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Fragmentation dynamics of frozen-thawed ram sperm DNA is modulated by sperm concentration
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Theriogenology 74 (2010) 1362–1370 www.theriojournal.com
Fragmentation dynamics of frozen-thawed ram sperm DNA is modulated by sperm concentration C. López-Fernándeza, S.D. Johnstonb,*, J.L. Fernándezc, R.J. Wilsond, J. Gosálveza a
Departamento de Biología, Unidad de Genética, Universidad Autónoma de Madrid (UAM). 20849-Madrid. España. b School of Animal Studies, The University of Queensland, Gatton, 4343, Australia c INIBIC-Genética, Complejo Hospitalario Universitario Juan Canalejo, As Xubias, A Coruña, Spain d School of Mathematics and Physics, The University of Queensland, St Lucia, 4072, Australia Received 8 October 2009; received in revised form 1 June 2010; accepted 1 June 2010
Abstract This study investigated the hypothesis that post-thaw incubation of ram sperm at high concentrations results in a faster rate of DNA fragmentation than when sperm are incubated at a lower concentration. Ejaculates from 10 rams were frozen-thawed, prepared in sperm concentrations of 100, 50, 25, 12, and 6 ⫻ 106 sperm/mL, and incubated for 6 h at 37 °C. Sperm DNA fragmentation was assessed using the sperm chromatin dispersion test (Sperm-Halomax®) at 1, 3, 4, and 6 h of incubation at 37 °C. On fitting a binary logistic regression with a cubic over time and treating ram and dilution levels as factors, there were significant effects with respect to the ram, dilution and time (all P-values were very much smaller than 0.001). Therefore, DNA fragmentation dynamics of incubated frozen-thawed ram sperm were not only dependent on the inherent sperm DNA fragmentation expressed immediately after thawing, but also on the concentration of sperm incubated in the sample. Although there was evidence of individual ram variation in SDF during the incubation period, the general finding of the current study was that lower sperm concentrations resulted in a slower rate of DNA fragmentation These findings have important implications for the post-thaw manipulation of ram sperm used for AI and advanced reproductive procedures that use sperm at low concentrations. Our data also emphasised the highly dynamic nature of sperm DNA fragmentation and the importance of conducting the procedure in a standardised manner. © 2010 Elsevier Inc. All rights reserved. Keywords: Sperm DNA fragmentation; Semen cryopreservation; Sheep; Dilution; In vitro incubation
1. Introduction Three major hypotheses have been proposed to explain cellular mechanisms that result in the altered sperm DNA molecule. The first is related to torsional stress in unconstrained DNA supercoils and is a direct consequence of histone-protamine replacement during mid-spermiogenesis [1,2]. The second hypothesis re-
* Corresponding author. Tel.: 617 32026902; fax: 617 33655644. E-mail address: [email protected] (S. Johnston). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.06.006
gards DNA fragmentation as a consequence of oxidative stress in the male reproductive tract [3– 6]. The third hypothesis concerns apoptotic-related DNA strand breakage, similar to that which occurs in abortive apoptosis in somatic cells; the presence of caspase 9 in the midpiece and the occurrence of activated caspases 8, 1 and 3 in the postacrosomal region appeared to support this view [4,6,7]. However, the etiologies of DNA damage are many and varied, ranging from bacterial infections [8], chemical toxicity [9,10], elevated temperature [11], diabetes [12,13], age
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[14,15], body mass [16], and genetic background [17]. Whereas many of the factors resulting in sperm DNA fragmentation are typically unavoidable, certain types of induced iatrogenic sperm DNA damage can become exacerbated when sperm are inappropriately manipulated in the laboratory. For example, we have previously shown how changes in temperature excursions can affect the rate of sperm DNA fragmentation during in vitro incubation [18]. Successful artificial insemination (AI) of frozenthawed semen relies on the delivery of sufficient numbers of sperm into the female reproductive tract, which are morphologically normal, have a functional capacity in terms of motility, and are able to undergo an acrosome reaction and to successfully participate in syngamy. Although numerous studies have examined post-thaw survival characteristics (motility, membrane integrity, and acrosomal status) of incubated mammalian sperm, only recently has there been interest in the effect of post-thaw incubation on sperm DNA fragmentation and the potential importance of this phenomenon to the success of AI programs [19,20]. Cryopreservation induced capacitation-like changes in ram sperm, which resulted in membrane destablisation and cell death [21]; compounding this phenomenon, excessive dilution of sperm and the consequent removal of seminal plasma has also been shown to be associated with a loss of motility and viability [22]. In addition, highly diluted frozen-thawed ram sperm recovered from swim-up procedures had changes in their motility and chlortetracycline staining patterns which were indicative of an increase in the incidence of capacitation [23,24]. A study on canine sperm revealed that both motility and membrane integrity was improved with increasing dilution of the frozen-thawed sample, but that the proportion of reacted acrosomes was similar [25]. In the bull, dilution and incubation of thawed sperm at high and low concentrations at room temperature for 24 h resulted in little difference between these populations in terms of viability or susceptibility to osmotic stress, although sperm preparations with lower sperm concentration had a higher proportion of viable cells with reacted acrosomes [26]. Surprisingly, the effect of dilution on sperm DNA fragmentation of incubated frozen-thawed sperm has not been investigated. Therefore, the objective of the current study was to analyze the effect of sperm concentration and post-thaw incubation on the integrity of ram sperm DNA. This experiment was based on the hypothesis that the rate of DNA fragmentation of frozen-thawed ram sperm is dependent on the initial sperm concentra-
