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In vitro fertilizing capacity and chromatin condensation of deep frozen boar semen packaged in 0.5 and 5 ml straws
Theriogenology 57 (2002) 2119±2128
In vitro fertilizing capacity and chromatin condensation of deep frozen boar semen packaged in 0.5 and 5 ml straws Alejandro CoÂrdovaa, Jose FeÂlix PeÂrez-GutieÂrrezb,*, BeleÂn LleoÂb, Carlos GarcõÂa-Artigab, Alberto Alvarezc, Volodymyr Drobchakb, Santiago MartõÂn-Rillob,1 a
Departamento de ProduccioÂn AgrõÂcola y Animal, Universidad AutoÂnoma Metropolitana, Xochimilco, Mexico b Facultad de Veterinaria, Dpto. PatologõÂa Animal IIÐReproduccioÂn, Universidad Complutense U.C.M., Avda. Puerta de Hierro s/n, 28040 Madrid, Spain c Centro de CitometrõÂa de Flujo y MicroscopõÂa Confocal, Universidad Complutense, Madrid, Spain Received 12 June 2001; accepted 19 November 2001
Abstract The effect of the straw volume employed for semen freezing was studied in 14 ejaculates from seven boars, by evaluating the viability, IVF capacity and chromatin state of spermatozoa. Frozen± thawed semen from 0.5 and 5 ml straws was compared to fresh semen. The chromatin condensation degree was determined by ¯ow cytometry, using propidium iodide as ¯uorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. The results obtained for IVF, motility and normal apical ridge (NAR) were: 91.64, 78.14 and 81.47% sperm penetration, 80.78, 68.38 and 70.83% monospermy, 10.86, 9.76 and 10.64% polyspermy, 87.14, 50.71 and 47.86% motility, 79.14, 56.14 and 53.36% NAR, for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively. Frozen±thawed spermatozoa showed signi®cantly increased (P < 0:05) chromatin compactness compared to fresh spermatozoa (55.42, 48.41 and 47.08 ¯uorescence units (MIFU), for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively). Chromatin was signi®cantly more unstable (P < 0:05) in spermatozoa frozen in 0.5 ml straws (174.7 MIFU) compared to those frozen in 5 ml straws (155.53 MIFU) or to those in fresh semen (149.74 MIFU). # 2002 Elsevier Science Inc. All rights reserved. Keywords: Sperm chromatin; In vitro fertilization; Cryopreservation; Porcine
* Corresponding author. Tel.: 34-91-3943-798; fax: 34-91-3943-808. E-mail address: [email protected] (J.F. PeÂrez-GutieÂrrez). 1 Deceased.
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 7 0 1 - X
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1. Introduction The use of frozen boar semen for improving pig population genetics is limited, and the swine industry is focusing on improving cryopreservation techniques in order to increase production. The lower conception rate after arti®cial insemination with cryostored spermatozoa, despite the apparent good post thaw motility, and even when using an insemination dose with a higher number of spermatozoa than with fresh semen [1,2], raises questions about cellular damage to spermatozoa during the freezing±thawing process. Among other factors, the shape and volume of the container can in¯uence the quality of frozen semen. Boar semen is usually frozen in 5 ml straws. Due to low heat transfer, cooling is fast in the peripheral regions and slow in the center of the straw, which can induce acrosome and membrane damage, injuries that can be reduced by lowering the straw volume [3]. Some authors have studied the effect of the straw volume on semen quality by evaluating in vitro motility rates and normal apical ridge (NAR) percentages as well as in vivo fertility rates [4±6]. However, other parameters related to fertility can be measured in vitro, including monospermy and polyspermy rates [7], as well as the chromatin state. The formation of a compact structure in sperm chromatin is necessary for DNA transient inactivation and protection [8]. In mature spermatozoa, the normal nucleosomal packaging of DNA found in somatic cells is transformed into a highly condensed form of chromatin, which consists mostly of nucleoprotamines [9]. Protamines contain a large number of cysteine residues, which are initially present as thiol groups [10,11]. These thiol groups are partially oxidized to form disulphide bonds during sperm maturation [12]. After ejaculation, sperm chromatin is stabilised by semen plasma, probably due to the presence of zinc, that binds free thiol groups [13,14]. Incubation of spermatozoa in semen plasma or under capacitation conditions induces a higher stability of chromatin, which has been attributed to a gradual depletion of zinc that leads to the exposure of free thiol groups, and allows the formation of additional disulphide bonds [13,15±17]. Previous studies in human and porcine species have shown that sperm chromatin can undergo important changes after the freezing±thawing procedure, resulting in a greater compactness of spermatozoa nuclei [18±21]. On the other hand, hyperstability of the sperm chromatin could delay paternal nuclear formation during fertilization [17,22], which can induce early embryonic death or lower embryonic development, frequently observed after arti®cial insemination with frozen boar semen [23]. Thus, chromatin condensation and chromatin stability of thawed spermatozoa may be critical factors to consider in assisted reproduction. The aim of this study was to evaluate the effect of straw volume on motility, NAR, chromatin condensation, chromatin stability and IVF capacity on frozen boar semen. 2. Materials and methods Unless otherwise stated, reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
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2.1. Semen collection Semen was collected on a weekly basis, from seven Duroc boars at the Agronomy School of the Universidad Complutense of Madrid, Spain. The boars were 2 years old and their fertility had been previously proven at this experimental farm. Ejaculates were obtained by the gloved hand method [24]. Only the sperm rich fraction was collected. Gel particles were removed by ®ltration through gauze, and volume, sperm concentration, motility and NAR were assessed immediately after collection. All the samples were evaluated by the same observer. Ejaculates showing a motility higher than 80%, a motility quality score higher than 3 and NAR higher than 70% were used. A total of 14 ejaculates (2 from each of the 7 boars) were used. Each ejaculate was divided into three fractions, one to be used as fresh semen and the other two to be frozen in 0.5 and 5 ml straws. 2.2. Assessment of concentration Cell concentration was assessed on fresh semen, in formol saline ®xed samples (1:100 dilution) using a BuÈker chamber. 2.3. Assessment of sperm motility Sperm motility was evaluated using a phase contrast microscope by placing a drop of the sample, without further dilution, on a microscope slide at 42 8C and covering with a cover slip. Samples were evaluated at 200 magni®cation, by counting progressively motile spermatozoa [25]. Motility was expressed as the percentage of motile spermatozoa per 100 sperm cells. Motility quality was assessed by the rate of forward progression, on a scale from 0 to 5. 2.4. Assessment of the NAR The evaluation of acrosomal integrity was done by examining formol saline ®xed samples with a phase contrast microscope, at 1000 magni®cation. A minimum of 100 acrosomes were examinated per sample. Damage to the acrosomal cap was classi®ed by the scoring system reported by Pursel et al. [26]. 2.5. Semen freezing The semen was processed and frozen by the straw freezing method, originally described by Westendorf et al. [27] and later modi®ed by MartõÂn [28]. Shortly after collection, the sperm rich fraction was diluted (1:4) with a commercial extender preparation, MR-A1 diluent (Kubus, S.A., Madrid, Spain), at 23 8C. After 1 h equilibration at 20 8C, a dose containing 6 109 spermatozoa (double dose), was prepared in a 50 ml centrifuge ¯ask. The sperm suspension was kept at 15 8C for 3 h and then centrifuged at 800 g for 10 min. The supernatant was then removed and the concentrated semen was diluted to a volume of 5 ml with the cooling extender (11% lactose and 20% egg yolk in distilled water) at 15 8C.
