as the percentage of motile spermatozoa.
Animals
Concentration
Nine Duroc boars, aged 1–3 years old were kept in individual pens in an evaporative housing system. Animals were fed twice a day with commercial feed and water was provided ad libitum via a water nipple. The boars had proven fertility and were used for routine artificial insemination (AI).
Sperm concentration was assessed by a Neubauer hemocytometer after diluting semen with formal saline to 1:100 (v/v) and evaluation by phase contrast microscopy at 400× magnification. Sperm concentration was expressed as sperm ×106 sperm/mL.
© 2015 Japanese Society of Animal Science
Animal Science Journal (2015) 86, 863–868
CURCUMIN IMPROVES FROZEN BOAR SEMEN QUALITIES
Viability (viable and non-viable spermatozoa) The sperm viability was assessed as described earlier by Chanapiwat et al. (2009). Briefly, 10 μL of diluted semen was mixed with 2.7 μL of SYBR-14 and 10 μL of Ethidiumhomodimer-1 (EthD-1). After incubation at 37°C for 15 min, at least 200 sperms were assessed under fluorescence microscope at 400× magnification. The nuclei of live sperm with intact plasma membrane was stained as a green color with SYBR-14, and sperm with damaged plasma membrane or dead sperm stained as a red color with EthD-1. The sperm viability was expressed as the percentage of live sperm with intact plasma membrane.
Acrosome integrity The evaluation of sperm acrosome integrity was conducted by using fluorescein isothiocyanate labeled peanut (Arachis hypogaea) agglutinin (FITC-PNA) staining (Chanapiwat et al. 2009). Briefly, 10 μL of diluted semen was mixed with 10 μL of EthD-1, and incubated at 37°C for 15 min before smearing 5 μL of the mixture on a glass slide and air drying. It was fixed with 95% ethanol for 30 s and air dried. Fifty microliters of FITC-PNA solution (diluted FITC-PNA with phosphate buffered saline (PBS) 1:10 v/v) was spread over the slides and kept in a moist chamber at 4°C for 30 min. Then, slides were rinsed with cold PBS and air dried. At least 200 sperm were assessed by fluorescence microscope at 1000× magnification (Axioskop 40; Carl Zeiss, Inc., Oberkochen, Germany) and classified as intact acrosomes and non-intact acrosomes. The results were presented as the percentage of live sperm with intact acrosome.
Statistical analysis The statistical analysis was performed using SPSS version 19.0 (SPSS 19.0; SPSS Inc., Chicago, IL, USA). Sperm parameters consisting of sperm progressive motility, sperm viability and acrosome integrity were evaluated. All parameters were tested for normal distribution with Shapiro-Wilk Test. The sperm parameters not normally distributed (i.e. sperm motility and sperm viability) were transformed using log transformation. Sperm parameters were analyzed using one-way analysis of variance (ANOVA) according to Randomized Complete Block Design (RCBD). Each treatment (different concentrations of curcumin in freezing extender I) was the fixed factor and the boars as random block factor. The comparison of sperm parameters among treatment groups were performed by Duncan’s new multiple range test. All statistical analyses were performed as a General Linear Model (GLM) and the statistically significant difference was considered as P < 0.05.
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centration and volume of fresh semen were varied between 121–867 × 106 sperm/mL and 80–190 mL, respectively.
Post-thaw semen analysis Progressive motility There was a significantly higher percentage of progressive motility in the treatment groups than in the control group (P < 0.001). A higher percentage of progressive motility was found in groups C (33.3%) and D (36.1%) (supplementation of curcumin at 0.25 and 0.50 mmol/L, respectively) than in the other groups (Fig. 1). Sperm viability No significant difference in percentage of sperm viability was found between the control group (30.9%) and treatment groups (range 30.9–36.4%). However, a higher percentage of sperm viability was found in groups C (35.3%) and D (36.4%) when compared with other groups (Fig. 2). Acrosome integrity There was a significantly higher percentage of acrosome integrity in the treatment groups than in the control group (P < 0.01). A higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (Fig. 3).
