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Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa
Theriogenology 67 (2007) 1150–1157 www.theriojournal.com
Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa B.D. Corcuera a, P. Marigorta b, A. Sagu¨e´s a, F. Saiz-Cidoncha b, J.F. Pe´rez-Gutie´rrez c,* a Kubus, S.A., Polı´gono Industrial Euro´polis C/E, n20, Las Rozas, 28230 Madrid, Spain Departamento de Reproduccio´n Animal, INIA, Ctra. de la Corun˜a km 5.9, 28040 Madrid, Spain c Departamento de Medicina y Cirugı´a Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Av. Puerta de Hierro s/n, 28040 Madrid, Spain b
Received 12 June 2006; received in revised form 3 January 2007; accepted 14 January 2007
Abstract The effect of lactose and glycerol concentration, as well as the equilibration time with glycerol was studied on motility, normal apical ridge (NAR), and chromatin state of boar spermatozoa after the freezing and thawing process. In the first experiment, samples were frozen in first and second extenders containing different concentrations of lactose (11, 12 and 14%). In the second experiment samples were frozen using second extenders with different concentrations of glycerol (4, 6, 8 and 10%) and were incubated at 5 8C for 0 and 30 min. Motility, motility after caffeine treatment, NAR, chromatin condensation and stability (susceptibility to decondense after heparin treatment) were evaluated. The results indicated that freezing spermatozoa in extenders with increasing concentrations of lactose adversely affected motility but provided a protective effect on acrosomes. Increased lactose concentration induced higher chromatin condensation but maintained the same stability. Increasing the glycerol concentration in the second extender from 4–6 to 8% led to higher motility and NAR as well as lower chromatin condensation and stability. When 30 min equilibration time was allowed after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. The longer equilibration time was detrimental for motility when freezing in the 8% glycerol extender but favourable when using the 4% glycerol extender. Compared to the 8% glycerol, spermatozoa frozen in the 10% glycerol extender showed similar motility and increased chromatin condensation and stability, as well as low values of NAR that did not improve by longer incubation time. # 2007 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Cryoprotectants; Sperm motility; Chromatin; Porcine
1. Introduction The freezing and thawing of boar spermatozoa causes considerable cell damage leading to severe
* Corresponding author. Tel.: +34 913943798; fax: +34 913943808. E-mail address: [email protected] (J.F. Pe´rez-Gutie´rrez). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.01.002
reductions in farrowing rates and litter size after artificial insemination with frozen semen [1]. A number of mechanisms have been proposed to explain injury to spermatozoa following freezing and thawing. It has been proposed that upon freezing, ice forms first in the extracellular medium resulting in hyperosmolal concentration of solutes outside the cell, pH changes and solute precipitation that could cause extreme cell
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shrinkage up to irreversible membrane damage [2,3]. Intracellular ice crystal formation can lead to mechanical damage of the plasma membrane and organelles causing oxidative stress as well as alterations in sperm metabolism [2–4]. During the last decades, many studies have focused on preventing cryodamage by using cryoprotective agents [5–12]. Non-permeable cryoprotectants such as sugars protect the membrane from the volume changes the cell experience upon freezing and thawing. Permeable cryoprotectants such as glycerol is very effective in lowering the intracellular water freezing point and reduce intracellular ice crystal formation. The combination of both types of cryoprotectants is commonly used. It has been shown that freezing at moderately hypertonic conditions can improve the survival of bull [6,7], ram [8,9] and boar [5,10,11] spermatozoa. In addition, it has been reported that reducing the exposure time to glycerol can improve bull spermatozoa motility and NAR upon freezing and thawing procedure [6,7]. However, the effects of the glycerol equilibration time on boar spermatozoa have shown different results [12,13]. Most of these studies have evaluated post thaw viability by assessing motility, membrane and acrosome integrity. However, there are other parameters related to fertility such as chromatin condensation and stability that could also be evaluated [14,15]. Previous studies in human, bovine and porcine species have shown that sperm chromatin can undergo important changes after the freezing–thawing procedure [15–18]. Since spermatozoa undergo sustained swelling or shrinkage when exposed to hypotonic or hypertonic solutions [19–21], it is possible that the osmolality of freezing media could alter boar sperm chromatin state. Up to our knowledge there are no studies related to the effect of cryoprotectants on boar chromatin state. The aim of this study was to evaluate the effect of extenders with different concentrations of lactose and glycerol, varying in osmolality, as well as the prefreezing exposure time to glycerol on boar sperm motility, NAR and chromatin state upon freezing and thawing. 2. Material and methods 2.1. Experimental design Two independent experiments were performed with two different sets of ejaculates, one to study the effect of lactose (experiment 1) and another one to study the
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effect of glycerol (experiment 2). For experiment 1, samples were subjected to three different treatments (11, 12 and 14% lactose). For experiment 2, samples were subjected to eight different treatments (4, 6, 8, 10% glycerol, incubated for 0 and 30 min). For each experiment, 10 ejaculates were collected from each of 5 boars, giving a total of 50 ejaculates. Each ejaculate was divided into identical fractions that were subjected to the different treatments. For each experiment, the 50 samples were frozen and, after thawing, they were pooled as follows: the 5 samples corresponding to the first ejaculates of the 5 boars, the 5 samples corresponding to the second ejaculates of the 5 boars, . . ., the 5 samples corresponding to the tenth ejaculates of the 5 boars. Therefore, for each treatment we obtained 10 pooled samples, each one being a mixed sample of the 5 boars. Each pooled sample was analyzed once. 2.2. Semen collection Semen was collected on a weekly basis, from five Large White boars, aged 1.5–2 years, whose fertility had been previously proven. The boars were housed in individual pens at the Instituto Nacional de Investigaciones Agrarias (Madrid, Spain) and fed with a commercial standard diet. The ejaculates (only the sperm rich fraction) were obtained by the gloved hand method [22]. Gel particles were removed by filtration through a sterile gauze [23]. A subsample from each ejaculate was taken for preliminary motility and NAR evaluation [24]. All samples were evaluated by only one technician. Ejaculates showing motility higher than 80%, and NAR higher than 70% were used. The rest of the ejaculate was processed and subjected to each of the freezing treatments in 5 mL straws. 2.3. Semen freezing The freezing procedure involved the dilution with extenders of different concentrations of lactose and glycerol as described in experiments 1 and 2. 2.3.1. Experiment 1 The concentration of lactose was changed in both the first and the second extenders. The first extender was composed of 20% hen egg yolk and varying concentrations of lactose: 11% (Ext. 1a), 12% (Ext. 1b) and 14% (Ext. 1c). The osmolality for groups 1a, 1b and 1c was 320, 360 and 420 mOsm/kg, respectively. Osmolality was determined by using a freezing-point depression osmometer (Model 3D2, Advanced Instruments, Need-