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The diluted semen was gently mixed and cooled to 5 8C for 1.5 h. The semen was further diluted (1:1) with freezing extender (11% lactose, 20% egg yolk, 4% glycerol and 0.5% lauryl sodium sulfate in distilled water) to 10 ml. Then 5 and 0.5 ml straws were ®lled and sealed manually, using metallic sealing balls and polyvinyl alcohol. Straws were wiped dry and the air bubble was brought to the center of the straw. The 5 and 0.5 ml straws, containing 3000 106 and 300 106 spermatozoa, respectively, were then placed in contact with nitrogen vapor for 20 min, about 3 cm above the nitrogen liquid level, and then plunged in the liquid nitrogen tank and stored until used. 2.6. Thawing Thawing of straws was done after 6 months of storage in liquid nitrogen. The 5 and 0.5 ml straws were thawed in a water bath at 42 8C for 12 and 45 s, respectively. Motility and NAR were immediately assessed. 2.7. In vitro capacitation Fresh and thawed semen samples were centrifuged at 500 g for 10 min. The pellets were diluted with Tyrode's medium, supplemented with 0.25 mM sodium pyruvate, 6 mg/ml BSA, 21.6 mM sodium lactate and 10 mM HEPES (TALP±HEPES) [29], at 37 8C, to a ®nal volume of 1.5 ml. Spermatozoa were allowed to swim up in this medium during a 30 min incubation at 37 8C. In order to select the motile sperm fraction the upper 0.5 ml of medium was collected [30]. This fraction was further diluted with TALP±HEPES to a ®nal concentration of 75 106 spermatozoa/ml. Aliquots of 0.5 ml were incubated in wells of a 4 well-plate (Nunc, Roskilde, Denmark) for 1±1.5 h, at 37 8C, in a humidi®ed 5% CO2 atmosphere. 2.8. In vitro fertilization Swine ovaries were obtained from a local slaughterhouse and transported, in less than 1 h, to the laboratory, in 0.157 M NaCl at 37 8C. Oocytes, with cumulus cells, were aspirated from 3 to 6 mm diameter follicles with a 21 gauge needle attached to a 5 ml syringe. Thirty to 35 oocytes were incubated in a 100 ml drop of maturation medium: TC 199 medium supplemented with 10% inactivated fetal calf serum, 1 mg/ml glucose, 2 IU/ ml pergonal (LH, FSH), 0.25 mM sodium pyruvate, 10 mg/ml gentamycin and 1 mg/ml b-estradiol. The microdrops were placed in 4 well-plates (Nunc) and covered with paraf®n oil. Oocytes were incubated for 48 h, at 37 8C, in a humidi®ed atmosphere with 5% CO2, and then coincubated with 5 104 spermatozoa per well in a TALP fertilization medium microdrop [31] for 12±16 h, under the same conditions as above. 2.9. IVF evaluation The oocytes were ®xed in methanol:acetic acid (3:1) for 24 h, stained with 2% acetic orcein and examined at 100 magni®cation under a bright ®eld microscope to assess fertilization. Those oocytes showing two pronuclei were considered fertilized.
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2.10. Analysis of sperm chromatin condensation and stability The chromatin condensation degree was determined by ¯ow cytometry, employing as ¯uorochrome propidium iodide (PI), an intercalating compound whose accessibility to DNA was measured by ¯uorescence. Two determinations were done. First, chromatin condensation was determined. Second, the susceptibility of chromatin to decondensation, or chromatin stability, was determined by inducing ®rst decondensation with EDTA plus SDS, and then determining the chromatin condensation state. 2.11. Nuclear sperm chromatin treatment Sperm chromatin was stained with propidium iodide, according to Molina et al. [32], using the Cycle Test PLUS DNA reagent kit from Becton Dickinson (San Jose, CA). For determining chromatin condensation, samples were prepared by resuspending fresh and frozen semen for 5 min in capacitation medium to a concentration of 1± 2 106 spermatozoa per sample. After centrifugation at 500 g for 10 min, supernatant was discarded. The sperm membrane was permeabilized, using trypsin (0.5%) in a spermine-tetrahydrochloride detergent buffer, for 10 min at room temperature. To stop trypsin action and to remove double stranded RNA, trypsin inhibitor and ribonuclease A (12 mg/ml) were added and incubated for a further 10 min. Subsequently, ice-cold propidium iodide (50 mg/ml) and spermine-tetrahydrochloride in citrate stabilizing buffer, were added. Samples were incubated for 1 h in the dark before ¯ow cytometer analysis. For determining chromatin stability, decondensation of chromatin was induced by resuspending fresh and frozen±thawed semen, for 5 min, in a capacitation medium solution, containing 6 mM EDTA and 1% SDS in borate buffer (0.05 M, pH 9.0) [22], to a concentration of 1±2 106 spermatozoa per sample. Samples were then treated as for chromatin condensation analysis, as described above. 2.12. Flow cytometric measurements Flow cytometer analysis was performed by using a FACscan ¯ow cytometer (Becton Dickinson, S.A., San Jose, CA). Propidium iodide was excited by an argon-ion laser emitting at 488 nm and PI ¯uorescence was measured at 605 nm with using a 600/40 bandpass ®lter. Data were analyzed using Cell Quest software from Becton Dickinson. Measurements were expressed as mean intensity ¯uorescence units, arbitrary units (MIFU) related to the PI uptake by DNA. 2.13. Statistical analysis Results were analyzed by ANOVA and multiple comparison test using SAS software (SAS Institute Inc., Cary, NC).