DISCUSSION The statistical analysis of fresh semen samples such as progressive motility, viability and acrosome integrity was not significantly different among boars, showing
RESULTS Fresh semen analysis Sperm parameters, such as progressive motility, sperm viability, acrosome integrity and morphology were evaluated. The percentage of progressive motility, sperm viability, acrosome integrity and morphology were 83 (range 70–95), 76 (range 50–88), 92 (range 77–99), and 88.5 (range 84–92), respectively. However, no significant differences in fresh semen qualities were found among boars (n = 9). The conAnimal Science Journal (2015) 86, 863–868
Figure 1 Progressive motility of spermatozoa in frozen semen from Duroc boars (n = 9) in different groups: grade A = control, B to F added with 0.125, 0.25, 0.50, 0.75 and 1 mmol/L of curcumin, respectively, which are presented as bars (mean ± SD). Bars marked by different letters are significantly different (P < 0.001).
© 2015 Japanese Society of Animal Science
866 P. CHANAPIWAT and K. KAEOKET
Figure 2 Sperm viability of spermatozoa in frozen semen from Duroc boars (n = 9) in different groups: grade A = control, B to F added with 0.125, 0.25, 0.50, 0.75 and 1 mmol/L of curcumin, respectively which are presented as bars (mean ± SD).
Figure 3 Acrosome integrity of spermatozoa in frozen semen from Duroc boars (n = 9) in different groups: grade A = control, B to F added with 0.125, 0.25, 0.50, 0.75 and 1 mmol/L of curcumin, respectively which are presented as bars (mean ± SD). Bars marked by different letters are significantly different (P < 0.01).
that boar semen samples used in this study were uniform. The supplementation of curcumin at 0.25 or 0.50 mmol/L in the freezing extender yields better frozen-thawed boar semen qualities in terms of progressive motility and acrosome integrity when compared with control and other treatment groups. To our knowledge, the present study is the first report demonstrating the beneficial effect of adding curcumin during cryopreservation on the qualities of frozenthawed boar semen. The present results might be supported by the fact that boar sperm is able to uptake substances such as antioxidants and utilize them for © 2015 Japanese Society of Animal Science
protecting its plasma membrane from those ROS which occur both in an intracellular and extracellular milieu during cryopreservation (Maldjian et al. 2005; Kaeoket et al. 2010a, b). The results in the present study are in agreement with those of Bucak et al. (2010) in that an addition of curcumin could improve progressive motility and acrosome integrity of frozen-thawed Angora goat semen and also improve the progressive motility and functional integrity of the sperm plasma membrane of frozen bull semen (Bucak et al. 2012). The improvement in the qualities of frozen-thawed boar semen found in the present study might be explained by the fact that curcumin, a polyphenolic compound, a natural antioxidant, has the protective effects against cold shock and oxidative damage by inhibition of ROS formation (Reddy & Lokesh 1994) and lipid peroxidation (Sreejayan & Rao 1994), as a result of its scavenging property against free radicals, such as superoxide anion, hydroxyl radicals (Motterlini et al. 2000) and nitric oxide (Ak & GÜlcin 2008) as shown in an in vitro study (Jayaprakasha et al. 2006). In addition, it has been demonstrated that curcumin increased the activity of superoxide dismutase (SOD, an enzymatic antioxidant) and also the total glutathione (tGSH, non-enzymatic antioxidant) level during cryopreservation of Angora goat semen (Bucak et al. 2010) and bovine semen (Bucak et al. 2012), respectively. These two antioxidants (i.e. SOD and GSH) have been described as serving as a defense mechanism against lipid peroxidation and ROS (Aitken & Clarkson 1988; Agarwal et al. 2005), thus may protect sperm from oxidative damage. However, the cellular mechanisms of the anti-oxidative effect of curcumin during boar semen cryopreservation need further investigation. With regard to the biphasic effect on the concentration of curcumin in the present study, it is in agreement with previous reports in that the qualities of frozen-thawed boar semen depend on the concentration of antioxidants (Chanapiwat et al. 2009; Kaeoket et al. 2010b) and too high a concentration may not be expedient if sperm has restrictive antioxidant uptake (Kaeoket et al. 2010c, 2012). In accordance with the present results, it has been demonstrated that curcumin caused a concentration-dependent decrease in human and murine sperm progressive motility, capacitation/acrosome reaction and fertilisation in an in vitro study and if administered intra-vaginally, produced a significant reduction in fertility (Rithaporn et al. 2003; Naz 2011). In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen. Animal Science Journal (2015) 86, 863–868
CURCUMIN IMPROVES FROZEN BOAR SEMEN QUALITIES
ACKNOWLEDGMENTS The authors are grateful to Dr. Surasak Jittakhot PhD (Animal Nutrition), Faculty of Veterinary Science, Mahidol University for advice in the statistical analysis. Drs. Saowanee Kwansuk, Jutaporn Sunghan, Maythawee Singhabutra are thanked for technical assistant. Financial support was provided by Faculty of Veterinary Science, Mahidol University.