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ham Heights, MA). The second extender was composed of 20% hen egg yolk, 4% glycerol, 0.5% lauryl sodium sulfate and varying concentrations of lactose: 11% (Ext. 2a), 12% (Ext. 2b) and 14% (Ext. 2c). The osmolality for groups 2a, 2b and 2c was 1000, 1080 and 1150 mOsm/kg, respectively. Ext. 1a, 1b and 1c were used with Ext. 2a, 2b and 2c, respectively. 2.3.2. Experiment 2 The first extender (Ext. 1a) was used with second extenders with different glycerol contents. The composition of the second extenders was 11% lactose, 20% egg yolk, 0.5% lauryl sulfate and varying concentrations of glycerol: 4, 6, 8 and 10%, with osmolalities of 1000, 1500, 1880 and 2320 mOsm/kg, respectively. For these samples, incubation with the second extender was done at 5 8C for 0 and 30 min. The semen was processed and frozen by the straw freezing method, originally described by Westendorf et al. [25] with some modifications. After collection, the sperm rich fraction was diluted 1:4 with a commercial extender (MR-A1 diluent, Kubus, S.A., Madrid, Spain) at 23 8C. After 1 h equilibration at 23 8C, each ejaculate was divided into doses containing 6 109 spermatozoa (at a concentration of 6 108 spermatozoa/mL) to be subjected to each of the treatments, three for experiment 1 and eight for experiment 2. Doses were kept at 15 8C for 3 h and centrifuged at 800 g for 10 min. After supernatant removal the concentrated semen was diluted to 5 mL at 15 8C with the first extender. The diluted semen was gently mixed and cooled at 5 8C for 1.5 h. Samples were further diluted (1:2) to 10 mL at 5 8C with the second extender. In the experiment 1, doses were immediately packaged whereas in the experiment 2, doses were incubated with the second extender for 0 and 30 min at 5 8C before being packaged in 5 mL straws. Straws were manually sealed with metallic balls, wiped dry and the air bubble was brought to the center of the straw. Finally, straws were placed in contact with nitrogen vapor, about 3 cm above the nitrogen liquid level for 20 min, and plunged into the liquid nitrogen tank and stored until used. 2.4. Thawing Thawing of the straws was carried out after 12 months of storage in liquid nitrogen. Straws were thawed in a water bath at 42 8C for 40 s. Immediately after thawing, the five seminal doses from the different boars that were subjected to the same treatment were pooled. Ten heterospermic groups per treatment were examined, after 15 min of incubation at room temperature.
2.5. Semen evaluation 2.5.1. Concentration and sperm motility assessment Sperm concentration was estimated using a Bu¨rker counting chamber. One millilitre of semen was diluted (1:100) in a 40% formaldehyde physiological solution. One drop of this dilution was placed into the Bu¨rker chamber and the number of spermatozoa was assessed under the microscope (40) by counting the number of cells found inside 40 small squares. Total sperm motility was assessed by placing a sample on a prewarmed microscope slide (42 8C) overlaid with a coverslip and determining, under a light microscope (20), the percentage of spermatozoa that showed movement of the flagellum. Motility with caffeine was determined as described previously after adding a drop of isotonic sodium citrate solution containing 0.1% caffeine to the diluted semen sample [26]. 2.5.2. Assessment of NAR The evaluation of acrosomal integrity was done by examining citrate:formol (1:10) fixed samples with a phase contrast microscope, at 100. A minimum of 100 acrosomes was examined per sample. Acrosomal damage was evaluated according to Pursel et al. [27]. 2.5.3. Analysis of sperm chromatin condensation and stability The chromatin was assessed as chromatin condensation and chromatin stability. Degree of chromatin condensation was determined by using a radioisotope method based on the specific affinity of tritium labelled actinomicyn D (3H-AMD) to bind the base pair guanine-cytosine [28]. 3H-AMD binding to chromatin was estimated by the total counts taken up by the spermatozoa, according to Strzezek et al. [29]. The increase in total counts is related to higher 3H-AMD uptake and, therefore, to decreased chromatin compactness. Chromatin stability was assessed by determining chromatin condensation after inducing decondensation with heparin. Samples were prepared by resuspending thawed semen in 250 mM Tris-citrate-fructose buffer to a final concentration of 50 106 spermatozoa/mL in each sample. After centrifugation at 12,500 g for 15 min, the supernatant was discarded. For determining chromatin condensation, each pellet was resuspended in 10 mL Tris-citrate buffer with 0.1 mCi/mL of 3HAMD (Amersham International, Amersham, UK). For determining chromatin stability, pellets were resuspended in 10 mL of the same buffer containing 200 IU
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Table 1 Sperm motility, NAR, chromatin condensation and stability of frozen boar semen in first extenders with different concentrations of lactose and second extenders with the same concentrations of lactose and 4% glycerol Lactose level (%)
11 12 14
Motility (%)*
a
23.3 5.1 28.6 6.4 b 22.9 7.0 a
Motility with caffeine (%)* a
40.0 7.9 42.9 6.0 a 35.7 9.4 b
NAR (%)*
Chromatin 3H-AMD uptake (cpm)** Heparin (for condensation)
a
35.4 8.7 39.4 4.5 b 40.6 7.7 b
a
28.5 2.4 22.5 4.1 b 23.9 2.1 b
+ Heparin (for stability) 33.3 5.7 33.0 2.5a 30.7 3.0a
Values are expressed as means S.D. (n = 10). Values within each column with different superscripts (a and b) are significantly different (P < 0.05). Units in percentage and related to the total number of spermatozoa (*) or in total counts (cpm 103) taken up by 50 106 spermatozoa incubated in the absence (for chromatin condensation) or presence of heparin (for chromatin stability) (**). Smaller counts values mean higher chromatin compactness.