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Table 1 IVF parameters, motility, NAR, chromatin condensation and stability for fresh and frozen semen in 0.5 and 5 ml straws. Fresh semen
Frozen±thawed semen, packed in 0.5 ml straws
Penetration (%) Monospermy (%) Polyspermy (%) Motility (%) NAR (%) Chromatin condensation (MIFU) Chromatin stability (MIFU)
91.64 80.78 10.86 87.14 79.14 55.42 149.74
a
0.99 0.78a 0.40a 1.25a 1.15a 0.92a 8.69a
78.14 68.38 9.76 50.71 56.14 48.41 174.70
5 ml straws b
1.00 0.90b 0.38a 1.64b 2.12b 1.25b 9.40b
81.47 70.83 10.64 47.86 53.36 47.08 155.53
0.93c 0.67c 0.44a 1.55b 1.32b 1.22c 7.21a
Values are expressed as mean S:E:M:, n 14. Values in each row with the same superscript are not signi®cantly different (P < 0:05). Units: percentages (%) related to the total number of spermatozoa (*) or the total number of oocytes (**); MIFU: mean intensity ¯uorescence units, related to propidium iodide uptake by DNA. Chromatin condensation refers to sperm resuspended in TALP±HEPES medium and chromatin stability refers to the chromatin condensation of sperm after treatment with EDTA plus SDS.
3. Results 3.1. Effect of straw volume on the viability of frozen semen After freezing and thawing, viable spermatozoa were assessed by motility, NAR and IVF parameters, i.e. monospermy, polyspermy and penetration (Table 1). All of these parameters, except polyspermy, decreased when semen was frozen in either 0.5 or 5 ml straws. However, no signi®cant differences were found between 0.5 and 5 ml straws, regarding both motility and NAR. No signi®cant differences between the two straw volumes for polyspermy were found, and both penetration and monospermy were slightly higher for frozen semen in 5 ml straws. 3.2. Effect of straw volume on sperm chromatin condensation and stability The results obtained for chromatin condensation and stability for fresh and frozen semen in 0.5 and 5 ml straws are presented in Table 1. Frozen±thawed spermatozoa showed a signi®cant decrease in PI uptake, indicating increased chromatin compactness, compared to fresh spermatozoa. Spermatozoa frozen in 5 ml straws showed a slightly lower PI uptake (i.e. higher chromatin condensation) than those frozen in 0.5 ml straws. Chromatin stability refers to the chromatin condensation of sperm after treatment with EDTA plus SDS. A higher PI uptake is related to easier decondensation of the chromatin and, therefore, to its higher instability. Frozen samples in 0.5 ml showed signi®cant less stability compared to fresh or to frozen samples in 5 ml straws.
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4. Discussion 4.1. Effect of straw volume on the viability of frozen semen As expected, motility, NAR, monospermy and penetration decreased upon freezing of semen regardless the straw volume used. It is generally accepted that increasing the volume of straws is detrimental for both motility and NAR due to a slower heat transfer [3,4,33]. However, no differences between 0.5 and 5 ml straws for motility, NAR and polyspermy were found and, even though penetration and monospermy were higher for frozen semen in 5 ml straws, the differences were very small. These results corroborate previous studies [7] that have shown, using the same procedure, no detrimental effects on viability and IVF capacity with the use of 5 ml straws compared to 0.5 ml straws. 4.2. Effect of straw volume on sperm chromatin condensation and stability After freezing and thawing, boar spermatozoa showed a more compact chromatin than in fresh samples (Table 1). This increase in chromatin condensation after freezing and thawing is in agreement with previous studies [19±21]. Furthermore, spermatozoa frozen in 5 ml straws showed a slightly higher condensation than those frozen in 0.5 ml straws. Hypercondensation in frozen samples, however, was not related to a higher stability of chromatin. In fact, after inducing decondensation with EDTA plus SDS, chromatin in frozen samples did show equal or higher instability. In addition, while spermatozoa frozen in 5 ml straws did not show different chromatin stability compared to fresh semen, there was a signi®cant difference in frozen spermatozoa packaged in 0.5 ml straws, which displayed less stability. This difference was maintained when the results for the two straw volumes were compared for each of the samples employed (results not shown). Therefore, these results could not be due to natural differences among samples, but rather to an effect of straw volume or the procedure, which undergoes minor changes depending on the straw volume used. The effect of straw volume could be due to the slower heat transfer in 5 ml straws, while the effects of the procedure could be due to thawing at 42 8C for different times, 12 and 45 s, for 0.5 and 5 ml straws, respectively (see Section 2). Various mechanisms, such as binding between DNA and nucleoprotamines or the relative number of disul®de bonds may be involved in the condensation state of chromatin [34±37]. However, the mechanisms that induce changes in chromatin stability are still unknown. Jager et al. [38] have suggested that decondensation is more a physico-chemical rather than an enzymatic process and it depends on temperature, time and pH. However, there is growing evidence suggesting that reactive oxygen species (ROS) increase during freezing and thawing of spermatozoa and can play an important role in fertility [39±41]. Oxidative stress can be induced by freezing and it has been suggested that prolonged exposure of spermatozoa to even mild oxidation conditions could lead to overoxidation of thiol groups and hypercondensation of DNA [42]. On the other hand, ROS can also induce breaks in the DNA [43]. The presence of DNA strand breaks has been found to be related to an increased susceptibility of DNA to denaturation [44,45].