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Reddy AC, Lokesh BR. 1994. Studies on the inhibitory effects of curcumin and eugenol on the formation of reactive oxygen species and the oxidation of ferrous iron. Molecular and Cellular Biochemistry 137, 1–8. Rithaporn T, Monga M, Rajasekaran M. 2003. Curcumin: a potential vaginal contraceptive. Contraception 68, 219– 223. Sreejayan N, Rao MN. 1994. Curcuminoids as potent inhibitors of lipid peroxidation. Journal of Pharmaceutics and Pharmacology 46, 1013–1016.
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Waterhouse KE, Hofmo PO, Tverdal A, Miller RR. 2006. Within and between breed differences in freezing tolerance and plasma membrane fatty acid composition of boar sperm. Reproduction 131, 887–894. Watson PF. 1995. Recent developments and concepts in the cryopreservation of spermatozoa and the assessment of their post-thawing function. Reproduction Fertility and Development 7, 871–891.
Animal Science Journal (2015) 86, 863–868
Animal Science Journal (2015) 86, 863–868
doi: 10.1111/asj.12395
R A P I D C O M M U N I C AT I O N The effect of Curcuma longa extracted (curcumin) on the quality of cryopreserved boar semen Panida CHANAPIWAT and Kampon KAEOKET Semen Laboratory, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Phuttamonthon, Nakhon-Pathom, Thailand
ABSTRACT The aim of this study was to determine the optimal concentration of curcumin needed for cryopreservation of boar semen. Semen samples (n = 9) were collected from nine Duroc boars which having proven fertility were used for routine artificial insemination. Semen samples were collected and divided into six groups (groups A-F) according to various concentrations of curcumin in freezing extender (i.e. 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively). The semen was frozen by traditional liquid nitrogen vapor method and stored at −196°C in the liquid nitrogen tank. After storage, frozen semen samples were thawed at 50°C for 12 s and evaluated for progressive motility, viability and acrosome integrity. The present results indicated that the addition of curcumin at 0.25 (group C) or 0.50 mmol/L curcumin (group D) yielded the higher percentage of progressive motility (33.3 and 36.1%, respectively) (P < 0.001). A significantly higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (P < 0.01). However, there was no significant difference in percentage of viability among groups. In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen.
Key words: antioxidant, boar semen, cryopreservation, curcumin.
