SHORT COMMUNICATION
Effect of egg yolk plasma on dog sperm cryopreservation C. D. Corcini1, K. L. Goularte3, D. C. Bongalhardo2, T. Lucia Jr1, R. D. Jardim3 & A. S. Varela Junior3 1 ReproPel, Faculdade de Veterin aria, Universidade Federal de Pelotas, Pelotas, Brazil; 2 Instituto de Biologia, Universidade Federal de Pelotas, Pelotas, Brazil; 3 Instituto de Ci^ encias Biologicas – Reproducao Animal Comparada-Universidade Federal de Rio Grande, Rio Grande, Brasil
Keywords Acrosome integrity—membrane integrity—motility Correspondence Antonio Sergio Varela Junior, Universidade Federal do Rio Grande - Reproducß~ ao Animal Comparada, Campus Carreros, predio 06, Rio Grande, RS 96201900, Brazil. Tel.: 55 53 32935186; E-mail: [email protected] Accepted: January 11, 2015 doi: 10.1111/and.12411
Summary This study evaluated the quality of frozen-thawed dog spermatozoon after the inclusion of egg yolk plasma (EYP) instead of whole egg yolk (EY) in the cryopreservation extender and after distinct periods of exposure to EYP. Seven mongrel dogs were used as sperm donors, and EYP was obtained by centrifugation. In Experiment 1, post-thawing sperm motility (MOT) and integrity of membrane (INT) and acrosome (ACR) were superior for spermatozoon extended with 20% EYP T2 than with 20% EY (P < 0.05), although normal sperm morphology (MOR) did not differ (P > 0.05). In Experiment 2, after ejaculates extended with 20% EYP were cooled at 5°C for 2, 6 and 10 h before freezing, MOT, INT and ACR were similar among periods (P > 0.05). Thus, dog spermatozoon extended with 20% EYP can be kept cooled for up to 10 h prior to freezing, achieving post-thawing quality greater than that obtained with the inclusion of EY in freezing extenders.
Introduction
Material and methods
The cryoprotecting effect of egg yolk (EY) against cold shock that occurs during freezing and thawing of spermatozoa is attributed to its content of low-density lipoprotein (LDL), which adheres to the sperm membrane, causing influx of phospholipids and cholesterol into the membrane. Also, LDL forms complexes with seminal plasma proteins that get in contact with spermatozoon during ejaculation, avoiding efflux of phospholipids and cholesterol from the sperm membrane (Therien et al., 1999; Bergeron et al., 2004). However, maximum LDL influx into the sperm membrane requires cooling at 5°C for nearly 8 h, which is not considered ideal in most sperm freezing protocols (Bergeron et al., 2004). As EY contains substances such as granules, minerals and high-density lipoproteins that make sperm respiration difficult and may lead to decreased sperm motility (Pace & Graham, 1974), their removal would benefit sperm cryopreservation. The inclusion of purified LDL in extenders is beneficial for dog sperm cryopreservation (Varela Junior et al., 2009), but it is unfeasible because of the long purification process. The objectives of this study were to evaluate the effect of egg yolk plasma (EYP) on dog sperm quality after thawing and to establish the maximum feasible cooling period for dog spermatozoon before freezing.
A total of 112 ejaculates were collected from seven mongrel dogs (16 per dog). The dogs were 3–4 years old. All sperm quality evaluations were performed by the same trained technician. Ejaculates with less than 80% motility and 80% normal morphology were discarded. Samples were extended in Tris-glucose (Iguer-Ouada & Verstegen, 2001) with 5% glycerol, composing treatments including 20% EY (T1) and 20% EYP (T2). The EYP was separated using 20 ml of egg yolk diluted in 80 ml of Trisglucose (McBee & Cotterill, 1979). Three successive centrifugations were performed, at 10 0009 g for 45 min. Each time, mineral granules, cell debris and yolk by-products contained in the pellet were discarded and the final supernatant contained EYP. Ejaculates were diluted 1/1 (v/ v) to a final concentration of 2 9 108 spermatozoa per ml. In Experiment 1, spermatozoon was cooled up to 20°C for 1 h and then kept at 5°C for 2 h. In Experiment 2, three cooling periods were compared: 2 h; 6 h; and 10 h. After cooling, spermatozoon was packed in 0.25 ml straws and placed at 5 cm above liquid nitrogen for 10 min before immersion. Thawing was performed in water bath at 70°C for 8 s (Varela Junior et al., 2009). Thawed samples were maintained in water bath at 39°C for 10 min. Sperm morphology was evaluated using a phase contrast microscope (Olympus BX, Olympus