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Therefore, the increased compactness observed in frozen samples could be due to an increased formation of disul®de bonds or to stronger noncovalent interactions. On the other hand, the increased instability of chromatin found in spermatozoa frozen in 0.5 ml straws could be the result of more profound molecular changes, possibly be related to protamine conformational changes, induced by enhanced covalent or noncovalent interactions, or to an increased DNA strand breakage. The lower penetration and monospermy found in frozen samples compared to fresh semen was accompanied by a decreased motility and NAR, as well as an increased condensation of chromatin, while a signi®cant decrease in chromatin stability was observed only in 0.5 ml straws. When chromatin condensation and stability results were plotted against IVF results we did not ®nd any correlation. Therefore, the chromatin state seemed to have little impact on IVF parameters. However, studies in different species have shown that alterations in the chromatin structure are related to a low in vivo fertility [46±48]. This is consistent with the generally accepted notion that chromatin packaging disorder correlates more with developmental disturbances rather than with a decrease in IVF capacity [49±52]. Because of the impact of the chromatin state on fertility rates this is an important parameter to evaluate in conjunction with IVF and sperm viability. In summary, the freezing and thawing process altered chromatin organization. It increased chromatin condensation, and chromatin instability was signi®cantly higher when spermatozoa were frozen in 0.5 ml compared to 5 ml straws. In fact, chromatin stability was the parameter which changed more signi®cantly when the two straw volume samples were analyzed. Further studies are needed to assess the in vivo signi®cance of this instability. Acknowledgements We wish to thank project CDTI 98-0185 (Spain), the company KUBUS (Madrid, Spain) and DivisioÂn de Ciencias BioloÂgicas de la Salud, UAM (Xochimilco, Mexico). We also thank Agencia EspanÄola de CooperacioÂn Internacional for a fellowship to V.D. References [1] Almlid T, Stavne SE, Johnson LA. Fertility evaluation of the straw freezing technique for boar semen under practical A.I. conditions. Zuchthygiene (Beri) 1987;22:193±202. [2] Johnson LA, Aalbers JG, Willems CMT, Sybema W. Use of boar spermatozoa for arti®cial insemination: I. Fertilizing capacity of fresh and frozen spematozoa in sows in 36 farms. J Anim Sci 1981;52:1130±6. [3] Weitze KF, Rath D, Baron G. Deep freezing of boar semen in plastic straws. Dstch TieraÈrtzl Wschr 1987;94:485±8. [4] Bwanga CO, Braganca MM, Einarsson S, Rodriguez-Martinez H. Cryopreservation of boar semen in mini and maxi-straws. J Vet Med A 1990;37:651±8. [5] Bwanga CO, Hofmo PO, Grevle IS, Einarsson S, Rodriguez-Martinez H. In vivo fertilizing capacity of deep frozen boar semen packaged in plastic bags and maxi-straws. J Vet Med A 1991;38:281±6. [6] Hammit DG, Martin PA. Fertility of frozen±thawed porcine semen following controlled-rate freezing in straws. Theriogenology 1989;32:359±68. [7] CoÂrdova A, PeÂrez JF, Lleo B, GarcõÂa Artiga C, MartõÂn Rillo S. In vitro fertilizing capacity of deep frozen boar semen packaged in 0.5 and 5 ml straws. Reprod Domest Anim 2001;36:199±202.
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[33] Berger B, Fischerleitner F. On deep freezing of boar semen: investigations on the effects of different straw volumes. Methods of freezing and thawing extenders. Reprod Domest Anim 1992;27:266±70. [34] Miller MA, Mausi Y. Changes in the stainability and sulphydril level in the sperm nucleus during sperm± oocyte interaction in mice. Gam Res 1982;5:167±79. [35] Zirkin BR, Soucek DA, Chang TSK, Perreault SD. In vitro and in vivo studies of mammalian sperm nuclear decondensation. Gam Res 1985;11:349±65. [36] Huret JL. Nuclear decondensation of human sperm: a review. Arch Androl 1986;16:97±109. [37] Perreault SD, Naish SJ, Zirkin BR. The timing of hamster sperm nuclear decondensation and male pronucleus formation is related to sperm nuclear disul®de bond content. Biol Reprod 1987;36:239±44. [38] Jager S, Wijchman J, Kremer J. In vitro swelling of the human sperm nucleus in the presence of sodium dodecyl sulphate. Arch Androl 1983;10:201±8. [39] Bilodeau JF, Chatterjee S, Sirard MA, Gagnon C. Levels of antioxidant defenses are decreased in bovine spermatozoa after a cycle of freezing and thawing. Mol Reprod Dev 2000;55:282±8. [40] Mazzilli F, Rossi T, Sabatini L, Pulcinelli FM, Rapone S, Dondero F, et al. Human sperm cryopreservation and reactive oxygen species (ROS) production. Acta Eur Fertil 1995;26:145±8. [41] Alvarez JG, Storey BT. Evidence of increased lipid peroxidative damage and loss of superoxide dismutase activity as a mode of sub-lethal cryodamage to human sperm during cryopreservation. J Androl 1992; 13:232±41. [42] Rufas O, Fisch B, Seligman J, Tadir Y, Ovadia J, Shalgi R. Thiol status in human sperm. Mol Reprod Dev 1991;29:282±8. [43] Twigg JP, Irvine DS, Aitken RJ. Oxidative damage to DNA in human spermatozoa does not preclude pronucleus formation at intracytoplasmatic sperm injection. Hum Reprod 1998;13:1864±71. [44] Evenson DP, Sailer BL, Jost LK. Relationship between stallion sperm deoxyribonucleic acid (DNA) susceptibility to denaturation in situ and presence of DNA strand breaks: implications for fertility and embryo viability. Biol Reprod Mono 1995;1:655±9. [45] Evenson DP, Thompson L, Jost L. Flow cytometric evaluation of boar semen by the sperm chromatin structure assay as related to cryopreservation and fertility. Theriogenology 1994;41:637±51. [46] Sailer BL, Jost LK, Evenson DP. Bull sperm head morphometry related to abnormal chromatin structure and fertility. Cytometry 1996;24:167±73. [47] Ibrahim ME, Moussa MA, Pedersen H. Sperm chromatin heterogeneity as an infertility factor. Arch Androl 1988;21:129±33. [48] Evenson DP, Darzynkienwicz Z, Melamed MR. Relation of mammalian sperm chromatin heterogeneity to fertility. Science 1980;240:1131±3. [49] Filatov MV, Semenova EV, Vorobeva OA, Leonteva OA, Drobchenko EA. Relationship between abnormal sperm chromatin packing and IVF results. Mol Hum Reprod 1999;5:825±30. [50] Hammadeh ME, Askari AS, Gerog T, Rosenbaum P, Schmidt W. Effect of freeze±thawing procedure on chromatin stability, morphological alteration and membrane integrity of human spermatozoa in fertile and subfertile men. Int J Androl 1999;22:155±62. [51] Jager S. Sperm nuclear stability and male infertility. Arch Androl 1990;25:253±9. [52] Carrell DT, Emery BR, Peterson CM. The correlation of sperm chromatin decondensation following in vitro exposure to heparin and sperm penetration rates. J Assist Reprod Genet 1998;15:560±4.