INTRODUCTION Cryopreservation is an effective technique for preservation and distribution of top-quality genetic materials, targeting improvement of pig herd productivity. However, during boar semen cryopreservation processes, detrimental effects such as oxidative stress, cold shock, osmotic changes and lipid–protein reorganisations within the cell membranes occur and are able to damage spermatozoa, consequently causing low fertilization rate, low farrowing rate and small litter size (Eriksson et al. 2002). Nevertheless, in the last decade, many studies have been reported on field fertility trials in which different techniques were applied, for example, using seminal plasma during the thawing process and/or seminal plasma during artificial insemination (AI) (Kaeoket et al. 2010a, 2011; Okazaki & Shimada 2012; Chanapiwat et al. 2014), adding oxytocin during AI (Okazaki et al. 2014), with compromised results. Sperm plasma membrane consists of phospholipids, sterols, saturated fatty acids and polyunsaturated fatty acids (PUFA). Viability of sperm during cryopreser© 2015 Japanese Society of Animal Science
vation is related to alteration of amounts of PUFA in membrane lipids (Waterhouse et al. 2006; Kaeoket et al. 2010b). It is well documented that oxidative stress (i.e. the detrimental effect during cryopreservation) is an imbalance between the production of reactive oxygen species (ROS) and the scavenging capacity of the antioxidants in a particular milieu (Baumber et al. 2000). When the production of ROS exceeds the antioxidant defense system, this substantial ROS results in the oxidative damage of membrane lipids, proteins and DNA in the sperm which leads to an impairment of sperm plasma membrane (loss of function), decreased motility and viability (Watson 1995; Baumber et al. 2000; Hernandez et al. 2007; Jeong & Kim 2009). It has
Correspondence: Kampon Kaeoket, Semen Laboratory, Department of Clinical Sciences and Public Health, Faculty of Veterinary Science, Mahidol University, Phuttamonthon, Nakhon-pathom 73170, Thailand. (Email: vskkk@mahidol .ac.th) Received 9 October 2014; accepted for publication 24 December 2014.
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also been shown that addition of antioxidants such as vitamin E and glutathione (Kaeoket et al. 2008), L-cysteine (Chanapiwat et al. 2009; Kaeoket et al. 2010a), Gamma-oryzanol (Kaeoket et al. 2012) and Docosahexaenoic acid (DHA) from fish oil (Kaeoket et al. 2010c) to the freezing extender improved frozenthawed boar semen qualities (i.e. motility, viability and acrosome integrity) by minimizing the harmful effects of ROS (Maldjian et al. 2005). Currently, natural antioxidants are gaining popularity for medical and cosmetic purposes because they are commercially available and have the least side effects (Khopde et al. 1999). Curcumin, a yellow pigment from turmeric (Curcuma longa) extract, a polyphenolic compound, is dietary spice turmeric that has been used as a food ingredient, cosmetic and medical treatment for many years (Cousins et al. 2007). Curcumin is one of the natural antioxidants which act as an antiinflammatory, antitoxic and anticancer agent for medical treatment (Motterlini et al. 2000; Jang et al. 2009). Reddy and Lokesh (1994) and Jayaprakasha et al. (2006) demonstrated, in the experimental study of inhibition of ROS formation, that curcumin showed eight times more potent than vitamin E as a free radical scavenger. In addition, Sreejayan and Rao (1994) proved that curcumin exhibits protective effects against cold shock and oxidative damage by inhibiting lipid peroxidation, as a result of its powerful scavenging activity against free radicals, such as superoxide anion, hydroxyl radicals and nitric oxide in in vitro assays (Ak & GÜlcin 2008). With regard to its biological, pharmacological and antioxidant activities, curcumin has been shown to improve frozen-thawed semen qualities when supplemented in freezing extender for cryopreservation of goat (Bucak et al. 2010) and bovine (Bucak et al. 2012) semen. No previous study has been performed for investigating the significant roles of curcumin on the qualities (i.e. progressive motility, viability and acrosome integrity) of cryopreserved boar semen. The aim of this study was, therefore, to investigate the optimal concentration of curcumin needed for cryopreservation of boar semen.