of heparin. All samples were gently shaken, transferred to glass tubes and incubated at 37 8C. One-millilitre solutions were taken at 0 and 60 min and centrifuged (12500 g, 10 min). Three aliquots of 0.1 mL were taken from each supernatant and placed in scintillation vials. Subsequently, 9 mL of scintillation fluid was added to each sample. Counts were obtained from a liquid scintillation counter B 1215 Rachbeta II (LKD Wallac, Turku, Finland) and corrected for background and the appropriate dilution factor. Results were presented as the total counts taken up by 50 106 spermatozoa, obtained from the difference in cpm of the supernatants corresponding to the sample incubated for 60 and 0 min.
motility, motility after caffeine treatment, NAR, chromatin condensation and stability (experiment 1): Y i jk ¼ M þ Ri þ J j þ Oi j þ ei jk where Yijk is the observation; M the overall mean of the population; Ri the random effect of the repetition (i = 1, 2, . . ., 10); Jj the fixed effect of the lactose concentration in the first extender (j = 1, 2, 3); Oij the effect of the interaction within the previously described factors; eijk is the residual effect. The effect glycerol concentration as well as the equilibration time with glycerol on the sperm motility, NAR and chromatin state (experiment 2) were analyzed according to the following model:
2.6. Statistical analysis Y i jkl ¼ M þ Ri þ J j þ Pk þ Oi j þ T ik þ S jk þ OTSi jk The following model was used to test the effects of different concentrations of lactose on the sperm
þ ei jkl
Table 2 Sperm motility, NAR, chromatin condensation and stability of frozen boar semen in a first extender with 11% lactose and second extenders with the same concentration of lactose and different concentrations of glycerol, after 0 and 30 min of incubation with the second extender at 5 8C Glycerol level (%)
Motility (%) *
Motility with caffeine (%) *
NAR (%)*
0
4 6 8 10
28.6 8.3 ad 27.1 7.0 ad 40.0 9.7 bd 35.7 7.3 bd
32.9 9.8 ad 33.0 7.5 ad 45.7 7.3 bd 41.4 6.4 bd
30
4 6 8 10
24.3 4.9 ad 30.0 7.6 bd 30.7 5.3 be 28.6 3.5 ae
37.1 6.9 ae 30.9 5.3 bd 35.7 4.9 ae 35.8 5.3 ae
Glycerol exposure time (min)
Chromatin 3H-AMD uptake (cpm) ** + Heparin (for condensation)
Heparin (for stability)
25.7 4.7ad 24.0 7.2ad 15.3 4.0bd 15.0 3.7bd
21.6 2.7ad 20.8 8.6ad 26.4 7.6bd 23.8 3.2ad
30.4 7.2ad 32.9 6.4ad 38.1 5.9bd 30.6 5.2ad
31.1 7.3ae 22.3 3.7bd 25.4 5.4be 13.4 4.0cd
24.0 2.3ad 24.5 2.5ad 25.0 5.7ad 26.2 7.1ad
36.2 2.9ae 37.6 4.1ae 39.9 6.6ad 29.8 4.8bd
Values are expressed as means S.D. (n = 10). Different supercripts (a–c) denote significant statistical difference (P < 0.05) within the same incubation time. Different superscripts (d, e) denote significant statistical difference (P < 0.05) between different incubation times. Units in percentage and related to the total number of spermatozoa (*) or in total counts (cpm 103) taken up by 50 106 spermatozoa incubated in the absence (for chromatin condensation) or presence of heparin (for chromatin stability) (**). Smaller values of counts mean higher chromatin compactness.
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where Yijkl is the observation; M the overall mean of the population; Ri the random effect of the repetition (i = 1, 2, . . ., 10); Jj the fixed effect of the glycerol concentration in the second extender (j = 1, 2, 3, 4); Pk the fixed effect of the incubation time with glycerol at 5 8C (k = 1, 2); Oij, Tik, Sjk the effect of the interaction of two factors; OTSijk the effect of the interaction of three factors; eijkl is the residual effect. Data were first analyzed by Harvey’s least squares maximum likelihood analysis of variance [30]. The least squares means and standard deviations of untransformed percentages are presented in the Tables 1 and 2. For the statistical analysis, percentages values were subjected to arcsim transformation before applying the unpaired student t-test to means and standard deviations for each datum point [31]. Results from the first and second experiments were analyzed by one-way analysis of variance using SAS software [32]. A probability level of P < 0.05 was considered to be statistically significant. 3. Results 3.1. Effect of the different concentrations of lactose on motility, NAR and chromatin state The results obtained for the experiment 1 (Table 1) showed that immediately after thawing, the motility of spermatozoa frozen in the 12% lactose extender was significantly higher than spermatozoa frozen in either 11 or 14% lactose extenders. When motility was evaluated after the caffeine treatment, spermatozoa frozen in 14% lactose extender showed significantly lower motility than those frozen in 11 or 12% lactose extenders. Spermatozoa frozen in 14% lactose extender showed significantly lower motility values in presence and absence of caffeine. The percentage of spermatozoa showing normal apical ridge was significantly higher in those groups frozen in the 12 and 14% lactose extenders compared to those frozen in the 11% lactose extender. Analysis of chromatin condensation on boar spermatozoa frozen in 11% lactose extender showed significantly higher total counts compared to those spermatozoa frozen in either 12 or 14% lactose extenders. An increase in total counts, related to easier accessibility of DNA, was found after the exposure to heparin. No significant differences (P > 0.05) in chromatin stability were observed among spermatozoa frozen in extenders containing different lactose concentrations.