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tion at which the thawed sample was extended. The results of this investigation will provide important insights and practical applications into the way in which thawed ram sperm should be processed and diluted prior to its use in laparoscopic AI or other advanced reproductive technologies. 2. Materials and methods 2.1. Animals, semen collection and sperm cryopreservation Semen was collected by artificial vagina from 10 sexually mature (2– 4 y) rams housed at the Ovigen Centre, Zamora, Spain; these animals were kept under controlled feeding and photoperiodic conditions for maximal reproductive performance and were clinically healthy throughout the semen collection period. In preparation for cryopreservation, the fresh ejaculate was diluted to a final concentration of 1000 ⫻ 106 total sperm in a commercial egg yolk-based extender, (Triladyl; Minitube Canada, Woodstock, ON, Canada) loaded into 0.25 mL PVC straws, and frozen in a programmable freezing chamber (Minitube Iberica, Tarragona, Spain). 2.2. Dilution and incubation of thawed sperm Semen straws were thawed by plunging in a waterbath at 37 °C for approximately 30 to 60 s [27], after which time each sample was immediately diluted in a pre-warmed (37 °C) commercial extender (INRA 96, IMV Technologies, L’Aigle, France) to give a sperm concentration of ⬃200 ⫻ 106 sperm/mL. At this time, the post-thaw survival of the thawed sperm was assessed; only samples with high post-thaw survival (⬎50% normal acrosomes, ⬎35% positive to the HOS test, and ⬎35% progressive motility) were used for subsequent incubation experiments. The semen sample was then allowed to cool to 15 °C over 30 min to reduce metabolism prior to being centrifuged (300 ⫻ g for 10 min); this step was included to remove the potential harmful influence of cryomedia which may have inadvertently affected DNA integrity during incubation. The supernatant was gently removed and resuspended with fresh diluent (INRA 96) to provide a standardised working sperm concentration of 100 ⫻ 106 sperm/mL (C100). Sperm DNA fragmentation (SDF) was assessed at this stage of post-thaw incubation and designated time T0. This diluted semen sample was subsequently serially diluted in INRA 96 to produce sperm concentrations of 50 (C50), 25 (C25), 12 (C12), and 6
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Fig. 1. Ram sperm DNA fragmentation following the sperm chromatin dispersion test [18]. (a) Ram sperm showing absence of sperm DNA damage—all sperm have a small halo of chromatin dispersion; (b) The sperm chromatin dispersion after 6 h of incubation. Note large haloes of dispersed chromatin representing sperm with fragmented DNA.
(C6) ⫻ 106 sperm/mL; these sperm populations were then incubated at 37 °C in 5% CO2 for 6 h and SDF assessed at 1 (T1), 3 (T3), 4 (T4), and 6 h (T6) of incubation. 2.3. Assessment of sperm DNA fragmentation The incidence of SDF throughout the incubation period was assessed by removing an aliquot of the semen sample and further diluting it in INRA 96 to a concentration of approximately 10 ⫻ 106 sperm/mL; this dilution step was conducted immediately before processing the sperm for the Halomax assay, to ensure that the semen sample was not overly concentrated for microscopic analysis. Sperm DNA fragmentation was assessed using the sperm chromatin dispersion test as previously described and validated for the ram ([18]; Sperm-Halomax®; Halotech DNA, Madrid, Spain). Following the SCD procedure, fragmented and nonfragmented sperm within the microgel were stained with a dual emission DNA-blue/Protein-green fluorochrome combination. Nucleoids were stained using the DNA-specific stain, 4=,6-diamidine-2= phenylindole dihydrochloride (DAPI; Sigma Aldridge, Irvine, UK), whereas 2,7-dibromo-4-hydroxy mercurifluorescein (Sigma Aldridge, Irvine, UK) was used for protein staining. Ram sperm with fragmented DNA were identified as
large fluorescent halos of dispersed chromatin, whereas those with non-fragmented DNA had small fluorescent halos and a compact nuclear core (Fig. 1). Images were captured using a black-and-white cooled Leica DCF 300 camera mounted onto an epifluoresencence Motic A-400 microscope using a single band pass filter (Cy54040A-492/516; Semrock, Rochester, NY, USA). Three hundred sperm were observed for SDF and the number of fragmented sperm reported as a percentage. 2.4. Statistical analysis As the objective of this study was to identify systematic changes in the expected (cumulative) rate of SDF as dilution increases, a binary logistic cubic regression over time [28] was fitted to the percentage of fragmented cells in each ram semen sample over time and where each sample consisted of an assessment of 300 cells. The logistic regression model used assumed that the probability of SDF for a single cell by a given time depended on the ram, the dilution factor and time, with the relationship with time being a cubic in time, with the distribution assumed for the number of cells to fragment for each combination of ram, dilution and time to be the binomial distribution. For each ram, a common initial expected rate of SDF was assumed for all dilutions, as the pre-incubation fragmentation of
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Fig. 2. Observed changes in % SDF of ram sperm following post-thaw incubation of samples at various sperm concentrations (6, 12, 25, 50, and 100 ⫻ 106 sperm/mL) for 6 h at 37 °C.
each individual ram was the same. Using the most diluted concentration as a base cubic, parameterisation was defined in terms of differences of dilution effect on the coefficients of the terms in the cubic polynomials over time as dilution decreased. This allowed for a backwards elimination method to be used to reduce the dilution effects in the cubic models over time and so identify significant differences in the expected cumulative rate as dilution decreased. Consequently, it could be determined if dilution had a significant effect on the expected rate of SDF and in which direction such an effect might be. The full binary logistic cubic regression over time over all dilutions and rams was used to evaluate whether there were significant differences in expected rate of SDF over rams, dilution and time.