Semen collection and preparation The semen samples (n = 9) from boars were collected by using the glove-hand technique (Kaeoket et al. 2005) with a vinyl glove. Only sperm rich fraction was collected and evaluated within 20 min following collection. Fresh semen parameters including semen volume, concentration, pH and progressive motility were evaluated. Only ejaculates with progressive motility and normal morphology, more than 80%, were used for cryopreservation. After evaluation, fresh semen was diluted with Modena™ extender to 1:1 (v/v) (Swine Genetics International, Ltd, Cambridge, IA, USA) and transported by cell incubator (Micom control system 20Q, Continental plastic CORP, Delavan, WI, USA) at 15°C to the semen laboratory, Faculty of Veterinary Science, Mahidol University.
Semen freezing and thawing process The freezing protocol was performed according to Chanapiwat et al. (2009). Briefly, after incubation at 15°C for 120 min, diluted semen was transferred into 50 mL centrifuge tubes and centrifuged at 800 × g, 15°C (Hettich Universal 32R, Tuttlingen, Germany) for 10 min. After centrifugation, the supernatant was discarded and the sperm pellet was re-suspended with freezing extender I (20% of egg yolk in 11% lactose solution) to a concentration of 1.5 × 109 sperm/mL. Freezing extender I was divided into six groups (A-F) according to the different concentrations of curcumin in 0.5% dimethyl sulfoxide (DMSO) (Curcumin c1386; Sigma-Aldrich Chemical, Buchs, Switzerland) as follows: 0, 0.125, 0.25, 0.50, 0.75 and 1.0 mmol/L, respectively. After cooling at 5°C for 90 min, diluted semen was mixed with freezing extender II (89.5% freezing extender I with 9% (v/v) glycerol and 1.5% (v/v) Equex-STM® (Nova Chemical Sales Inc., Scituate, MA, USA) to a final concentration of 1.0 × 109 sperm/mL (Gadea et al. 2005; Kaeoket et al. 2010b). The processed semen were loaded into 0.50 mL polyvinyl chloride medium straws (Bio-Vet, Z.I. Le Berdoulet, France), and were placed on a rack in an expandable polystyrene box (20.5 cm × 31 cm × 18.5 cm) which contained liquid nitrogen and all the straw was left in contact with nitrogen vapor at 4 cm above the liquid nitrogen level for 20 min and was plunged into liquid nitrogen tank (at −196°C) for storage (Chanapiwat et al. 2009). After storage for a week, thawing was done at 50°C for 12 s (Kaeoket et al. 2010b). The thawed sample was diluted with ModenaTM extender (1:4 v/v) and kept in an incubator at 37°C until semen evaluation.
Evaluation of sperm MATERIALS AND METHODS
Progressive motility
This research project was approved by the Faculty of Veterinary Science – Animal Care and Use Committee, Mahidol University, Phuttamonthon, Thailand (FVS-ACUC-Protocol No. MUVS-2013-03).
Progressive sperm motility was evaluated at 37°C under a phase contrast microscope at 100× and 400× magnification and progressive motility was expressed as the percentage of motile spermatozoa.
Animals
Concentration
Nine Duroc boars, aged 1–3 years old were kept in individual pens in an evaporative housing system. Animals were fed twice a day with commercial feed and water was provided ad libitum via a water nipple. The boars had proven fertility and were used for routine artificial insemination (AI).
Sperm concentration was assessed by a Neubauer hemocytometer after diluting semen with formal saline to 1:100 (v/v) and evaluation by phase contrast microscopy at 400× magnification. Sperm concentration was expressed as sperm ×106 sperm/mL.
© 2015 Japanese Society of Animal Science
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Viability (viable and non-viable spermatozoa) The sperm viability was assessed as described earlier by Chanapiwat et al. (2009). Briefly, 10 μL of diluted semen was mixed with 2.7 μL of SYBR-14 and 10 μL of Ethidiumhomodimer-1 (EthD-1). After incubation at 37°C for 15 min, at least 200 sperms were assessed under fluorescence microscope at 400× magnification. The nuclei of live sperm with intact plasma membrane was stained as a green color with SYBR-14, and sperm with damaged plasma membrane or dead sperm stained as a red color with EthD-1. The sperm viability was expressed as the percentage of live sperm with intact plasma membrane.