In vitro fertilizing capacity and chromatin condensation of deep frozen boar semen packaged in 0.5 and 5 ml straws Alejandro CoÂrdovaa, Jose FeÂlix PeÂrez-GutieÂrrezb,*, BeleÂn LleoÂb, Carlos GarcõÂa-Artigab, Alberto Alvarezc, Volodymyr Drobchakb, Santiago MartõÂn-Rillob,1 a
Departamento de ProduccioÂn AgrõÂcola y Animal, Universidad AutoÂnoma Metropolitana, Xochimilco, Mexico b Facultad de Veterinaria, Dpto. PatologõÂa Animal IIÐReproduccioÂn, Universidad Complutense U.C.M., Avda. Puerta de Hierro s/n, 28040 Madrid, Spain c Centro de CitometrõÂa de Flujo y MicroscopõÂa Confocal, Universidad Complutense, Madrid, Spain Received 12 June 2001; accepted 19 November 2001
Abstract The effect of the straw volume employed for semen freezing was studied in 14 ejaculates from seven boars, by evaluating the viability, IVF capacity and chromatin state of spermatozoa. Frozen± thawed semen from 0.5 and 5 ml straws was compared to fresh semen. The chromatin condensation degree was determined by ¯ow cytometry, using propidium iodide as ¯uorochrome, and the chromatin stability was evaluated by inducing its decondensation with SDS and EDTA. The results obtained for IVF, motility and normal apical ridge (NAR) were: 91.64, 78.14 and 81.47% sperm penetration, 80.78, 68.38 and 70.83% monospermy, 10.86, 9.76 and 10.64% polyspermy, 87.14, 50.71 and 47.86% motility, 79.14, 56.14 and 53.36% NAR, for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively. Frozen±thawed spermatozoa showed signi®cantly increased (P < 0:05) chromatin compactness compared to fresh spermatozoa (55.42, 48.41 and 47.08 ¯uorescence units (MIFU), for fresh semen, thawed semen in 0.5 and 5 ml straws, respectively). Chromatin was signi®cantly more unstable (P < 0:05) in spermatozoa frozen in 0.5 ml straws (174.7 MIFU) compared to those frozen in 5 ml straws (155.53 MIFU) or to those in fresh semen (149.74 MIFU). # 2002 Elsevier Science Inc. All rights reserved. Keywords: Sperm chromatin; In vitro fertilization; Cryopreservation; Porcine
* Corresponding author. Tel.: 34-91-3943-798; fax: 34-91-3943-808. E-mail address: [email protected] (J.F. PeÂrez-GutieÂrrez). 1 Deceased.
0093-691X/02/$ ± see front matter # 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 3 - 6 9 1 X ( 0 2 ) 0 0 7 0 1 - X
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1. Introduction The use of frozen boar semen for improving pig population genetics is limited, and the swine industry is focusing on improving cryopreservation techniques in order to increase production. The lower conception rate after arti®cial insemination with cryostored spermatozoa, despite the apparent good post thaw motility, and even when using an insemination dose with a higher number of spermatozoa than with fresh semen [1,2], raises questions about cellular damage to spermatozoa during the freezing±thawing process. Among other factors, the shape and volume of the container can in¯uence the quality of frozen semen. Boar semen is usually frozen in 5 ml straws. Due to low heat transfer, cooling is fast in the peripheral regions and slow in the center of the straw, which can induce acrosome and membrane damage, injuries that can be reduced by lowering the straw volume [3]. Some authors have studied the effect of the straw volume on semen quality by evaluating in vitro motility rates and normal apical ridge (NAR) percentages as well as in vivo fertility rates [4±6]. However, other parameters related to fertility can be measured in vitro, including monospermy and polyspermy rates [7], as well as the chromatin state. The formation of a compact structure in sperm chromatin is necessary for DNA transient inactivation and protection [8]. In mature spermatozoa, the normal nucleosomal packaging of DNA found in somatic cells is transformed into a highly condensed form of chromatin, which consists mostly of nucleoprotamines [9]. Protamines contain a large number of cysteine residues, which are initially present as thiol groups [10,11]. These thiol groups are partially oxidized to form disulphide bonds during sperm maturation [12]. After ejaculation, sperm chromatin is stabilised by semen plasma, probably due to the presence of zinc, that binds free thiol groups [13,14]. Incubation of spermatozoa in semen plasma or under capacitation conditions induces a higher stability of chromatin, which has been attributed to a gradual depletion of zinc that leads to the exposure of free thiol groups, and allows the formation of additional disulphide bonds [13,15±17]. Previous studies in human and porcine species have shown that sperm chromatin can undergo important changes after the freezing±thawing procedure, resulting in a greater compactness of spermatozoa nuclei [18±21]. On the other hand, hyperstability of the sperm chromatin could delay paternal nuclear formation during fertilization [17,22], which can induce early embryonic death or lower embryonic development, frequently observed after arti®cial insemination with frozen boar semen [23]. Thus, chromatin condensation and chromatin stability of thawed spermatozoa may be critical factors to consider in assisted reproduction. The aim of this study was to evaluate the effect of straw volume on motility, NAR, chromatin condensation, chromatin stability and IVF capacity on frozen boar semen. 2. Materials and methods Unless otherwise stated, reagents were purchased from Sigma Chemical Co. (St. Louis, MO).