3. Results The effects of post-thaw dilution and incubation on ram sperm DNA fragmentation for each ram are shown (Fig. 2). The SDF dynamics of all individual rams showed a generalised consistent pattern of dynamics whereby the rate of SDF was lower in samples with lower sperm concentration. In addition to fitting a binary logistic regression with a cubic over time and treating ram ID and dilution levels as factors (see Fig. 3 for two examples), there were significant effects with respect to the ram, dilution and time (all P-values were very much smaller than 0.001). As there were significant differences from ram to ram in the pattern of differences over time and dilutions (i.e., the interactions
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Fig. 3. Comparison of observed (raw) and fitted (model) changes in % SDF of ram sperm (Rams 4 and 8) following post-thaw incubation of samples at various sperm concentrations (6, 12, 25, 50, and 100 ⫻ 106 sperm/mL) for 6 h at 37 °C.
between ram, dilution and time), the results for each ram need to be considered separately in order to determine the nature of the relationship of fragmentation to dilution over time. Backward elimination (with a 5% significance level) was used to remove non-significant terms in the binary logistic full cubic model over time, to determine which dilutions were not significantly different over time for each ram, and where lower order polynomials could be used in part of the model. For some rams, the cubic was reduced to a lower order polynomial for the base polynomial (see results for the C6 concentration). Results indicating which dilutions gave non-significantly dif-
ferent patterns of SDF and those that were significantly different are shown in Table 1 (the P-values provided some indication of the significance of the eliminated terms for that ram), with the corresponding fitted SDF values predicted by the model. As described in Figure 2 and from the results of the statistical analysis in Table 1, there was variability in the pattern of SDF over time and dilution by ram. Despite this variation, and apart from the results for Ram 8, there was a non-strict ordering by dilution, so that each incubation time and for each ram, the amount of DNA fragmentation typically did not increase as the dilution increases. Furthermore, apart from the results
Table 1 Results of backward elimination for the binary logistic regression to determine the effect of sperm concentration on the integrity of ram sperm DNA. Ram
Non-significantly different pairs of dilutions
Significantly different pairs of dilutions
P-value for eliminated terms
1 2 3 4 5 6 7 8 9 10
(C6,C12) (C25,C100) (C25,C50) (C50,C100) — (C25,C50) (C25,C50) — (C50,C100) — (C50,C100) (C6,C12) (C12,C25) (C25, C50) (C50, C100)
(C12,C25) (C25,C50) (C6,C12) (C12,C25) (C6,C12) (C12,C25) (C25,C50) (C50,C100) (C6,C12) (C12,C25) (C50,C100) (C6,C12) (C12,C25) (C50,C100) (C6,C12) (C12,C25) (C25,C50) (C50,C100) (C6,C12) (C12,C25) (C25,C50) (C6,C12) (C12,C25) (C25,C50) (C50,C100) (C6,C12) (C12,C25) (C25,C50) —
0.2378 0.6612 0.1196 0.1301 0.4572 0.5118 0.1441 0.7168 0.8099 0.4423
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reported for Ram 10, there was at least one time in the observation period for which the amount of DNA fragmentation decreased significantly as the dilution increased, although the dilution and time for which this occurs varied for each ram. Two exceptions to the general pattern of observations were found in Rams 8 and 10. Ram 8 appeared to have a slower expected rate of SDF for the straw concentration (C100) than for the other concentrations, suggesting that it was an outlier when compared the majority of other rams in this study. There were no significant differences at any times between the expected rate of SDF for all dilutions of semen from Ram 10 (Fig. 2). Given the variation in patterns in SDF across the rams, it is worthwhile highlighting the results of the individual rams in further detail. In seven of the 10 rams (R1, R2, R4, R5, R7, R9, and R10), the final SDF after 6 h incubation for most sperm concentrations appeared to be similar, except for the lowest sperm concentration (C6) which typically had lower SDF (exceptions were R1 and R2). The SDF of the C6 dilution in Rams 4, 5, 7, and 9 after 6 h incubation was substantially lower than that for samples with higher sperm concentrations incubated for the same time period. The rate of SDF in Rams 3 and 6 showed a slightly different pattern, in that there was a larger range of SDF observed across the various dilutions after 6 h incubation. This phenomenon was particularly illustrated in Ram 6, where the SDF of C6 after 6 h incubation was the same as that reported at the commencement of the incubation period. The sperm of Rams 6 and 8 also had lower values of fitted SDF across all dilutions after 6 h incubation ranging from 7– 63 and 41– 64 respectively, when compared to the other rams (Rams 3, 4, 5, 7, and 8) which collectively ranged from 63–90. 4. Discussion In this study, DNA fragmentation dynamics of incubated frozen-thawed ram sperm were not only dependent on the inherent sperm DNA fragmentation expressed immediately after thawing, but also on the concentration of sperm incubated in the sample over time. Although there was evidence of individual ram variation in SDF during the incubation period, the general finding was that lower sperm concentrations resulted in a slower rate of DNA fragmentation. An example of how diluting the semen may slow down the rate of post-thaw SDF was found in Ram 6, where the SDF of the lowest sperm concentration (6.3 ⫻ 106
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sperm/mL) remained unchanged ⬃ 10% throughout the entire 6 h incubation period, whereas sperm incubated at 100 ⫻ 106 sperm/mL had an SDF of 60%. Interestingly, the model that we have developed in this study to help explain the frozen-thawed sperm DNA fragmentation dynamics of the ram, predicted that highly concentrated sperm samples frozen and thawed without post-thaw dilution were likely to be more vulnerable to an increased rate of DNA fragmentation; perhaps this phenomenon might help to further explain the relatively poor success of intra-cervical AI in the ewe, which typically employs a significantly more concentrated frozen-thawed sperm sample than that used for laparoscopic AI [29]. In this respect, more sperm may not necessarily be best and an over abundance of sperm may actually be detrimental to the survival of the sperm population as a whole, at least prior to the dilution of the thawed inseminate dose in the female reproductive tract. If the effect of sperm concentration has a direct impact on the rate of DNA damage and this rate is dependent on the animal, it would be interesting to know whether there is an “optimal effective sperm concentration” for each individual ram, in terms of preventing DNA damage following cryopreservation. The standard recommended dose for the use of cryopreserved ram semen in laparoscopic AI is 25 ⫻ 106 sperm/mL [27,30,31]. In the current experiment, the majority of ram semen samples diluted at 25 ⫻ 106 sperm/mL had approximately 50% of sperm with fragmented DNA after 3 h incubation, compared to 12 ⫻ 106 and 6 ⫻ 106 sperm/mL, which had approximately 35 and 30% SDF respectively. Therefore, we inferred that lowering the insemination dose of frozen-thawed sperm may be beneficial to the integrity of sperm DNA, but it remains to be seen whether such a lower concentration of sperm would reduce conception rate. Even though the semen samples used in the current study were all considered to be of high quality prior to cryopreservation, Ram 10 initially had a high level of SDF that rapidly increased within 1 h of incubation. Interestingly, the SDF of this ram was unaffected by dilution, suggesting that the DNA damage was likely to be an inherent feature of this individual animal; one might predict that this ram would have comparatively low fertility. At the other end of the spectrum was Ram 8, which had a consistently lower level of SDF irrespective of sperm concentration; we inferred that Ram 8 had an inherently low level of SDF and consequently might be associated with increased fertility.
