ORIGINAL ARTICLE
Effect of Camellia sinensis supplementation and increasing holding time on quality of cryopreserved boar semen lez & F. Martınez I. Gale, L. Gil, C. Malo, N. Gonza Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain
Keywords Antioxidants—boar semen—cryopreservation—holding time Correspondence C. Malo, Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, C/Miguel Servet, 177, 50013 Zaragoza, Spain. Tel.: +976761544; Fax: +976761612; E-mail: [email protected] Accepted: March 29, 2014 doi: 10.1111/and.12293
Summary Cryopreservation of boar semen is still considered suboptimal due to the low fertility when compared with fresh semen. This study was performed to evaluate the effects of green tea (Camellia sinensis) supplementation of the freezing extender at different concentration (0, 2.5%, 5%, 10%) and also to determine the influence of increasing holding time from 2 to 24 h at 15 °C. Seventeen ejaculates from nine boars were used to make pools of three of them and then cryopreserved. Sperm motility, viability, acrosome integrity, membrane functionality (HOST) and capacitation status were determined before freezing and at 0, 30, 60, 90 and 120 min after thawing. Lipid peroxidation was evaluated just after thawing. The main findings emerging from this study were the following: (i) no improvement in quality of thawed spermatozoa with addition of tea to the freezing extender, (ii) no improvement in quality of thawed spermatozoa with prolonged holding time, (iii) lower peroxidation rate in presence of tea 5% and (iv) a decrease in the number of uncapacited viable spermatozoa with any tea supplementation. We conclude that amplification of holding time in semen cryopreservation process does not vary results, facilitating freezing protocol. Tea supplementation reduces lipoxidation but did not improve quality parameters.
Introduction In recent years, the development of freezing techniques for semen cryopreservation has become a major source for the preservation of genetic material in domestic animals (Songsasen & Leibo, 1997; Cremades et al., 2005). However, the use of frozen-thawed boar semen is limited due to the low fertility outcomes compared with extended fresh semen (Buranaamnuay et al., 2009). The detrimental effect of cryopreservation on sperm includes coldshock, osmotic stress and oxidative stress from reactive oxygen substances (ROS) production which decreases lipid content of the membranes. Boar spermatozoa is very susceptible to ROS due to the high concentration of polyunsaturated fatty acids (PUFA) within the lipid membrane (Buhr et al., 1994). ROS have a dual effect on sperm function; at low concentrations ROS induces sperm capacitation, hyperactivation and fertilisation; mean while excessive ROS production reduces sperm motility (Guthrie & Welch, 2006), causes © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 505–512
damage DNA (Bennetts & Aitken, 2005) and decrease sperm-oocyte fusion (Watson, 1995). Some antioxidants, particularly vegetable antioxidants, have been employed in sperm cryopreservation. Zhao et al. (2009) reduced lipoxidation of spermatozoa during cryopreservation by incorporation of Rodhiola sacra aqueous extract (RSAE), a genus of Chinese herb. Interestingly, in recent studies from our laboratory, rosemary has been successfully used to freeze boar (Malo et al., 2010), ovine (Gil et al., 2010) and canine (Gonzalez et al., 2010) sperm, improving post-thaw motility and in vitro fertilisation parameters: Fennel also showed protective effect in boar semen cryopreservation (Malo et al., 2012). However, no studies have been published on boar semen cryopreservation using green tea. Green tea is a well-known alimentary antioxidant substance; this effect is largely attributed to polyphenols, chemicals with potent antioxidant properties. In fact, green tea polyphenols are more potent antioxidants than Vitamin C, Vitamin E, or rosemary extract (Wittayarat et al., 2012). 505
Cryopreservation boar semen
Main polyphenols found in green tea are theaflavins, thearubigins and catechins. Green tea contains four primary catechin (compounds which are a form of flavanol monomers): epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and apigallocatechin- 3- gallate (EGCG). EGCG is the most studied polyphenol component in green tea, being the most active (Hara, 1997), it has a powerful antioxidant effect and prevents oxidative damage in healthy cells (Westerterp-Platenga, 2010; Singh et al., 2011). Green tea also contains alkaloids including theobromine, theophylline and caffeine, that stimulate motility of dog and domestic cat frozen-thawed spermatozoa (Milani et al., 2010), without affecting longevity. Holding time is the period during which an ejaculate, either in a raw or diluted state, is held at 15 °C before further processing for cold state. In boars, longer holding times positively influence sperm quality parameters including sperm (Eriksson et al., 2001; Casas & Althouse, 2013). Therefore, the main aim of this study was to evaluate green tea as a cryomedium supplement for the cryopreservation of boar semen and determine the influence of longer holding times before cryopreservation on semen quality after thawing. Materials and methods Experimental extenders Unless otherwise indicated, all chemicals were from Sigma-Aldrich Co. (Alcobendas, Madrid, Spain). Orvus ES Paste (OEP) was from Minitub. Green tea sources were from La Flor del Pirineo, S.L., Manresa, Spain. The medium used for semen collection was Dulbeco0 s phosphate buffered saline (DPBS) medium composed of 136.89 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4 and 1.46 mM CaCl22H2O. Lactose–egg yolk extender containing 20% (v:v) egg yolk, 80% of 11% L-lactose solution (290 mM) and 100 lg ml1 of kanamycin sulphate was used as base extender. Fennel extenders were prepared according to the procedure for antioxidant extraction described by Malo et al. (2010). We extracted the green tea essence by soaking dry commercial herbs. After heating 100 ml of water to 100 °C, leaves at different concentrations [2.5 g (T2.5); 5 g (T5); 10 g (T10)] were added to the water and maintained for 10 min at this temperature. Once the water had cooled to 25 °C, the solution was filtered to remove the leaves. The resulting solution had a pH of 7 0.4. Sperm dilution was performed in a two-step procedure, fraction 1 (F1) and fraction 2 (F2), F2 being the one with the addition of glycerol (9%) and OEP (1.5%).