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2.1. Semen collection Semen was collected on a weekly basis, from seven Duroc boars at the Agronomy School of the Universidad Complutense of Madrid, Spain. The boars were 2 years old and their fertility had been previously proven at this experimental farm. Ejaculates were obtained by the gloved hand method [24]. Only the sperm rich fraction was collected. Gel particles were removed by ®ltration through gauze, and volume, sperm concentration, motility and NAR were assessed immediately after collection. All the samples were evaluated by the same observer. Ejaculates showing a motility higher than 80%, a motility quality score higher than 3 and NAR higher than 70% were used. A total of 14 ejaculates (2 from each of the 7 boars) were used. Each ejaculate was divided into three fractions, one to be used as fresh semen and the other two to be frozen in 0.5 and 5 ml straws. 2.2. Assessment of concentration Cell concentration was assessed on fresh semen, in formol saline ®xed samples (1:100 dilution) using a BuÈker chamber. 2.3. Assessment of sperm motility Sperm motility was evaluated using a phase contrast microscope by placing a drop of the sample, without further dilution, on a microscope slide at 42 8C and covering with a cover slip. Samples were evaluated at 200 magni®cation, by counting progressively motile spermatozoa [25]. Motility was expressed as the percentage of motile spermatozoa per 100 sperm cells. Motility quality was assessed by the rate of forward progression, on a scale from 0 to 5. 2.4. Assessment of the NAR The evaluation of acrosomal integrity was done by examining formol saline ®xed samples with a phase contrast microscope, at 1000 magni®cation. A minimum of 100 acrosomes were examinated per sample. Damage to the acrosomal cap was classi®ed by the scoring system reported by Pursel et al. [26]. 2.5. Semen freezing The semen was processed and frozen by the straw freezing method, originally described by Westendorf et al. [27] and later modi®ed by MartõÂn [28]. Shortly after collection, the sperm rich fraction was diluted (1:4) with a commercial extender preparation, MR-A1 diluent (Kubus, S.A., Madrid, Spain), at 23 8C. After 1 h equilibration at 20 8C, a dose containing 6 109 spermatozoa (double dose), was prepared in a 50 ml centrifuge ¯ask. The sperm suspension was kept at 15 8C for 3 h and then centrifuged at 800 g for 10 min. The supernatant was then removed and the concentrated semen was diluted to a volume of 5 ml with the cooling extender (11% lactose and 20% egg yolk in distilled water) at 15 8C.
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The diluted semen was gently mixed and cooled to 5 8C for 1.5 h. The semen was further diluted (1:1) with freezing extender (11% lactose, 20% egg yolk, 4% glycerol and 0.5% lauryl sodium sulfate in distilled water) to 10 ml. Then 5 and 0.5 ml straws were ®lled and sealed manually, using metallic sealing balls and polyvinyl alcohol. Straws were wiped dry and the air bubble was brought to the center of the straw. The 5 and 0.5 ml straws, containing 3000 106 and 300 106 spermatozoa, respectively, were then placed in contact with nitrogen vapor for 20 min, about 3 cm above the nitrogen liquid level, and then plunged in the liquid nitrogen tank and stored until used. 2.6. Thawing Thawing of straws was done after 6 months of storage in liquid nitrogen. The 5 and 0.5 ml straws were thawed in a water bath at 42 8C for 12 and 45 s, respectively. Motility and NAR were immediately assessed. 2.7. In vitro capacitation Fresh and thawed semen samples were centrifuged at 500 g for 10 min. The pellets were diluted with Tyrode's medium, supplemented with 0.25 mM sodium pyruvate, 6 mg/ml BSA, 21.6 mM sodium lactate and 10 mM HEPES (TALP±HEPES) [29], at 37 8C, to a ®nal volume of 1.5 ml. Spermatozoa were allowed to swim up in this medium during a 30 min incubation at 37 8C. In order to select the motile sperm fraction the upper 0.5 ml of medium was collected [30]. This fraction was further diluted with TALP±HEPES to a ®nal concentration of 75 106 spermatozoa/ml. Aliquots of 0.5 ml were incubated in wells of a 4 well-plate (Nunc, Roskilde, Denmark) for 1±1.5 h, at 37 8C, in a humidi®ed 5% CO2 atmosphere. 2.8. In vitro fertilization Swine ovaries were obtained from a local slaughterhouse and transported, in less than 1 h, to the laboratory, in 0.157 M NaCl at 37 8C. Oocytes, with cumulus cells, were aspirated from 3 to 6 mm diameter follicles with a 21 gauge needle attached to a 5 ml syringe. Thirty to 35 oocytes were incubated in a 100 ml drop of maturation medium: TC 199 medium supplemented with 10% inactivated fetal calf serum, 1 mg/ml glucose, 2 IU/ ml pergonal (LH, FSH), 0.25 mM sodium pyruvate, 10 mg/ml gentamycin and 1 mg/ml b-estradiol. The microdrops were placed in 4 well-plates (Nunc) and covered with paraf®n oil. Oocytes were incubated for 48 h, at 37 8C, in a humidi®ed atmosphere with 5% CO2, and then coincubated with 5 104 spermatozoa per well in a TALP fertilization medium microdrop [31] for 12±16 h, under the same conditions as above. 2.9. IVF evaluation The oocytes were ®xed in methanol:acetic acid (3:1) for 24 h, stained with 2% acetic orcein and examined at 100 magni®cation under a bright ®eld microscope to assess fertilization. Those oocytes showing two pronuclei were considered fertilized.