3.2. Effect of the different concentrations of glycerol on motility, NAR and chromatin state The results obtained for the experiment 2 (Table 2) showed significantly higher post-thaw motility in spermatozoa frozen immediately (0 min) after dilution in 8 and 10% than those frozen after dilution in 4 and 6% glycerol extenders. As previously observed in experiment 1, the caffeine treatment induced an increase in post thaw sperm motility values. The results were similar in presence or absence of caffeine. The samples frozen in 8 and 10% glycerol showed a significantly higher post-thaw motility than those frozen in 4 and 6% glycerol extenders. The integrity of the apical ridge was affected by the concentration of glycerol of the extender when samples were frozen immediately after the addition of glycerol. The percentage of spermatozoa showing normal apical ridge was higher when diluted in 4 and 6% than in 8 and 10% glycerol extenders. When comparing post-thaw motility among incubation times (0 and 30 min), it was observed that increasing incubation time reduced the post thaw motility of those spermatozoa frozen in 8 and 10% glycerol. These results were similar in caffeine treated and non-treated samples. Increasing the time of exposure to 6% glycerol did not affect motility parameters. However, at 4% glycerol, motility increased in the presence of caffeine. The post thaw NAR values either were not altered or significantly increased after 30 min of exposure to glycerol, with respect to those spermatozoa frozen immediately after dilution with glycerol. When analyzing the chromatin state it was observed that spermatozoa frozen immediately after dilution in the 8% glycerol extender showed higher counts, thus less condensed chromatin, than frozen in lower glycerol concentrations. After heparin treatment, significantly higher counts were also observed in spermatozoa frozen in 8% glycerol than in lower concentrations, indicating that the former had less stable chromatin. A further increase of the concentration to 10% caused more condensed and more stable chromatin. When the time of exposure to glycerol was 30 min no significant differences on counts related to chromatin condensation or stability were found among spermatozoa frozen in 4, 6 and 8% glycerol. These values were close to those obtained at 0 min for 8% glycerol. Furthermore, when the concentrations of glycerol were 4 and 6%, a significant change towards less stable chromatin was observed when increasing the incubation time with glycerol from 0 to 30 min. On the other hand,
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higher concentrations (10%) of glycerol resulted in more stable chromatin regardless the incubation time. 4. Discussion 4.1. Effect of cryoprotectants on frozen sperm motility and NAR Among the lactose concentrations used in this study, the best motility results were obtained when freezing in 12% lactose extenders while higher concentrations (14%) showed a negative effect. This agrees with previous studies that have reported that high levels of lactose exert a detrimental effect on motility [33–35]. It was also shown that a number of spermatozoa apparently immotile had actually the potential to move as evidenced by the caffeine treatment. The causes of this reversible loss of motility are not clear. It has been shown that the reversible loss of motility may be due to temporary metabolic failure leading to changes in ATP content and calcium uptake that are highly correlated with motility [11,36]. A reduction of intracellular water content could also be the cause of the reversible inhibition of motility since it could increase the friction in tail causing an inhibition of sliding of microtubules or other structural elements in the flagellum [37,38]. On the other hand, increasing the lactose concentration from 11 to 12 and 14% improved NAR values. This agrees with previous studies that have reported the protective effect of lactose on the acrosome membrane of boar spermatozoa [33–35], that could be due to the formation of hydrogen bonds with the phosphate groups on membrane phospholipids [38,39]. This study confirms that high levels of lactose exert a negative effect on motility but a positive effect on the proportion of normal acrosomes [33–35]. Among the conditions used, 12% lactose showed the best results for motility and NAR. Results showed that increasing concentration of glycerol improved the post thaw motility of boar spermatozoa but decreased the percentage of normal acrosomes. The best glycerol concentrations for motility were 8 and 10% whereas for acrosomal integrity (NAR) were 4 and 6%. These results corroborates previous dose dependent studies that have shown that increasing glycerol concentration in the range of 0 to 7% improved motility but negatively affected acrosome integrity [40,41]. However, they differ from Bamba and Cran [42] that have observed, in addition to the negative effect on NAR, a decrease in motility when glycerol exceeded 6%. These differences could be attributed to differences in the experimental
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procedures, since it has been shown that the concentration of glycerol required for optimum motility and NAR are determined by several factors including the cooling rate and the temperature and time of exposure to glycerol [38,43]. In general terms, increasing the glycerol exposure time to 30 min either did not alter or was beneficial for motility at lower glycerol concentration (4–6%) but was detrimental for higher concentrations (8–10%). The 30 min exposure to glycerol either improved or maintained NAR values. These results agree with previous results which showed a beneficial effect of increasing the exposure time to glycerol (0–6%) for motility and NAR [13,44]. Considering the results for glycerol, the best motility and NAR values were obtained for boar spermatozoa frozen in 4% glycerol extender (2% final concentration) and allowed to equilibrate for 30 min. 4.2. Effect of cryoprotectants on frozen sperm chromatin This study showed that a higher concentration of lactose led to increased chromatin compactness but did not show differences regarding the stability. Glycerol, however, showed different behavior. In general, those