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The model we have produced is interesting from a biological perspective, as it suggests that there is some point during in vitro storage where DNA fragmentation accelerates, giving rise to an exponential increase. Perhaps metabolic by-products or toxins that accumulate in the diluent prior to insemination approached a critical concentration after which the sperm DNA rapidly degenerated; this is perhaps best illustrated in the 6.25% dilution for Ram 6. Nevertheless, we might expect a slowing down in the cumulative rate of DNA fragmentation as it approached 100%, simply because there are not many sperm left to fragment at this point. The interesting aspect is that the asymptote seemed to be less than 100% in some cases, suggesting the presence of a more robust subpopulation with a higher tolerance to DNA fragmentation. If these sperm could be effectively identified, harvested and used, they would likely be associated with increased fertility. Using the sperm chromatin structure assay (SCSA), Peris et al [32] assessed the sperm chromatin damage of frozen-thawed sperm from six ejaculates diluted to 50 ⫻ 106 sperm/mL after 6 and 24 h incubation at 37 °C and reported a slight increase in DFI (from approximately 14 to 18%). López-Fernández et al [18] also examined 25 frozen-thawed ejaculates of 10 to 15 ⫻ 106 sperm/mL at 5 and 24 h incubation using the sperm chromatin dispersion assay (as used in the current study), and reported a mean SDF of approximately 65 and 87% respectively. Although it is obvious that different rams were used in these three studies, the results of the current study seemed generally similar to those found by López-Fernández et al [18], and indicate a somewhat disparate assessment of ram sperm DNA fragmentation using the SCD assay and SCSA; this difference requires further examination and validation. Oxidative stress has been implicated as a primary mechanism of DNA fragmentation in sperm [4] and given that cryopreservation has also been shown to increase the level of reactive oxygen species in sperm [6,33], it is likely also a primary cause of DNA fragmentation in cryopreserved sperm [6,34]. Although studies such as Thomson et al [6] have examined oxidative damage immediately after cryopreservation, dead or dying sperm are likely to substantially contribute to ROS generation [35] via membrane alteration and through the loss of active enzymes into the media. The accumulation of toxic metabolic products and active free enzymes such as those found in the acrosome, will ultimately lead to the degeneration of the accompanying surviving sperm; therefore, as sperm degrade,
they should do so cumulatively and in an exponential manner. This phenomenon may help to explain the DNA fragmentation dynamics in the current experiment, as more concentrated sperm were more likely to be exposed to ROS than those incubated at a low concentration. Therefore, we inferred that removal of dead or dying sperm immediately after cryopreservation via swim-up or Percol gradient isolation could be beneficial to sperm DNA integrity. The variable nature of the changing rate of SDF over time and with sperm concentration shown in this study, demonstrated that SDF is not a static concept, and that any assessment of DNA fragmentation must be conducted in the context of a standardized protocol. Therefore, we recommend that DNA fragmentation be assessed under standardized conditions of incubation time and sperm concentration, as a failure to do so will most likely produce erroneous interpretations when results from various laboratories are compared. Due care in the assessment of sperm DNA is particularly important in species with sperm predisposed to high rates of sperm DNA damage, including sheep [18], humans [36], and fish [37]. This effect seems to be less critical in other species, e.g. pigs and cattle, where sperm have much lower rates of DNA fragmentation following cryopreservation [38,39]. The information gained from our experiments will be particularly relevant to those reproductive technologies such as IVF, ICSI, intra-cytoplasmic morphologically selected sperm (IMSI), Physiologic ICSI (PICSI) and XY sperm sorting, all of which require that sperm to be processed for extended intervals and used in low concentration; in these cases, the use of highly diluted semen could reduce the incidence of DNA damage and provide better fertility outcomes. Acknowledgements The authors thank F. Arroyo and A. Gosálbez for their technical help. All frozen straws of ram semen were kindly provided by Ovigen, Zamora, Spain. This work was supported by the Ministry of Education and Science, Spain - Grant BFU 2007-66340/BFI and the Gothenburg Stochastic Center and the Swedish Foundation for Strategic Research, through the Gothenburg Mathematical Modelling Center. References [1] McPherson S, Longo F. Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev 1992;31:268 –79.