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2.10. Analysis of sperm chromatin condensation and stability The chromatin condensation degree was determined by ¯ow cytometry, employing as ¯uorochrome propidium iodide (PI), an intercalating compound whose accessibility to DNA was measured by ¯uorescence. Two determinations were done. First, chromatin condensation was determined. Second, the susceptibility of chromatin to decondensation, or chromatin stability, was determined by inducing ®rst decondensation with EDTA plus SDS, and then determining the chromatin condensation state. 2.11. Nuclear sperm chromatin treatment Sperm chromatin was stained with propidium iodide, according to Molina et al. [32], using the Cycle Test PLUS DNA reagent kit from Becton Dickinson (San Jose, CA). For determining chromatin condensation, samples were prepared by resuspending fresh and frozen semen for 5 min in capacitation medium to a concentration of 1± 2 106 spermatozoa per sample. After centrifugation at 500 g for 10 min, supernatant was discarded. The sperm membrane was permeabilized, using trypsin (0.5%) in a spermine-tetrahydrochloride detergent buffer, for 10 min at room temperature. To stop trypsin action and to remove double stranded RNA, trypsin inhibitor and ribonuclease A (12 mg/ml) were added and incubated for a further 10 min. Subsequently, ice-cold propidium iodide (50 mg/ml) and spermine-tetrahydrochloride in citrate stabilizing buffer, were added. Samples were incubated for 1 h in the dark before ¯ow cytometer analysis. For determining chromatin stability, decondensation of chromatin was induced by resuspending fresh and frozen±thawed semen, for 5 min, in a capacitation medium solution, containing 6 mM EDTA and 1% SDS in borate buffer (0.05 M, pH 9.0) [22], to a concentration of 1±2 106 spermatozoa per sample. Samples were then treated as for chromatin condensation analysis, as described above. 2.12. Flow cytometric measurements Flow cytometer analysis was performed by using a FACscan ¯ow cytometer (Becton Dickinson, S.A., San Jose, CA). Propidium iodide was excited by an argon-ion laser emitting at 488 nm and PI ¯uorescence was measured at 605 nm with using a 600/40 bandpass ®lter. Data were analyzed using Cell Quest software from Becton Dickinson. Measurements were expressed as mean intensity ¯uorescence units, arbitrary units (MIFU) related to the PI uptake by DNA. 2.13. Statistical analysis Results were analyzed by ANOVA and multiple comparison test using SAS software (SAS Institute Inc., Cary, NC).
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Table 1 IVF parameters, motility, NAR, chromatin condensation and stability for fresh and frozen semen in 0.5 and 5 ml straws. Fresh semen
Frozen±thawed semen, packed in 0.5 ml straws
Penetration (%) Monospermy (%) Polyspermy (%) Motility (%) NAR (%) Chromatin condensation (MIFU) Chromatin stability (MIFU)
91.64 80.78 10.86 87.14 79.14 55.42 149.74
a
0.99 0.78a 0.40a 1.25a 1.15a 0.92a 8.69a
78.14 68.38 9.76 50.71 56.14 48.41 174.70
5 ml straws b
1.00 0.90b 0.38a 1.64b 2.12b 1.25b 9.40b
81.47 70.83 10.64 47.86 53.36 47.08 155.53
0.93c 0.67c 0.44a 1.55b 1.32b 1.22c 7.21a
Values are expressed as mean S:E:M:, n 14. Values in each row with the same superscript are not signi®cantly different (P < 0:05). Units: percentages (%) related to the total number of spermatozoa (*) or the total number of oocytes (**); MIFU: mean intensity ¯uorescence units, related to propidium iodide uptake by DNA. Chromatin condensation refers to sperm resuspended in TALP±HEPES medium and chromatin stability refers to the chromatin condensation of sperm after treatment with EDTA plus SDS.
3. Results 3.1. Effect of straw volume on the viability of frozen semen After freezing and thawing, viable spermatozoa were assessed by motility, NAR and IVF parameters, i.e. monospermy, polyspermy and penetration (Table 1). All of these parameters, except polyspermy, decreased when semen was frozen in either 0.5 or 5 ml straws. However, no signi®cant differences were found between 0.5 and 5 ml straws, regarding both motility and NAR. No signi®cant differences between the two straw volumes for polyspermy were found, and both penetration and monospermy were slightly higher for frozen semen in 5 ml straws. 3.2. Effect of straw volume on sperm chromatin condensation and stability The results obtained for chromatin condensation and stability for fresh and frozen semen in 0.5 and 5 ml straws are presented in Table 1. Frozen±thawed spermatozoa showed a signi®cant decrease in PI uptake, indicating increased chromatin compactness, compared to fresh spermatozoa. Spermatozoa frozen in 5 ml straws showed a slightly lower PI uptake (i.e. higher chromatin condensation) than those frozen in 0.5 ml straws. Chromatin stability refers to the chromatin condensation of sperm after treatment with EDTA plus SDS. A higher PI uptake is related to easier decondensation of the chromatin and, therefore, to its higher instability. Frozen samples in 0.5 ml showed signi®cant less stability compared to fresh or to frozen samples in 5 ml straws.