conditions that favoured higher intracellular glycerol levels, such as the use of extenders with higher glycerol concentration or a longer equilibration time, showed a less condensed and less stable chromatin. However, this effect was observed for concentrations between 4 and 8% glycerol, since higher concentrations (10% glycerol) showed more condensed and more stable chromatin. Previous studies have shown increased chromatin condensation upon freezing and thawing compared to fresh semen species [14,17,43]. The state of chromatin condensation depends on the nature and degree of interactions among its components. Protamines play an important role in these interactions, that include disulphide bonds (–SS–), interactions of thiol groups with zinc ions and non-covalent interactions [45,46]. Stronger interactions could be involved in those samples showing more condensed chromatin. On the other hand, there is increasing evidence suggesting that DNA breaks and overoxidation of thiol groups in protamines can play an important role in chromatin destabilisation after freezing and thawing [14,18]. Less stable chromatin found after heparin treatment may be related to more damaged DNA. However, it can also be due to decreased interactions among chromatin components, particularly –SS– bonds
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or interactions of different nature than those mediated by zinc, since heparin acts as strong polyanion that bind (chelate) zinc cations [47]. A possible though speculative explanation of these results lies on the water binding capacity of lactose and glycerol, which could alter the physicochemical interactions among proteins, water and ions and, therefore, may influence chromatin condensation. Lactose is a non-penetrating agent that binds water in the extracellular space. A partial loss of intracellular water may favour stronger interactions among protamines and increased chromatin condensation. Glycerol is a penetrating agent that can bind and retain water within the cell, which could favour lower chromatin condensation. 4.3. Overall effects of cryoprotectants on frozen boar spermatozoa The results obtained in this study showed that the nature and concentration of cryoprotectants in the extenders caused changes in motility and NAR, as expected, but also on the chromatin state of frozen boar spermatozoa. Taking together all parameters, it was shown that increasing the concentration of lactose in the extenders from 11 to 12% caused an increase in motility, NAR and chromatin compactness, while increasing it further to 14% was detrimental only for motility. Chromatin stability was not affected by the lactose concentration. Increasing the glycerol concentration in the second extender from 4 to 6% and freezing spermatozoa immediately after dilution did not show any changes but increasing it further to 8% led to higher motility and lower NAR as well as lower chromatin condensation and stability. When a 30 min incubation time was used after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. This incubation time was detrimental for motility when freezing with the 8% but favourable with the 4% glycerol extender. Increasing the glycerol concentration from 8 to 10% showed spermatozoa with no significant changes in motility but increased chromatin condensation and stability. The spermatozoa frozen in 10% glycerol showed low values of NAR that did not improve by longer incubation time. While the impact of the changes in motility and NAR on fertility can be foreseen, the significance of the chromatin changes is difficult to assess. Sperm chromatin compactness is necessary for DNA inactivation and protection [48]. On the other hand, hyperstability of chromatin could delay paternal nuclear
formation during fertilization [49,50] that can induce early embryonic death or lower embryonic development frequently observed after artificial insemination with frozen boar semen [51]. Further in vitro and in vivo studies would provide a better understanding of the significance of these findings. Acknowledgements We wish to thank to the Department of Statistics (CSIC) for their assistance, to Prof. J. Strzez´ek for his critical advice and manuscript reading and to the late Prof. S. Martı´n Rillo for his support. BDC was supported by a fellowship from the Ministerio de Educacio´n y Ciencia (Spain) under the Programa de Cooperacio´n Cientı´fica con Iberoame´rica. References [1] Johnson LA, Aalbers JG, Willems CMT, Sybesma W. Use of boar semen for artificial insemination. Fertility capacity of fresh and frozen spermatozoa in sows on 36 farms. J Anim Sci 1981;52:1130–6. [2] Holt WV, North RD. Effects of temperature and restoration of osmotic equilibrium during thawing on the induction of plasma membrane damage in cryopreserved ram spermatozoa. Biol Reprod 1994;51:414–24. [3] Meryman HT. Osmotic stress as a mechanism of freezing injury. Cryobiology 1971;8:489–500. [4] Muldrew K, McGann LE. The osmotic rupture hypothesis of intracellular freezing injury. Biophys J 1994;66:532–41. [5] Caiza De La Cueva FI, Rigau T, Rosa P, Piedrafita J, Ro´driguez GE. Resistance to hyperosmotic stress in boar spermatozoa: the role of the ionic pumps and the relatioship with cryosurvival. Anim Reprod Sci 1997;48:301–15. [6] Lui Z, Foote RT, Brockett CC. Survival of bull sperm frozen at different rates in media varying in osmolality. Cryobiology 1998;37:219–30. [7] Lui Z, Foote RT. Osmotic effects on volume and motility of bull sperm exposed to membrane permeable and nonpermeable agents. Cryobiology 1998;37:207–18. [8] Abdelhakeam AA, Graham EF, Va´zquez IA. Studies on the absence of glycerol in unfrozen and frozen ram semen. Cryobiology 1991;28:43–9. [9] Fiser PS, Fairfull RW. Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa. Theriogenology 1986; 25:473–84. [10] Zeng WX, Shimada M, Isobe N, Terada T. Survival of boar spermatozoa frozen in diluents of varying osmolality. Theriogenology 2001;56:447–58. [11] Fraser L, Gorszczaruk K, Strzezek J. Relationship between motility and membrane integrity of boar spermatozoa in media varying in osmolality. Reprod Dom Anim 2001;36:325–9. [12] Almid T, Johnson LA. Effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J Anim Sci 1988;66:2899–905.