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Theriogenology 74 (2010) 1362–1370 www.theriojournal.com
Fragmentation dynamics of frozen-thawed ram sperm DNA is modulated by sperm concentration C. López-Fernándeza, S.D. Johnstonb,*, J.L. Fernándezc, R.J. Wilsond, J. Gosálveza a
Departamento de Biología, Unidad de Genética, Universidad Autónoma de Madrid (UAM). 20849-Madrid. España. b School of Animal Studies, The University of Queensland, Gatton, 4343, Australia c INIBIC-Genética, Complejo Hospitalario Universitario Juan Canalejo, As Xubias, A Coruña, Spain d School of Mathematics and Physics, The University of Queensland, St Lucia, 4072, Australia Received 8 October 2009; received in revised form 1 June 2010; accepted 1 June 2010
Abstract This study investigated the hypothesis that post-thaw incubation of ram sperm at high concentrations results in a faster rate of DNA fragmentation than when sperm are incubated at a lower concentration. Ejaculates from 10 rams were frozen-thawed, prepared in sperm concentrations of 100, 50, 25, 12, and 6 ⫻ 106 sperm/mL, and incubated for 6 h at 37 °C. Sperm DNA fragmentation was assessed using the sperm chromatin dispersion test (Sperm-Halomax®) at 1, 3, 4, and 6 h of incubation at 37 °C. On fitting a binary logistic regression with a cubic over time and treating ram and dilution levels as factors, there were significant effects with respect to the ram, dilution and time (all P-values were very much smaller than 0.001). Therefore, DNA fragmentation dynamics of incubated frozen-thawed ram sperm were not only dependent on the inherent sperm DNA fragmentation expressed immediately after thawing, but also on the concentration of sperm incubated in the sample. Although there was evidence of individual ram variation in SDF during the incubation period, the general finding of the current study was that lower sperm concentrations resulted in a slower rate of DNA fragmentation These findings have important implications for the post-thaw manipulation of ram sperm used for AI and advanced reproductive procedures that use sperm at low concentrations. Our data also emphasised the highly dynamic nature of sperm DNA fragmentation and the importance of conducting the procedure in a standardised manner. © 2010 Elsevier Inc. All rights reserved. Keywords: Sperm DNA fragmentation; Semen cryopreservation; Sheep; Dilution; In vitro incubation
1. Introduction Three major hypotheses have been proposed to explain cellular mechanisms that result in the altered sperm DNA molecule. The first is related to torsional stress in unconstrained DNA supercoils and is a direct consequence of histone-protamine replacement during mid-spermiogenesis [1,2]. The second hypothesis re-
* Corresponding author. Tel.: 617 32026902; fax: 617 33655644. E-mail address: [email protected] (S. Johnston). 0093-691X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.06.006
gards DNA fragmentation as a consequence of oxidative stress in the male reproductive tract [3– 6]. The third hypothesis concerns apoptotic-related DNA strand breakage, similar to that which occurs in abortive apoptosis in somatic cells; the presence of caspase 9 in the midpiece and the occurrence of activated caspases 8, 1 and 3 in the postacrosomal region appeared to support this view [4,6,7]. However, the etiologies of DNA damage are many and varied, ranging from bacterial infections [8], chemical toxicity [9,10], elevated temperature [11], diabetes [12,13], age
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[14,15], body mass [16], and genetic background [17]. Whereas many of the factors resulting in sperm DNA fragmentation are typically unavoidable, certain types of induced iatrogenic sperm DNA damage can become exacerbated when sperm are inappropriately manipulated in the laboratory. For example, we have previously shown how changes in temperature excursions can affect the rate of sperm DNA fragmentation during in vitro incubation [18]. Successful artificial insemination (AI) of frozenthawed semen relies on the delivery of sufficient numbers of sperm into the female reproductive tract, which are morphologically normal, have a functional capacity in terms of motility, and are able to undergo an acrosome reaction and to successfully participate in syngamy. Although numerous studies have examined post-thaw survival characteristics (motility, membrane integrity, and acrosomal status) of incubated mammalian sperm, only recently has there been interest in the effect of post-thaw incubation on sperm DNA fragmentation and the potential importance of this phenomenon to the success of AI programs [19,20]. Cryopreservation induced capacitation-like changes in ram sperm, which resulted in membrane destablisation and cell death [21]; compounding this phenomenon, excessive dilution of sperm and the consequent removal of seminal plasma has also been shown to be associated with a loss of motility and viability [22]. In addition, highly diluted frozen-thawed ram sperm recovered from swim-up procedures had changes in their motility and chlortetracycline staining patterns which were indicative of an increase in the incidence of capacitation [23,24]. A study on canine sperm revealed that both motility and membrane integrity was improved with increasing dilution of the frozen-thawed sample, but that the proportion of reacted acrosomes was similar [25]. In the bull, dilution and incubation of thawed sperm at high and low concentrations at room temperature for 24 h resulted in little difference between these populations in terms of viability or susceptibility to osmotic stress, although sperm preparations with lower sperm concentration had a higher proportion of viable cells with reacted acrosomes [26]. Surprisingly, the effect of dilution on sperm DNA fragmentation of incubated frozen-thawed sperm has not been investigated. Therefore, the objective of the current study was to analyze the effect of sperm concentration and post-thaw incubation on the integrity of ram sperm DNA. This experiment was based on the hypothesis that the rate of DNA fragmentation of frozen-thawed ram sperm is dependent on the initial sperm concentra-