Acrosome integrity The evaluation of sperm acrosome integrity was conducted by using fluorescein isothiocyanate labeled peanut (Arachis hypogaea) agglutinin (FITC-PNA) staining (Chanapiwat et al. 2009). Briefly, 10 μL of diluted semen was mixed with 10 μL of EthD-1, and incubated at 37°C for 15 min before smearing 5 μL of the mixture on a glass slide and air drying. It was fixed with 95% ethanol for 30 s and air dried. Fifty microliters of FITC-PNA solution (diluted FITC-PNA with phosphate buffered saline (PBS) 1:10 v/v) was spread over the slides and kept in a moist chamber at 4°C for 30 min. Then, slides were rinsed with cold PBS and air dried. At least 200 sperm were assessed by fluorescence microscope at 1000× magnification (Axioskop 40; Carl Zeiss, Inc., Oberkochen, Germany) and classified as intact acrosomes and non-intact acrosomes. The results were presented as the percentage of live sperm with intact acrosome.
Statistical analysis The statistical analysis was performed using SPSS version 19.0 (SPSS 19.0; SPSS Inc., Chicago, IL, USA). Sperm parameters consisting of sperm progressive motility, sperm viability and acrosome integrity were evaluated. All parameters were tested for normal distribution with Shapiro-Wilk Test. The sperm parameters not normally distributed (i.e. sperm motility and sperm viability) were transformed using log transformation. Sperm parameters were analyzed using one-way analysis of variance (ANOVA) according to Randomized Complete Block Design (RCBD). Each treatment (different concentrations of curcumin in freezing extender I) was the fixed factor and the boars as random block factor. The comparison of sperm parameters among treatment groups were performed by Duncan’s new multiple range test. All statistical analyses were performed as a General Linear Model (GLM) and the statistically significant difference was considered as P < 0.05.
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centration and volume of fresh semen were varied between 121–867 × 106 sperm/mL and 80–190 mL, respectively.
Post-thaw semen analysis Progressive motility There was a significantly higher percentage of progressive motility in the treatment groups than in the control group (P < 0.001). A higher percentage of progressive motility was found in groups C (33.3%) and D (36.1%) (supplementation of curcumin at 0.25 and 0.50 mmol/L, respectively) than in the other groups (Fig. 1). Sperm viability No significant difference in percentage of sperm viability was found between the control group (30.9%) and treatment groups (range 30.9–36.4%). However, a higher percentage of sperm viability was found in groups C (35.3%) and D (36.4%) when compared with other groups (Fig. 2). Acrosome integrity There was a significantly higher percentage of acrosome integrity in the treatment groups than in the control group (P < 0.01). A higher percentage of acrosome integrity was found in groups B (29.7%), C (31.1%) and D (30.2%) than in the other groups (Fig. 3).
DISCUSSION The statistical analysis of fresh semen samples such as progressive motility, viability and acrosome integrity was not significantly different among boars, showing
RESULTS Fresh semen analysis Sperm parameters, such as progressive motility, sperm viability, acrosome integrity and morphology were evaluated. The percentage of progressive motility, sperm viability, acrosome integrity and morphology were 83 (range 70–95), 76 (range 50–88), 92 (range 77–99), and 88.5 (range 84–92), respectively. However, no significant differences in fresh semen qualities were found among boars (n = 9). The conAnimal Science Journal (2015) 86, 863–868
Figure 1 Progressive motility of spermatozoa in frozen semen from Duroc boars (n = 9) in different groups: grade A = control, B to F added with 0.125, 0.25, 0.50, 0.75 and 1 mmol/L of curcumin, respectively, which are presented as bars (mean ± SD). Bars marked by different letters are significantly different (P < 0.001).
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Figure 2 Sperm viability of spermatozoa in frozen semen from Duroc boars (n = 9) in different groups: grade A = control, B to F added with 0.125, 0.25, 0.50, 0.75 and 1 mmol/L of curcumin, respectively which are presented as bars (mean ± SD).