B.D. Corcuera et al. / Theriogenology 67 (2007) 1150–1157 [13] Fiser PS, Fairfull RW. Glycerol equilibration time revised. In: Rath D, Johnson LA, Weitze KF, editors. Boar semen preservation III Reprod Dom Anim, vol. 31. 1996. p. 141–6. [14] Co´rdova A, Pe´rez-Gutie´rrez JF, Lleo´ B, Garcı´a-Artiga C, Alvarez A, Drobchak V, et al. In vitro fertilizing capacity and chromatin condensation of deep frozen boar semen packaged in 0.5 and 5 mL straws. Theriogenology 2002;57:2119–28. [15] Madrid-Bury N, Pe´rez-Gutie´rrez JF, Pe´rez-Garnelo S, Moreira P, Pintando Sanjuanbenito B, Gutie´rrez-Ada´n A, et al. Relationship between non-return rate and chromatin condensation of deep frozen bull spermatozoa. Theriogenology 2005;64:232–41. [16] Kvist U, Kjelberg S, Bjo¨rndahl L, Hammar H, Roomans GM. Zinc in sperm chromatin stability in fertile men and men in barren unions. Scand J Urol Nephrol 1988;22:1–6. [17] Hamamah S, Royere D, Nicolle JC, Paquignon M, Lansac J. Effects of freezing–thawing on the spermatozoon nucleus: a comparative chromatin cytometric study in the porcine and human species. Reprod Nutr Dev 1990;30:59–64. [18] Co´rdova-Izquierdo A, Oliva JH, Lleo´ B, Garcı´a-Artiga C, Doris Corcuera BD, Pe´rez-Gutie´rrez JF. Effect of different thawing temperatures on the viability, in vitro fertilizing capacity and chromatin condensation of frozen boar semen packaged in 5 mL straws. Anim Reprod Sci 2006;92:145–54. [19] Bredderman PJ, Foote RH. Volume of stressed bull spermtozoa and protoplasmatic droplets, and the relationship of cell size to motility and fertility. J Anim Sci 1969;28:496–501. [20] Drevious LO. Bull spermatozoa as osmometers. J Reprod Fertil 1972;28:29–30. [21] Gilmore JA, McGann LE, Gao DY, Peter AT, Kleinhans FW, Cristner JK. Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol Reprod 1995;53:985–95. [22] Hancock JL, Hovell GJR. The collection of boar semen. Vet Rec 1959;71:664–5. [23] Einarsson S. Studies on the composition of epididimal content and semen in the boar. Acta Vet Scand Suppl 1971;36:1–80. [24] King GJ, Macpherson JW. A comparison of two methods for boar semen collection. J Anim Sci 1973;36:563–5. [25] Westendorf P, Richter L, Treu H. Zur Tiefgefrierung von Ebersperma Labor-und Besamungsergebnisse mit dem Hu¨lsenberger Pailletten-Verfahren. Dstch Tiera¨rtzl Wschr 1975;82:261–7. [26] Martı´n Rillo S, Martı´nez E, Garcı´a Artiga C, De Alba C. Boar semen evaluation in practice. Reprod Dom Anim 1996;31:519– 26. [27] Pursel VG, Johnson LA, Rampacek GB. Acrosome morphology of boar spermatozoa incubated before cold shock. J Anim Sci 1972;34:278–83. [28] Darzynkiewicz Z, Gledhill BL, Ringertz NR. Changes in deoxyribonucleo-protein during spermiogenesis in the bull. 3H-actinomycin D binding capacity. Exp Cell Res 1969;58:435–8. [29] Strzezek J, Kordan W, Glogowski J, Wysocki P, Borkowski K. Influence of semen-collection frequency on sperm quality in boars, with special reference to biochemical markers. Reprod Dom Anim 1995;30:85–94. [30] Harvey WR. Mixed model least squares and maximum likelihood computer program. In: PC-1 manual. Columbus, OH: WR Harvey; 1987. [31] Snedecor GW, Cochran WG. Statistical methods. Iowa State University Press; 1980. [32] SAS. User’s guide. Release 6.2. Cary, NC: SAS Institute Inc.; 1998. [33] Salamon S. Deep freezing of boar semen. III. Effects of centrifugation, diluent and dilution rate, pellet volume, and method
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of thawing on survival of boar spermatozoa. Aust J Biol Sci 1973;23:239–47. Wilmut I, Polge C. The low temperature preservation of boar spermatozoa. II. The motility and morphology of boar spermatozoa frozen and thawed in diluent which contained only sugar and egg yolk. Cryobioloby 1977; 14:483–91. Visser D, Salamon S. Effect of composition of Tris-based diluent on survival of boar spermatozoa following deep freezing. Aust J Biol Sci 1974;27:485–97. Januskauskas A, Rodriguez-Martinez H. Assesment of sperm viability by measurement of ATP, membrane integrity and motility in frozen/thawed bull semen. Acta Vet Scand 1995;36:571–4. Wolders H, Matthijs A, Engel B. Effects of trehalose and sucrose, osmolality of the freezing medium, and cooling rate on viability and intactness of bull sperm after freezing and thawing. Cryobiology 1997;35:93–105. Gao DY, Ashworth E, Watson PF, Kleinhans FW, Mazur P, Critser JK. Hyperosmotic tolerance of human spermatozoa: separate effects of glycerol, sodium cloride and sucrose on spermiolysis. Biol Reprod 1993;49:112–23. Anchordoguy TJ, Rudolph AS, Carpenter JF, Crowe JH. Modes of interaction of cryoprotectans with membrane phospholipids during freezing. Cryobiology 1987;24:324–31. Pursel VG, Schulman LL, Johson LA. Effect of glycerol concentration of frozen boar semen. Theriogenology 1978;9:305– 12. Scheid IR, Westendrof P, Treu H. Deep freezing of boar semen in plastic tubes ‘‘Hulsenberg-pilletten’’: effect of different glycerol concentrations. In: Proc VIth pig vet congr Copenhagen. 1980. p. 38. Bamba K, Cran DG. Further studies on rapid dilution and warming of boar semen. J Reprod Fertil 1988;82:509–18. Royere D, Barthelemy C, Hamamah S, Lansac J. Cryopreservation of spermatozoa: a 1996 review. Hum Reprod Update 1996;2:553–9. Johnson LA, Weitze KF, Fiser P, Maxwell WMC. Storage of boar semen. Anim Reprod Sci 2000;62:143–72. Huang TT, Kosover NS, Yanagimachi R. Localization of thiol and disulphide groups in guinea-pig spermatozoa during maturation and capacitation using bimane fluorescent labels. Biol Reprod 1984;31:797–809. Kvist U, Bjo¨rndahl L. Zinc preserves an inherent capacity for human sperm chromatin decondensation. Acta Physiol Scand 1985;124:195–200. Delgado NM, Huacuja L, Merchant H, Reyes R, Rosado A. Species-specific decondensation of human spermatozoa nuclei by heparin. Arch Androl 1980;4:305–13. Hunter JD. Single-strand nuclease action on heat-denatured spermiogenic chromatin. J Histochem Cytochem 1976; 24:901–7. Rosenborg L, Rao KM, Bjo¨rndahl L, Kvist U, Pousette A, Akerlo¨f E, et al. Changes in human sperm chromatin stability during preparation for in vitro fertilization. Int J Androl 1990;13:287–96. Huret JL. Variability of chromatin decondensation ability test on the human sperm. Arch Androl 1983;11:1–7. Lwoff L, Be´zard J, Paquignon M. Comparision de technologies de conservation des spermatozoides de verrant. Effect sur les me´canismes de la fe´condation. J Rech Porcine Fr 1987;19:79– 86.