© 2015 Blackwell Verlag GmbH Andrologia 2015, xx, 1–2
1
Egg yolk plasma on dog sperm cryopreservation
C. D. Corcini et al.
Table 1 Means (standard errors) for parameters of post-thawing quality of dog sperm conditioned in extenders including either 20% whole egg yolk (T1) or 20% egg yolk plasma (T2) and preserved with distinct cooling curves
Treatment
Motility (%)
T1 T2 * C1 * C2 * C3
45.2 61.4 55.1 60.8 57.4
Membrane integrity (%) 2.6b 2.6a 2.9 2.8 3.1
45.9 60.9 54.3 56.1 57.8
1.6b 1.1a 2.6 2.3 1.8
Acrosome integrity (%) 48.7 59.5 62.5 65.2 67.6
2.5b 3.1a 3.5 3.2 2.4
Normal morphology (%) 71.1 70.5 72.3 71.5 70.6
0.9a 1.9a 1.1 1.2 0.8
et al., 1999; Bergeron et al., 2004). However, LDL extraction requires approximately 48 h, whereas processing EYP reduces the period of extender preparation to nearly 3 h, using no ammonium sulphate, which can lead to losses in sperm quality when improperly removed. Experiment 2 showed no effects of the cooling periods on any variable analysed. Although the phospholipids’ influx plateau is reached after 8 h at 5°C (Bergeron et al., 2004), that period apparently is not necessary for maximum sperm protection. Thus, as any of the tested periods can be used, sperm collection in several dogs would be feasible, optimising time and labour.
a,b
Different letters indicated significant differences in the column (P < 0.001). * C1 cooled for 2 h at 5°C; C2 cooled for 6 h at 5°C; and C3 cooled for 10 h at 5°C.
America, Inc., Sao Paulo, SP, Brazil) at 10009. Post-thawing membrane and acrosome integrities were evaluated as described by Bencharif et al. (2010). Sperm quality parameters were compared among treatments and periods through repeated measures analysis of variance, with comparisons of means by Tukey test. All analyses were conducted with Statistixâ (2003). Results In Experiment 1, motility was 92.9 1.4% for fresh spermatozoon. After thawing, T2 presented greater motility, and membrane and acrosome integrities than T1 (P < 0.05), with no effect (P > 0.05) on normal sperm morphology (Table 1). In Experiment 2, there were no differences (P > 0.05) across cooling periods (Table 1). Discussion In Experiment 1, post-thawing sperm motility with EYP (42.5%) was more than 16% greater than with EY. This may have occurred because some substances that impair cellular respiration and sperm motility (Pace & Graham, 1974) were centrifuged out of EY. Sperm motility in T2 was greater than that reported by Bencharif et al. (2010)using the same extender, but with a different protocol. The improved acrosome and membrane integrities observed with EYP were likely due to LDL, which forms complexes with seminal plasma proteins, decreasing phospholipid and cholesterol efflux from the plasma membrane and enhancing its resistance to cold shock (Therien
2
Conclusion Egg yolk plasma is more efficient than whole egg yolk for dog sperm cryopreservation. Spermatozoon extended in egg yolk plasma can stay cooled for up to 10 h before freezing, without losses in sperm quality.
References Bencharif D, Amirat L, Pascal O, Anton M, Schmitt E, Desherces S, Delhomme G, Langlois M-L, Barriere P, Larrat M, Tainturier D (2010) The advantages of combining lowdensity lipoproteins with glutamine for cryopreservation of canine semen. Reprod Domest Anim 45:189–200. Bergeron A, Cr^ete MH, Brindle Y, Manjunath P (2004) Lowdensity lipoprotein fraction from hen‘s egg yolk decreases the binding of the major protein of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biol Reprod 70:708–717. Iguer-Ouada M, Verstegen JP (2001) Long-term preservation of chilled canine semen: Effect of commercial and laboratory prepared extenders. Theriogenology 55:671–684. McBee L, Cotterill O (1979) Ion exchange chromatography and electrophoresis of egg yolk. J Food Sci 44:656–660. Pace MM, Graham EF (1974) Components in egg yolk which protect bovine spermatozoa during freezing. J Anim Sci 39:1144–1149. Statistixâ (2003) Statistixâ 8 Analytical Software. User’s Manual. Tallahassee. 396 p. Therien I, Moreau R, Manjunath P (1999) Bovine seminal plasma phospholipid-binding Proteins stimulate phospholipid efflux from epididymal sperm. Biol Reprod 61:590–598. Varela Junior AS, Corcini CD, Ulguim RR, Alvarenga MVF, Bianchi I, Corr^ea MN, Lucia T Jr, Deschamps JC (2009) Effect of low density lipoprotein on the quality of cryopreserved dog semen. Anim Reprod Sci 115:323–327.