Figure 3 Acrosome integrity of spermatozoa in frozen semen from Duroc boars (n = 9) in different groups: grade A = control, B to F added with 0.125, 0.25, 0.50, 0.75 and 1 mmol/L of curcumin, respectively which are presented as bars (mean ± SD). Bars marked by different letters are significantly different (P < 0.01).
that boar semen samples used in this study were uniform. The supplementation of curcumin at 0.25 or 0.50 mmol/L in the freezing extender yields better frozen-thawed boar semen qualities in terms of progressive motility and acrosome integrity when compared with control and other treatment groups. To our knowledge, the present study is the first report demonstrating the beneficial effect of adding curcumin during cryopreservation on the qualities of frozenthawed boar semen. The present results might be supported by the fact that boar sperm is able to uptake substances such as antioxidants and utilize them for © 2015 Japanese Society of Animal Science
protecting its plasma membrane from those ROS which occur both in an intracellular and extracellular milieu during cryopreservation (Maldjian et al. 2005; Kaeoket et al. 2010a, b). The results in the present study are in agreement with those of Bucak et al. (2010) in that an addition of curcumin could improve progressive motility and acrosome integrity of frozen-thawed Angora goat semen and also improve the progressive motility and functional integrity of the sperm plasma membrane of frozen bull semen (Bucak et al. 2012). The improvement in the qualities of frozen-thawed boar semen found in the present study might be explained by the fact that curcumin, a polyphenolic compound, a natural antioxidant, has the protective effects against cold shock and oxidative damage by inhibition of ROS formation (Reddy & Lokesh 1994) and lipid peroxidation (Sreejayan & Rao 1994), as a result of its scavenging property against free radicals, such as superoxide anion, hydroxyl radicals (Motterlini et al. 2000) and nitric oxide (Ak & GÜlcin 2008) as shown in an in vitro study (Jayaprakasha et al. 2006). In addition, it has been demonstrated that curcumin increased the activity of superoxide dismutase (SOD, an enzymatic antioxidant) and also the total glutathione (tGSH, non-enzymatic antioxidant) level during cryopreservation of Angora goat semen (Bucak et al. 2010) and bovine semen (Bucak et al. 2012), respectively. These two antioxidants (i.e. SOD and GSH) have been described as serving as a defense mechanism against lipid peroxidation and ROS (Aitken & Clarkson 1988; Agarwal et al. 2005), thus may protect sperm from oxidative damage. However, the cellular mechanisms of the anti-oxidative effect of curcumin during boar semen cryopreservation need further investigation. With regard to the biphasic effect on the concentration of curcumin in the present study, it is in agreement with previous reports in that the qualities of frozen-thawed boar semen depend on the concentration of antioxidants (Chanapiwat et al. 2009; Kaeoket et al. 2010b) and too high a concentration may not be expedient if sperm has restrictive antioxidant uptake (Kaeoket et al. 2010c, 2012). In accordance with the present results, it has been demonstrated that curcumin caused a concentration-dependent decrease in human and murine sperm progressive motility, capacitation/acrosome reaction and fertilisation in an in vitro study and if administered intra-vaginally, produced a significant reduction in fertility (Rithaporn et al. 2003; Naz 2011). In conclusion, addition to the freezing extender of curcumin during cryopreservation at a concentration of 0.25 or 0.50 mmol/L is the optimal concentration of curcumin for improving the quality (i.e. increased progressive motility and acrosome integrity) of cryopreserved boar semen. Animal Science Journal (2015) 86, 863–868
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ACKNOWLEDGMENTS The authors are grateful to Dr. Surasak Jittakhot PhD (Animal Nutrition), Faculty of Veterinary Science, Mahidol University for advice in the statistical analysis. Drs. Saowanee Kwansuk, Jutaporn Sunghan, Maythawee Singhabutra are thanked for technical assistant. Financial support was provided by Faculty of Veterinary Science, Mahidol University.
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