Effect of lactose and glycerol on the motility, normal apical ridge, chromatin condensation and chromatin stability of frozen boar spermatozoa B.D. Corcuera a, P. Marigorta b, A. Sagu¨e´s a, F. Saiz-Cidoncha b, J.F. Pe´rez-Gutie´rrez c,* a Kubus, S.A., Polı´gono Industrial Euro´polis C/E, n20, Las Rozas, 28230 Madrid, Spain Departamento de Reproduccio´n Animal, INIA, Ctra. de la Corun˜a km 5.9, 28040 Madrid, Spain c Departamento de Medicina y Cirugı´a Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Av. Puerta de Hierro s/n, 28040 Madrid, Spain b
Received 12 June 2006; received in revised form 3 January 2007; accepted 14 January 2007
Abstract The effect of lactose and glycerol concentration, as well as the equilibration time with glycerol was studied on motility, normal apical ridge (NAR), and chromatin state of boar spermatozoa after the freezing and thawing process. In the first experiment, samples were frozen in first and second extenders containing different concentrations of lactose (11, 12 and 14%). In the second experiment samples were frozen using second extenders with different concentrations of glycerol (4, 6, 8 and 10%) and were incubated at 5 8C for 0 and 30 min. Motility, motility after caffeine treatment, NAR, chromatin condensation and stability (susceptibility to decondense after heparin treatment) were evaluated. The results indicated that freezing spermatozoa in extenders with increasing concentrations of lactose adversely affected motility but provided a protective effect on acrosomes. Increased lactose concentration induced higher chromatin condensation but maintained the same stability. Increasing the glycerol concentration in the second extender from 4–6 to 8% led to higher motility and NAR as well as lower chromatin condensation and stability. When 30 min equilibration time was allowed after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. The longer equilibration time was detrimental for motility when freezing in the 8% glycerol extender but favourable when using the 4% glycerol extender. Compared to the 8% glycerol, spermatozoa frozen in the 10% glycerol extender showed similar motility and increased chromatin condensation and stability, as well as low values of NAR that did not improve by longer incubation time. # 2007 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Cryoprotectants; Sperm motility; Chromatin; Porcine
1. Introduction The freezing and thawing of boar spermatozoa causes considerable cell damage leading to severe
* Corresponding author. Tel.: +34 913943798; fax: +34 913943808. E-mail address: [email protected] (J.F. Pe´rez-Gutie´rrez). 0093-691X/$ – see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2007.01.002
reductions in farrowing rates and litter size after artificial insemination with frozen semen [1]. A number of mechanisms have been proposed to explain injury to spermatozoa following freezing and thawing. It has been proposed that upon freezing, ice forms first in the extracellular medium resulting in hyperosmolal concentration of solutes outside the cell, pH changes and solute precipitation that could cause extreme cell
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shrinkage up to irreversible membrane damage [2,3]. Intracellular ice crystal formation can lead to mechanical damage of the plasma membrane and organelles causing oxidative stress as well as alterations in sperm metabolism [2–4]. During the last decades, many studies have focused on preventing cryodamage by using cryoprotective agents [5–12]. Non-permeable cryoprotectants such as sugars protect the membrane from the volume changes the cell experience upon freezing and thawing. Permeable cryoprotectants such as glycerol is very effective in lowering the intracellular water freezing point and reduce intracellular ice crystal formation. The combination of both types of cryoprotectants is commonly used. It has been shown that freezing at moderately hypertonic conditions can improve the survival of bull [6,7], ram [8,9] and boar [5,10,11] spermatozoa. In addition, it has been reported that reducing the exposure time to glycerol can improve bull spermatozoa motility and NAR upon freezing and thawing procedure [6,7]. However, the effects of the glycerol equilibration time on boar spermatozoa have shown different results [12,13]. Most of these studies have evaluated post thaw viability by assessing motility, membrane and acrosome integrity. However, there are other parameters related to fertility such as chromatin condensation and stability that could also be evaluated [14,15]. Previous studies in human, bovine and porcine species have shown that sperm chromatin can undergo important changes after the freezing–thawing procedure [15–18]. Since spermatozoa undergo sustained swelling or shrinkage when exposed to hypotonic or hypertonic solutions [19–21], it is possible that the osmolality of freezing media could alter boar sperm chromatin state. Up to our knowledge there are no studies related to the effect of cryoprotectants on boar chromatin state. The aim of this study was to evaluate the effect of extenders with different concentrations of lactose and glycerol, varying in osmolality, as well as the prefreezing exposure time to glycerol on boar sperm motility, NAR and chromatin state upon freezing and thawing. 2. Material and methods 2.1. Experimental design Two independent experiments were performed with two different sets of ejaculates, one to study the effect of lactose (experiment 1) and another one to study the
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effect of glycerol (experiment 2). For experiment 1, samples were subjected to three different treatments (11, 12 and 14% lactose). For experiment 2, samples were subjected to eight different treatments (4, 6, 8, 10% glycerol, incubated for 0 and 30 min). For each experiment, 10 ejaculates were collected from each of 5 boars, giving a total of 50 ejaculates. Each ejaculate was divided into identical fractions that were subjected to the different treatments. For each experiment, the 50 samples were frozen and, after thawing, they were pooled as follows: the 5 samples corresponding to the first ejaculates of the 5 boars, the 5 samples corresponding to the second ejaculates of the 5 boars, . . ., the 5 samples corresponding to the tenth ejaculates of the 5 boars. Therefore, for each treatment we obtained 10 pooled samples, each one being a mixed sample of the 5 boars. Each pooled sample was analyzed once. 2.2. Semen collection Semen was collected on a weekly basis, from five Large White boars, aged 1.5–2 years, whose fertility had been previously proven. The boars were housed in individual pens at the Instituto Nacional de Investigaciones Agrarias (Madrid, Spain) and fed with a commercial standard diet. The ejaculates (only the sperm rich fraction) were obtained by the gloved hand method [22]. Gel particles were removed by filtration through a sterile gauze [23]. A subsample from each ejaculate was taken for preliminary motility and NAR evaluation [24]. All samples were evaluated by only one technician. Ejaculates showing motility higher than 80%, and NAR higher than 70% were used. The rest of the ejaculate was processed and subjected to each of the freezing treatments in 5 mL straws. 