506
I. Gale et al.
H.P.L.C analysis of T10 extender showed 113 mg l1 of epicatechin, 3.4 mg l1 of catechin and 188 mg l1 of galenic acid. All were determined by chromatography method, with an Agilent’s ZORBAX Column. First, samples were filtered through a 0.45 lm filter, then 20 ll were injected in gradient, mobile phase for a reversedphase with mobile phases containing aqueous phase (water: formic acid) and organic phase (methanol: formic acid) in a ZORBAX Eclipse XDB-C18 column (4.6 9 150 mm, 5 lm). Measurement was carried out with UV detection for galenic acid with 313 nm wavelength and with fluorescence detection for epicatechin and catechin, 278 nm excitation. Semen collection, dilution and cryopreservation Eighteen ejaculates from 17 boars of proven motility and morphology were used (>85% and
Effect of Camellia sinensis supplementation and increasing holding time on quality of cryopreserved boar semen lez & F. Martınez I. Gale, L. Gil, C. Malo, N. Gonza Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, Zaragoza, Spain
Keywords Antioxidants—boar semen—cryopreservation—holding time Correspondence C. Malo, Department of Animal Pathology, Obstetrics and Reproduction Area, Faculty of Veterinary Medicine, Universidad de Zaragoza, C/Miguel Servet, 177, 50013 Zaragoza, Spain. Tel.: +976761544; Fax: +976761612; E-mail: [email protected] Accepted: March 29, 2014 doi: 10.1111/and.12293
Summary Cryopreservation of boar semen is still considered suboptimal due to the low fertility when compared with fresh semen. This study was performed to evaluate the effects of green tea (Camellia sinensis) supplementation of the freezing extender at different concentration (0, 2.5%, 5%, 10%) and also to determine the influence of increasing holding time from 2 to 24 h at 15 °C. Seventeen ejaculates from nine boars were used to make pools of three of them and then cryopreserved. Sperm motility, viability, acrosome integrity, membrane functionality (HOST) and capacitation status were determined before freezing and at 0, 30, 60, 90 and 120 min after thawing. Lipid peroxidation was evaluated just after thawing. The main findings emerging from this study were the following: (i) no improvement in quality of thawed spermatozoa with addition of tea to the freezing extender, (ii) no improvement in quality of thawed spermatozoa with prolonged holding time, (iii) lower peroxidation rate in presence of tea 5% and (iv) a decrease in the number of uncapacited viable spermatozoa with any tea supplementation. We conclude that amplification of holding time in semen cryopreservation process does not vary results, facilitating freezing protocol. Tea supplementation reduces lipoxidation but did not improve quality parameters.