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tion at which the thawed sample was extended. The results of this investigation will provide important insights and practical applications into the way in which thawed ram sperm should be processed and diluted prior to its use in laparoscopic AI or other advanced reproductive technologies. 2. Materials and methods 2.1. Animals, semen collection and sperm cryopreservation Semen was collected by artificial vagina from 10 sexually mature (2– 4 y) rams housed at the Ovigen Centre, Zamora, Spain; these animals were kept under controlled feeding and photoperiodic conditions for maximal reproductive performance and were clinically healthy throughout the semen collection period. In preparation for cryopreservation, the fresh ejaculate was diluted to a final concentration of 1000 ⫻ 106 total sperm in a commercial egg yolk-based extender, (Triladyl; Minitube Canada, Woodstock, ON, Canada) loaded into 0.25 mL PVC straws, and frozen in a programmable freezing chamber (Minitube Iberica, Tarragona, Spain). 2.2. Dilution and incubation of thawed sperm Semen straws were thawed by plunging in a waterbath at 37 °C for approximately 30 to 60 s [27], after which time each sample was immediately diluted in a pre-warmed (37 °C) commercial extender (INRA 96, IMV Technologies, L’Aigle, France) to give a sperm concentration of ⬃200 ⫻ 106 sperm/mL. At this time, the post-thaw survival of the thawed sperm was assessed; only samples with high post-thaw survival (⬎50% normal acrosomes, ⬎35% positive to the HOS test, and ⬎35% progressive motility) were used for subsequent incubation experiments. The semen sample was then allowed to cool to 15 °C over 30 min to reduce metabolism prior to being centrifuged (300 ⫻ g for 10 min); this step was included to remove the potential harmful influence of cryomedia which may have inadvertently affected DNA integrity during incubation. The supernatant was gently removed and resuspended with fresh diluent (INRA 96) to provide a standardised working sperm concentration of 100 ⫻ 106 sperm/mL (C100). Sperm DNA fragmentation (SDF) was assessed at this stage of post-thaw incubation and designated time T0. This diluted semen sample was subsequently serially diluted in INRA 96 to produce sperm concentrations of 50 (C50), 25 (C25), 12 (C12), and 6
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Fig. 1. Ram sperm DNA fragmentation following the sperm chromatin dispersion test [18]. (a) Ram sperm showing absence of sperm DNA damage—all sperm have a small halo of chromatin dispersion; (b) The sperm chromatin dispersion after 6 h of incubation. Note large haloes of dispersed chromatin representing sperm with fragmented DNA.
(C6) ⫻ 106 sperm/mL; these sperm populations were then incubated at 37 °C in 5% CO2 for 6 h and SDF assessed at 1 (T1), 3 (T3), 4 (T4), and 6 h (T6) of incubation. 2.3. Assessment of sperm DNA fragmentation The incidence of SDF throughout the incubation period was assessed by removing an aliquot of the semen sample and further diluting it in INRA 96 to a concentration of approximately 10 ⫻ 106 sperm/mL; this dilution step was conducted immediately before processing the sperm for the Halomax assay, to ensure that the semen sample was not overly concentrated for microscopic analysis. Sperm DNA fragmentation was assessed using the sperm chromatin dispersion test as previously described and validated for the ram ([18]; Sperm-Halomax®; Halotech DNA, Madrid, Spain). Following the SCD procedure, fragmented and nonfragmented sperm within the microgel were stained with a dual emission DNA-blue/Protein-green fluorochrome combination. Nucleoids were stained using the DNA-specific stain, 4=,6-diamidine-2= phenylindole dihydrochloride (DAPI; Sigma Aldridge, Irvine, UK), whereas 2,7-dibromo-4-hydroxy mercurifluorescein (Sigma Aldridge, Irvine, UK) was used for protein staining. Ram sperm with fragmented DNA were identified as
large fluorescent halos of dispersed chromatin, whereas those with non-fragmented DNA had small fluorescent halos and a compact nuclear core (Fig. 1). Images were captured using a black-and-white cooled Leica DCF 300 camera mounted onto an epifluoresencence Motic A-400 microscope using a single band pass filter (Cy54040A-492/516; Semrock, Rochester, NY, USA). Three hundred sperm were observed for SDF and the number of fragmented sperm reported as a percentage. 2.4. Statistical analysis As the objective of this study was to identify systematic changes in the expected (cumulative) rate of SDF as dilution increases, a binary logistic cubic regression over time [28] was fitted to the percentage of fragmented cells in each ram semen sample over time and where each sample consisted of an assessment of 300 cells. The logistic regression model used assumed that the probability of SDF for a single cell by a given time depended on the ram, the dilution factor and time, with the relationship with time being a cubic in time, with the distribution assumed for the number of cells to fragment for each combination of ram, dilution and time to be the binomial distribution. For each ram, a common initial expected rate of SDF was assumed for all dilutions, as the pre-incubation fragmentation of
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Fig. 2. Observed changes in % SDF of ram sperm following post-thaw incubation of samples at various sperm concentrations (6, 12, 25, 50, and 100 ⫻ 106 sperm/mL) for 6 h at 37 °C.
each individual ram was the same. Using the most diluted concentration as a base cubic, parameterisation was defined in terms of differences of dilution effect on the coefficients of the terms in the cubic polynomials over time as dilution decreased. This allowed for a backwards elimination method to be used to reduce the dilution effects in the cubic models over time and so identify significant differences in the expected cumulative rate as dilution decreased. Consequently, it could be determined if dilution had a significant effect on the expected rate of SDF and in which direction such an effect might be. The full binary logistic cubic regression over time over all dilutions and rams was used to evaluate whether there were significant differences in expected rate of SDF over rams, dilution and time.