© 2015 Blackwell Verlag GmbH Andrologia 2015, xx, 1–2
Effect of egg yolk plasma on dog sperm cryopreservation C. D. Corcini1, K. L. Goularte3, D. C. Bongalhardo2, T. Lucia Jr1, R. D. Jardim3 & A. S. Varela Junior3 1 ReproPel, Faculdade de Veterin aria, Universidade Federal de Pelotas, Pelotas, Brazil; 2 Instituto de Biologia, Universidade Federal de Pelotas, Pelotas, Brazil; 3 Instituto de Ci^ encias Biologicas – Reproducao Animal Comparada-Universidade Federal de Rio Grande, Rio Grande, Brasil
Keywords Acrosome integrity—membrane integrity—motility Correspondence Antonio Sergio Varela Junior, Universidade Federal do Rio Grande - Reproducß~ ao Animal Comparada, Campus Carreros, predio 06, Rio Grande, RS 96201900, Brazil. Tel.: 55 53 32935186; E-mail: [email protected] Accepted: January 11, 2015 doi: 10.1111/and.12411
Summary This study evaluated the quality of frozen-thawed dog spermatozoon after the inclusion of egg yolk plasma (EYP) instead of whole egg yolk (EY) in the cryopreservation extender and after distinct periods of exposure to EYP. Seven mongrel dogs were used as sperm donors, and EYP was obtained by centrifugation. In Experiment 1, post-thawing sperm motility (MOT) and integrity of membrane (INT) and acrosome (ACR) were superior for spermatozoon extended with 20% EYP T2 than with 20% EY (P < 0.05), although normal sperm morphology (MOR) did not differ (P > 0.05). In Experiment 2, after ejaculates extended with 20% EYP were cooled at 5°C for 2, 6 and 10 h before freezing, MOT, INT and ACR were similar among periods (P > 0.05). Thus, dog spermatozoon extended with 20% EYP can be kept cooled for up to 10 h prior to freezing, achieving post-thawing quality greater than that obtained with the inclusion of EY in freezing extenders.
Introduction
Material and methods
The cryoprotecting effect of egg yolk (EY) against cold shock that occurs during freezing and thawing of spermatozoa is attributed to its content of low-density lipoprotein (LDL), which adheres to the sperm membrane, causing influx of phospholipids and cholesterol into the membrane. Also, LDL forms complexes with seminal plasma proteins that get in contact with spermatozoon during ejaculation, avoiding efflux of phospholipids and cholesterol from the sperm membrane (Therien et al., 1999; Bergeron et al., 2004). However, maximum LDL influx into the sperm membrane requires cooling at 5°C for nearly 8 h, which is not considered ideal in most sperm freezing protocols (Bergeron et al., 2004). As EY contains substances such as granules, minerals and high-density lipoproteins that make sperm respiration difficult and may lead to decreased sperm motility (Pace & Graham, 1974), their removal would benefit sperm cryopreservation. The inclusion of purified LDL in extenders is beneficial for dog sperm cryopreservation (Varela Junior et al., 2009), but it is unfeasible because of the long purification process. The objectives of this study were to evaluate the effect of egg yolk plasma (EYP) on dog sperm quality after thawing and to establish the maximum feasible cooling period for dog spermatozoon before freezing.
A total of 112 ejaculates were collected from seven mongrel dogs (16 per dog). The dogs were 3–4 years old. All sperm quality evaluations were performed by the same trained technician. Ejaculates with less than 80% motility and 80% normal morphology were discarded. Samples were extended in Tris-glucose (Iguer-Ouada & Verstegen, 2001) with 5% glycerol, composing treatments including 20% EY (T1) and 20% EYP (T2). The EYP was separated using 20 ml of egg yolk diluted in 80 ml of Trisglucose (McBee & Cotterill, 1979). Three successive centrifugations were performed, at 10 0009 g for 45 min. Each time, mineral granules, cell debris and yolk by-products contained in the pellet were discarded and the final supernatant contained EYP. Ejaculates were diluted 1/1 (v/ v) to a final concentration of 2 9 108 spermatozoa per ml. In Experiment 1, spermatozoon was cooled up to 20°C for 1 h and then kept at 5°C for 2 h. In Experiment 2, three cooling periods were compared: 2 h; 6 h; and 10 h. After cooling, spermatozoon was packed in 0.25 ml straws and placed at 5 cm above liquid nitrogen for 10 min before immersion. Thawing was performed in water bath at 70°C for 8 s (Varela Junior et al., 2009). Thawed samples were maintained in water bath at 39°C for 10 min. Sperm morphology was evaluated using a phase contrast microscope (Olympus BX, Olympus