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4. Discussion 4.1. Effect of straw volume on the viability of frozen semen As expected, motility, NAR, monospermy and penetration decreased upon freezing of semen regardless the straw volume used. It is generally accepted that increasing the volume of straws is detrimental for both motility and NAR due to a slower heat transfer [3,4,33]. However, no differences between 0.5 and 5 ml straws for motility, NAR and polyspermy were found and, even though penetration and monospermy were higher for frozen semen in 5 ml straws, the differences were very small. These results corroborate previous studies [7] that have shown, using the same procedure, no detrimental effects on viability and IVF capacity with the use of 5 ml straws compared to 0.5 ml straws. 4.2. Effect of straw volume on sperm chromatin condensation and stability After freezing and thawing, boar spermatozoa showed a more compact chromatin than in fresh samples (Table 1). This increase in chromatin condensation after freezing and thawing is in agreement with previous studies [19±21]. Furthermore, spermatozoa frozen in 5 ml straws showed a slightly higher condensation than those frozen in 0.5 ml straws. Hypercondensation in frozen samples, however, was not related to a higher stability of chromatin. In fact, after inducing decondensation with EDTA plus SDS, chromatin in frozen samples did show equal or higher instability. In addition, while spermatozoa frozen in 5 ml straws did not show different chromatin stability compared to fresh semen, there was a signi®cant difference in frozen spermatozoa packaged in 0.5 ml straws, which displayed less stability. This difference was maintained when the results for the two straw volumes were compared for each of the samples employed (results not shown). Therefore, these results could not be due to natural differences among samples, but rather to an effect of straw volume or the procedure, which undergoes minor changes depending on the straw volume used. The effect of straw volume could be due to the slower heat transfer in 5 ml straws, while the effects of the procedure could be due to thawing at 42 8C for different times, 12 and 45 s, for 0.5 and 5 ml straws, respectively (see Section 2). Various mechanisms, such as binding between DNA and nucleoprotamines or the relative number of disul®de bonds may be involved in the condensation state of chromatin [34±37]. However, the mechanisms that induce changes in chromatin stability are still unknown. Jager et al. [38] have suggested that decondensation is more a physico-chemical rather than an enzymatic process and it depends on temperature, time and pH. However, there is growing evidence suggesting that reactive oxygen species (ROS) increase during freezing and thawing of spermatozoa and can play an important role in fertility [39±41]. Oxidative stress can be induced by freezing and it has been suggested that prolonged exposure of spermatozoa to even mild oxidation conditions could lead to overoxidation of thiol groups and hypercondensation of DNA [42]. On the other hand, ROS can also induce breaks in the DNA [43]. The presence of DNA strand breaks has been found to be related to an increased susceptibility of DNA to denaturation [44,45].
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Therefore, the increased compactness observed in frozen samples could be due to an increased formation of disul®de bonds or to stronger noncovalent interactions. On the other hand, the increased instability of chromatin found in spermatozoa frozen in 0.5 ml straws could be the result of more profound molecular changes, possibly be related to protamine conformational changes, induced by enhanced covalent or noncovalent interactions, or to an increased DNA strand breakage. The lower penetration and monospermy found in frozen samples compared to fresh semen was accompanied by a decreased motility and NAR, as well as an increased condensation of chromatin, while a signi®cant decrease in chromatin stability was observed only in 0.5 ml straws. When chromatin condensation and stability results were plotted against IVF results we did not ®nd any correlation. Therefore, the chromatin state seemed to have little impact on IVF parameters. However, studies in different species have shown that alterations in the chromatin structure are related to a low in vivo fertility [46±48]. This is consistent with the generally accepted notion that chromatin packaging disorder correlates more with developmental disturbances rather than with a decrease in IVF capacity [49±52]. Because of the impact of the chromatin state on fertility rates this is an important parameter to evaluate in conjunction with IVF and sperm viability. In summary, the freezing and thawing process altered chromatin organization. It increased chromatin condensation, and chromatin instability was signi®cantly higher when spermatozoa were frozen in 0.5 ml compared to 5 ml straws. In fact, chromatin stability was the parameter which changed more signi®cantly when the two straw volume samples were analyzed. Further studies are needed to assess the in vivo signi®cance of this instability. Acknowledgements We wish to thank project CDTI 98-0185 (Spain), the company KUBUS (Madrid, Spain) and DivisioÂn de Ciencias BioloÂgicas de la Salud, UAM (Xochimilco, Mexico). We also thank Agencia EspanÄola de CooperacioÂn Internacional for a fellowship to V.D. References [1] Almlid T, Stavne SE, Johnson LA. Fertility evaluation of the straw freezing technique for boar semen under practical A.I. conditions. Zuchthygiene (Beri) 1987;22:193±202. [2] Johnson LA, Aalbers JG, Willems CMT, Sybema W. Use of boar spermatozoa for arti®cial insemination: I. Fertilizing capacity of fresh and frozen spematozoa in sows in 36 farms. J Anim Sci 1981;52:1130±6. [3] Weitze KF, Rath D, Baron G. Deep freezing of boar semen in plastic straws. Dstch TieraÈrtzl Wschr 1987;94:485±8. [4] Bwanga CO, Braganca MM, Einarsson S, Rodriguez-Martinez H. Cryopreservation of boar semen in mini and maxi-straws. J Vet Med A 1990;37:651±8. [5] Bwanga CO, Hofmo PO, Grevle IS, Einarsson S, Rodriguez-Martinez H. In vivo fertilizing capacity of deep frozen boar semen packaged in plastic bags and maxi-straws. J Vet Med A 1991;38:281±6. [6] Hammit DG, Martin PA. Fertility of frozen±thawed porcine semen following controlled-rate freezing in straws. Theriogenology 1989;32:359±68. [7] CoÂrdova A, PeÂrez JF, Lleo B, GarcõÂa Artiga C, MartõÂn Rillo S. In vitro fertilizing capacity of deep frozen boar semen packaged in 0.5 and 5 ml straws. Reprod Domest Anim 2001;36:199±202.
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