3. Results The effects of post-thaw dilution and incubation on ram sperm DNA fragmentation for each ram are shown (Fig. 2). The SDF dynamics of all individual rams showed a generalised consistent pattern of dynamics whereby the rate of SDF was lower in samples with lower sperm concentration. In addition to fitting a binary logistic regression with a cubic over time and treating ram ID and dilution levels as factors (see Fig. 3 for two examples), there were significant effects with respect to the ram, dilution and time (all P-values were very much smaller than 0.001). As there were significant differences from ram to ram in the pattern of differences over time and dilutions (i.e., the interactions
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Fig. 3. Comparison of observed (raw) and fitted (model) changes in % SDF of ram sperm (Rams 4 and 8) following post-thaw incubation of samples at various sperm concentrations (6, 12, 25, 50, and 100 ⫻ 106 sperm/mL) for 6 h at 37 °C.
between ram, dilution and time), the results for each ram need to be considered separately in order to determine the nature of the relationship of fragmentation to dilution over time. Backward elimination (with a 5% significance level) was used to remove non-significant terms in the binary logistic full cubic model over time, to determine which dilutions were not significantly different over time for each ram, and where lower order polynomials could be used in part of the model. For some rams, the cubic was reduced to a lower order polynomial for the base polynomial (see results for the C6 concentration). Results indicating which dilutions gave non-significantly dif-
ferent patterns of SDF and those that were significantly different are shown in Table 1 (the P-values provided some indication of the significance of the eliminated terms for that ram), with the corresponding fitted SDF values predicted by the model. As described in Figure 2 and from the results of the statistical analysis in Table 1, there was variability in the pattern of SDF over time and dilution by ram. Despite this variation, and apart from the results for Ram 8, there was a non-strict ordering by dilution, so that each incubation time and for each ram, the amount of DNA fragmentation typically did not increase as the dilution increases. Furthermore, apart from the results
Table 1 Results of backward elimination for the binary logistic regression to determine the effect of sperm concentration on the integrity of ram sperm DNA. Ram
Non-significantly different pairs of dilutions
Significantly different pairs of dilutions
P-value for eliminated terms
1 2 3 4 5 6 7 8 9 10
(C6,C12) (C25,C100) (C25,C50) (C50,C100) — (C25,C50) (C25,C50) — (C50,C100) — (C50,C100) (C6,C12) (C12,C25) (C25, C50) (C50, C100)
(C12,C25) (C25,C50) (C6,C12) (C12,C25) (C6,C12) (C12,C25) (C25,C50) (C50,C100) (C6,C12) (C12,C25) (C50,C100) (C6,C12) (C12,C25) (C50,C100) (C6,C12) (C12,C25) (C25,C50) (C50,C100) (C6,C12) (C12,C25) (C25,C50) (C6,C12) (C12,C25) (C25,C50) (C50,C100) (C6,C12) (C12,C25) (C25,C50) —
0.2378 0.6612 0.1196 0.1301 0.4572 0.5118 0.1441 0.7168 0.8099 0.4423
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reported for Ram 10, there was at least one time in the observation period for which the amount of DNA fragmentation decreased significantly as the dilution increased, although the dilution and time for which this occurs varied for each ram. Two exceptions to the general pattern of observations were found in Rams 8 and 10. Ram 8 appeared to have a slower expected rate of SDF for the straw concentration (C100) than for the other concentrations, suggesting that it was an outlier when compared the majority of other rams in this study. There were no significant differences at any times between the expected rate of SDF for all dilutions of semen from Ram 10 (Fig. 2). Given the variation in patterns in SDF across the rams, it is worthwhile highlighting the results of the individual rams in further detail. In seven of the 10 rams (R1, R2, R4, R5, R7, R9, and R10), the final SDF after 6 h incubation for most sperm concentrations appeared to be similar, except for the lowest sperm concentration (C6) which typically had lower SDF (exceptions were R1 and R2). The SDF of the C6 dilution in Rams 4, 5, 7, and 9 after 6 h incubation was substantially lower than that for samples with higher sperm concentrations incubated for the same time period. The rate of SDF in Rams 3 and 6 showed a slightly different pattern, in that there was a larger range of SDF observed across the various dilutions after 6 h incubation. This phenomenon was particularly illustrated in Ram 6, where the SDF of C6 after 6 h incubation was the same as that reported at the commencement of the incubation period. The sperm of Rams 6 and 8 also had lower values of fitted SDF across all dilutions after 6 h incubation ranging from 7– 63 and 41– 64 respectively, when compared to the other rams (Rams 3, 4, 5, 7, and 8) which collectively ranged from 63–90. 4. Discussion In this study, DNA fragmentation dynamics of incubated frozen-thawed ram sperm were not only dependent on the inherent sperm DNA fragmentation expressed immediately after thawing, but also on the concentration of sperm incubated in the sample over time. Although there was evidence of individual ram variation in SDF during the incubation period, the general finding was that lower sperm concentrations resulted in a slower rate of DNA fragmentation. An example of how diluting the semen may slow down the rate of post-thaw SDF was found in Ram 6, where the SDF of the lowest sperm concentration (6.3 ⫻ 106
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sperm/mL) remained unchanged ⬃ 10% throughout the entire 6 h incubation period, whereas sperm incubated at 100 ⫻ 106 sperm/mL had an SDF of 60%. Interestingly, the model that we have developed in this study to help explain the frozen-thawed sperm DNA fragmentation dynamics of the ram, predicted that highly concentrated sperm samples frozen and thawed without post-thaw dilution were likely to be more vulnerable to an increased rate of DNA fragmentation; perhaps this phenomenon might help to further explain the relatively poor success of intra-cervical AI in the ewe, which typically employs a significantly more concentrated frozen-thawed sperm sample than that used for laparoscopic AI [29]. In this respect, more sperm may not necessarily be best and an over abundance of sperm may actually be detrimental to the survival of the sperm population as a whole, at least prior to the dilution of the thawed inseminate dose in the female reproductive tract. If the effect of sperm concentration has a direct impact on the rate of DNA damage and this rate is dependent on the animal, it would be interesting to know whether there is an “optimal effective sperm concentration” for each individual ram, in terms of preventing DNA damage following cryopreservation. The standard recommended dose for the use of cryopreserved ram semen in laparoscopic AI is 25 ⫻ 106 sperm/mL [27,30,31]. In the current experiment, the majority of ram semen samples diluted at 25 ⫻ 106 sperm/mL had approximately 50% of sperm with fragmented DNA after 3 h incubation, compared to 12 ⫻ 106 and 6 ⫻ 106 sperm/mL, which had approximately 35 and 30% SDF respectively. Therefore, we inferred that lowering the insemination dose of frozen-thawed sperm may be beneficial to the integrity of sperm DNA, but it remains to be seen whether such a lower concentration of sperm would reduce conception rate. Even though the semen samples used in the current study were all considered to be of high quality prior to cryopreservation, Ram 10 initially had a high level of SDF that rapidly increased within 1 h of incubation. Interestingly, the SDF of this ram was unaffected by dilution, suggesting that the DNA damage was likely to be an inherent feature of this individual animal; one might predict that this ram would have comparatively low fertility. At the other end of the spectrum was Ram 8, which had a consistently lower level of SDF irrespective of sperm concentration; we inferred that Ram 8 had an inherently low level of SDF and consequently might be associated with increased fertility.