© 2015 Blackwell Verlag GmbH Andrologia 2015, xx, 1–2
1
Egg yolk plasma on dog sperm cryopreservation
C. D. Corcini et al.
Table 1 Means (standard errors) for parameters of post-thawing quality of dog sperm conditioned in extenders including either 20% whole egg yolk (T1) or 20% egg yolk plasma (T2) and preserved with distinct cooling curves
Treatment
Motility (%)
T1 T2 * C1 * C2 * C3
45.2 61.4 55.1 60.8 57.4
Membrane integrity (%) 2.6b 2.6a 2.9 2.8 3.1
45.9 60.9 54.3 56.1 57.8
1.6b 1.1a 2.6 2.3 1.8
Acrosome integrity (%) 48.7 59.5 62.5 65.2 67.6
2.5b 3.1a 3.5 3.2 2.4
Normal morphology (%) 71.1 70.5 72.3 71.5 70.6
0.9a 1.9a 1.1 1.2 0.8
et al., 1999; Bergeron et al., 2004). However, LDL extraction requires approximately 48 h, whereas processing EYP reduces the period of extender preparation to nearly 3 h, using no ammonium sulphate, which can lead to losses in sperm quality when improperly removed. Experiment 2 showed no effects of the cooling periods on any variable analysed. Although the phospholipids’ influx plateau is reached after 8 h at 5°C (Bergeron et al., 2004), that period apparently is not necessary for maximum sperm protection. Thus, as any of the tested periods can be used, sperm collection in several dogs would be feasible, optimising time and labour.
a,b
Different letters indicated significant differences in the column (P < 0.001). * C1 cooled for 2 h at 5°C; C2 cooled for 6 h at 5°C; and C3 cooled for 10 h at 5°C.
America, Inc., Sao Paulo, SP, Brazil) at 10009. Post-thawing membrane and acrosome integrities were evaluated as described by Bencharif et al. (2010). Sperm quality parameters were compared among treatments and periods through repeated measures analysis of variance, with comparisons of means by Tukey test. All analyses were conducted with Statistixâ (2003). Results In Experiment 1, motility was 92.9 1.4% for fresh spermatozoon. After thawing, T2 presented greater motility, and membrane and acrosome integrities than T1 (P < 0.05), with no effect (P > 0.05) on normal sperm morphology (Table 1). In Experiment 2, there were no differences (P > 0.05) across cooling periods (Table 1). Discussion In Experiment 1, post-thawing sperm motility with EYP (42.5%) was more than 16% greater than with EY. This may have occurred because some substances that impair cellular respiration and sperm motility (Pace & Graham, 1974) were centrifuged out of EY. Sperm motility in T2 was greater than that reported by Bencharif et al. (2010)using the same extender, but with a different protocol. The improved acrosome and membrane integrities observed with EYP were likely due to LDL, which forms complexes with seminal plasma proteins, decreasing phospholipid and cholesterol efflux from the plasma membrane and enhancing its resistance to cold shock (Therien
2
Conclusion Egg yolk plasma is more efficient than whole egg yolk for dog sperm cryopreservation. Spermatozoon extended in egg yolk plasma can stay cooled for up to 10 h before freezing, without losses in sperm quality.
References Bencharif D, Amirat L, Pascal O, Anton M, Schmitt E, Desherces S, Delhomme G, Langlois M-L, Barriere P, Larrat M, Tainturier D (2010) The advantages of combining lowdensity lipoproteins with glutamine for cryopreservation of canine semen. Reprod Domest Anim 45:189–200. Bergeron A, Cr^ete MH, Brindle Y, Manjunath P (2004) Lowdensity lipoprotein fraction from hen‘s egg yolk decreases the binding of the major protein of bovine seminal plasma to sperm and prevents lipid efflux from the sperm membrane. Biol Reprod 70:708–717. Iguer-Ouada M, Verstegen JP (2001) Long-term preservation of chilled canine semen: Effect of commercial and laboratory prepared extenders. Theriogenology 55:671–684. McBee L, Cotterill O (1979) Ion exchange chromatography and electrophoresis of egg yolk. J Food Sci 44:656–660. Pace MM, Graham EF (1974) Components in egg yolk which protect bovine spermatozoa during freezing. J Anim Sci 39:1144–1149. Statistixâ (2003) Statistixâ 8 Analytical Software. User’s Manual. Tallahassee. 396 p. Therien I, Moreau R, Manjunath P (1999) Bovine seminal plasma phospholipid-binding Proteins stimulate phospholipid efflux from epididymal sperm. Biol Reprod 61:590–598. Varela Junior AS, Corcini CD, Ulguim RR, Alvarenga MVF, Bianchi I, Corr^ea MN, Lucia T Jr, Deschamps JC (2009) Effect of low density lipoprotein on the quality of cryopreserved dog semen. Anim Reprod Sci 115:323–327.
© 2015 Blackwell Verlag GmbH Andrologia 2015, xx, 1–2