Introduction In recent years, the development of freezing techniques for semen cryopreservation has become a major source for the preservation of genetic material in domestic animals (Songsasen & Leibo, 1997; Cremades et al., 2005). However, the use of frozen-thawed boar semen is limited due to the low fertility outcomes compared with extended fresh semen (Buranaamnuay et al., 2009). The detrimental effect of cryopreservation on sperm includes coldshock, osmotic stress and oxidative stress from reactive oxygen substances (ROS) production which decreases lipid content of the membranes. Boar spermatozoa is very susceptible to ROS due to the high concentration of polyunsaturated fatty acids (PUFA) within the lipid membrane (Buhr et al., 1994). ROS have a dual effect on sperm function; at low concentrations ROS induces sperm capacitation, hyperactivation and fertilisation; mean while excessive ROS production reduces sperm motility (Guthrie & Welch, 2006), causes © 2014 Blackwell Verlag GmbH Andrologia 2015, 47, 505–512
damage DNA (Bennetts & Aitken, 2005) and decrease sperm-oocyte fusion (Watson, 1995). Some antioxidants, particularly vegetable antioxidants, have been employed in sperm cryopreservation. Zhao et al. (2009) reduced lipoxidation of spermatozoa during cryopreservation by incorporation of Rodhiola sacra aqueous extract (RSAE), a genus of Chinese herb. Interestingly, in recent studies from our laboratory, rosemary has been successfully used to freeze boar (Malo et al., 2010), ovine (Gil et al., 2010) and canine (Gonzalez et al., 2010) sperm, improving post-thaw motility and in vitro fertilisation parameters: Fennel also showed protective effect in boar semen cryopreservation (Malo et al., 2012). However, no studies have been published on boar semen cryopreservation using green tea. Green tea is a well-known alimentary antioxidant substance; this effect is largely attributed to polyphenols, chemicals with potent antioxidant properties. In fact, green tea polyphenols are more potent antioxidants than Vitamin C, Vitamin E, or rosemary extract (Wittayarat et al., 2012). 505
Cryopreservation boar semen
Main polyphenols found in green tea are theaflavins, thearubigins and catechins. Green tea contains four primary catechin (compounds which are a form of flavanol monomers): epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and apigallocatechin- 3- gallate (EGCG). EGCG is the most studied polyphenol component in green tea, being the most active (Hara, 1997), it has a powerful antioxidant effect and prevents oxidative damage in healthy cells (Westerterp-Platenga, 2010; Singh et al., 2011). Green tea also contains alkaloids including theobromine, theophylline and caffeine, that stimulate motility of dog and domestic cat frozen-thawed spermatozoa (Milani et al., 2010), without affecting longevity. Holding time is the period during which an ejaculate, either in a raw or diluted state, is held at 15 °C before further processing for cold state. In boars, longer holding times positively influence sperm quality parameters including sperm (Eriksson et al., 2001; Casas & Althouse, 2013). Therefore, the main aim of this study was to evaluate green tea as a cryomedium supplement for the cryopreservation of boar semen and determine the influence of longer holding times before cryopreservation on semen quality after thawing. Materials and methods Experimental extenders Unless otherwise indicated, all chemicals were from Sigma-Aldrich Co. (Alcobendas, Madrid, Spain). Orvus ES Paste (OEP) was from Minitub. Green tea sources were from La Flor del Pirineo, S.L., Manresa, Spain. The medium used for semen collection was Dulbeco0 s phosphate buffered saline (DPBS) medium composed of 136.89 mM NaCl, 2.68 mM KCl, 8.1 mM Na2HPO4 and 1.46 mM CaCl22H2O. Lactose–egg yolk extender containing 20% (v:v) egg yolk, 80% of 11% L-lactose solution (290 mM) and 100 lg ml1 of kanamycin sulphate was used as base extender. Fennel extenders were prepared according to the procedure for antioxidant extraction described by Malo et al. (2010). We extracted the green tea essence by soaking dry commercial herbs. After heating 100 ml of water to 100 °C, leaves at different concentrations [2.5 g (T2.5); 5 g (T5); 10 g (T10)] were added to the water and maintained for 10 min at this temperature. Once the water had cooled to 25 °C, the solution was filtered to remove the leaves. The resulting solution had a pH of 7 0.4. Sperm dilution was performed in a two-step procedure, fraction 1 (F1) and fraction 2 (F2), F2 being the one with the addition of glycerol (9%) and OEP (1.5%).
506
I. Gale et al.
H.P.L.C analysis of T10 extender showed 113 mg l1 of epicatechin, 3.4 mg l1 of catechin and 188 mg l1 of galenic acid. All were determined by chromatography method, with an Agilent’s ZORBAX Column. First, samples were filtered through a 0.45 lm filter, then 20 ll were injected in gradient, mobile phase for a reversedphase with mobile phases containing aqueous phase (water: formic acid) and organic phase (methanol: formic acid) in a ZORBAX Eclipse XDB-C18 column (4.6 9 150 mm, 5 lm). Measurement was carried out with UV detection for galenic acid with 313 nm wavelength and with fluorescence detection for epicatechin and catechin, 278 nm excitation. Semen collection, dilution and cryopreservation Eighteen ejaculates from 17 boars of proven motility and morphology were used (>85% and