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The model we have produced is interesting from a biological perspective, as it suggests that there is some point during in vitro storage where DNA fragmentation accelerates, giving rise to an exponential increase. Perhaps metabolic by-products or toxins that accumulate in the diluent prior to insemination approached a critical concentration after which the sperm DNA rapidly degenerated; this is perhaps best illustrated in the 6.25% dilution for Ram 6. Nevertheless, we might expect a slowing down in the cumulative rate of DNA fragmentation as it approached 100%, simply because there are not many sperm left to fragment at this point. The interesting aspect is that the asymptote seemed to be less than 100% in some cases, suggesting the presence of a more robust subpopulation with a higher tolerance to DNA fragmentation. If these sperm could be effectively identified, harvested and used, they would likely be associated with increased fertility. Using the sperm chromatin structure assay (SCSA), Peris et al [32] assessed the sperm chromatin damage of frozen-thawed sperm from six ejaculates diluted to 50 ⫻ 106 sperm/mL after 6 and 24 h incubation at 37 °C and reported a slight increase in DFI (from approximately 14 to 18%). López-Fernández et al [18] also examined 25 frozen-thawed ejaculates of 10 to 15 ⫻ 106 sperm/mL at 5 and 24 h incubation using the sperm chromatin dispersion assay (as used in the current study), and reported a mean SDF of approximately 65 and 87% respectively. Although it is obvious that different rams were used in these three studies, the results of the current study seemed generally similar to those found by López-Fernández et al [18], and indicate a somewhat disparate assessment of ram sperm DNA fragmentation using the SCD assay and SCSA; this difference requires further examination and validation. Oxidative stress has been implicated as a primary mechanism of DNA fragmentation in sperm [4] and given that cryopreservation has also been shown to increase the level of reactive oxygen species in sperm [6,33], it is likely also a primary cause of DNA fragmentation in cryopreserved sperm [6,34]. Although studies such as Thomson et al [6] have examined oxidative damage immediately after cryopreservation, dead or dying sperm are likely to substantially contribute to ROS generation [35] via membrane alteration and through the loss of active enzymes into the media. The accumulation of toxic metabolic products and active free enzymes such as those found in the acrosome, will ultimately lead to the degeneration of the accompanying surviving sperm; therefore, as sperm degrade,
they should do so cumulatively and in an exponential manner. This phenomenon may help to explain the DNA fragmentation dynamics in the current experiment, as more concentrated sperm were more likely to be exposed to ROS than those incubated at a low concentration. Therefore, we inferred that removal of dead or dying sperm immediately after cryopreservation via swim-up or Percol gradient isolation could be beneficial to sperm DNA integrity. The variable nature of the changing rate of SDF over time and with sperm concentration shown in this study, demonstrated that SDF is not a static concept, and that any assessment of DNA fragmentation must be conducted in the context of a standardized protocol. Therefore, we recommend that DNA fragmentation be assessed under standardized conditions of incubation time and sperm concentration, as a failure to do so will most likely produce erroneous interpretations when results from various laboratories are compared. Due care in the assessment of sperm DNA is particularly important in species with sperm predisposed to high rates of sperm DNA damage, including sheep [18], humans [36], and fish [37]. This effect seems to be less critical in other species, e.g. pigs and cattle, where sperm have much lower rates of DNA fragmentation following cryopreservation [38,39]. The information gained from our experiments will be particularly relevant to those reproductive technologies such as IVF, ICSI, intra-cytoplasmic morphologically selected sperm (IMSI), Physiologic ICSI (PICSI) and XY sperm sorting, all of which require that sperm to be processed for extended intervals and used in low concentration; in these cases, the use of highly diluted semen could reduce the incidence of DNA damage and provide better fertility outcomes. Acknowledgements The authors thank F. Arroyo and A. Gosálbez for their technical help. All frozen straws of ram semen were kindly provided by Ovigen, Zamora, Spain. This work was supported by the Ministry of Education and Science, Spain - Grant BFU 2007-66340/BFI and the Gothenburg Stochastic Center and the Swedish Foundation for Strategic Research, through the Gothenburg Mathematical Modelling Center. References [1] McPherson S, Longo F. Localization of DNase I-hypersensitive regions during rat spermatogenesis: stage-dependent patterns and unique sensitivity of elongating spermatids. Mol Reprod Dev 1992;31:268 –79.
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