2.3. Semen freezing The freezing procedure involved the dilution with extenders of different concentrations of lactose and glycerol as described in experiments 1 and 2. 2.3.1. Experiment 1 The concentration of lactose was changed in both the first and the second extenders. The first extender was composed of 20% hen egg yolk and varying concentrations of lactose: 11% (Ext. 1a), 12% (Ext. 1b) and 14% (Ext. 1c). The osmolality for groups 1a, 1b and 1c was 320, 360 and 420 mOsm/kg, respectively. Osmolality was determined by using a freezing-point depression osmometer (Model 3D2, Advanced Instruments, Need-
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ham Heights, MA). The second extender was composed of 20% hen egg yolk, 4% glycerol, 0.5% lauryl sodium sulfate and varying concentrations of lactose: 11% (Ext. 2a), 12% (Ext. 2b) and 14% (Ext. 2c). The osmolality for groups 2a, 2b and 2c was 1000, 1080 and 1150 mOsm/kg, respectively. Ext. 1a, 1b and 1c were used with Ext. 2a, 2b and 2c, respectively. 2.3.2. Experiment 2 The first extender (Ext. 1a) was used with second extenders with different glycerol contents. The composition of the second extenders was 11% lactose, 20% egg yolk, 0.5% lauryl sulfate and varying concentrations of glycerol: 4, 6, 8 and 10%, with osmolalities of 1000, 1500, 1880 and 2320 mOsm/kg, respectively. For these samples, incubation with the second extender was done at 5 8C for 0 and 30 min. The semen was processed and frozen by the straw freezing method, originally described by Westendorf et al. [25] with some modifications. After collection, the sperm rich fraction was diluted 1:4 with a commercial extender (MR-A1 diluent, Kubus, S.A., Madrid, Spain) at 23 8C. After 1 h equilibration at 23 8C, each ejaculate was divided into doses containing 6 109 spermatozoa (at a concentration of 6 108 spermatozoa/mL) to be subjected to each of the treatments, three for experiment 1 and eight for experiment 2. Doses were kept at 15 8C for 3 h and centrifuged at 800 g for 10 min. After supernatant removal the concentrated semen was diluted to 5 mL at 15 8C with the first extender. The diluted semen was gently mixed and cooled at 5 8C for 1.5 h. Samples were further diluted (1:2) to 10 mL at 5 8C with the second extender. In the experiment 1, doses were immediately packaged whereas in the experiment 2, doses were incubated with the second extender for 0 and 30 min at 5 8C before being packaged in 5 mL straws. Straws were manually sealed with metallic balls, wiped dry and the air bubble was brought to the center of the straw. Finally, straws were placed in contact with nitrogen vapor, about 3 cm above the nitrogen liquid level for 20 min, and plunged into the liquid nitrogen tank and stored until used. 2.4. Thawing Thawing of the straws was carried out after 12 months of storage in liquid nitrogen. Straws were thawed in a water bath at 42 8C for 40 s. Immediately after thawing, the five seminal doses from the different boars that were subjected to the same treatment were pooled. Ten heterospermic groups per treatment were examined, after 15 min of incubation at room temperature.
2.5. Semen evaluation 2.5.1. Concentration and sperm motility assessment Sperm concentration was estimated using a Bu¨rker counting chamber. One millilitre of semen was diluted (1:100) in a 40% formaldehyde physiological solution. One drop of this dilution was placed into the Bu¨rker chamber and the number of spermatozoa was assessed under the microscope (40) by counting the number of cells found inside 40 small squares. Total sperm motility was assessed by placing a sample on a prewarmed microscope slide (42 8C) overlaid with a coverslip and determining, under a light microscope (20), the percentage of spermatozoa that showed movement of the flagellum. Motility with caffeine was determined as described previously after adding a drop of isotonic sodium citrate solution containing 0.1% caffeine to the diluted semen sample [26]. 2.5.2. Assessment of NAR The evaluation of acrosomal integrity was done by examining citrate:formol (1:10) fixed samples with a phase contrast microscope, at 100. A minimum of 100 acrosomes was examined per sample. Acrosomal damage was evaluated according to Pursel et al. [27]. 2.5.3. Analysis of sperm chromatin condensation and stability The chromatin was assessed as chromatin condensation and chromatin stability. Degree of chromatin condensation was determined by using a radioisotope method based on the specific affinity of tritium labelled actinomicyn D (3H-AMD) to bind the base pair guanine-cytosine [28]. 3H-AMD binding to chromatin was estimated by the total counts taken up by the spermatozoa, according to Strzezek et al. [29]. The increase in total counts is related to higher 3H-AMD uptake and, therefore, to decreased chromatin compactness. Chromatin stability was assessed by determining chromatin condensation after inducing decondensation with heparin. Samples were prepared by resuspending thawed semen in 250 mM Tris-citrate-fructose buffer to a final concentration of 50 106 spermatozoa/mL in each sample. After centrifugation at 12,500 g for 15 min, the supernatant was discarded. For determining chromatin condensation, each pellet was resuspended in 10 mL Tris-citrate buffer with 0.1 mCi/mL of 3HAMD (Amersham International, Amersham, UK). For determining chromatin stability, pellets were resuspended in 10 mL of the same buffer containing 200 IU
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Table 1 Sperm motility, NAR, chromatin condensation and stability of frozen boar semen in first extenders with different concentrations of lactose and second extenders with the same concentrations of lactose and 4% glycerol Lactose level (%)
11 12 14
Motility (%)*
a
23.3 5.1 28.6 6.4 b 22.9 7.0 a
Motility with caffeine (%)* a
40.0 7.9 42.9 6.0 a 35.7 9.4 b
NAR (%)*
Chromatin 3H-AMD uptake (cpm)** Heparin (for condensation)
a
35.4 8.7 39.4 4.5 b 40.6 7.7 b
a
28.5 2.4 22.5 4.1 b 23.9 2.1 b
+ Heparin (for stability) 33.3 5.7 33.0 2.5a 30.7 3.0a
Values are expressed as means S.D. (n = 10). Values within each column with different superscripts (a and b) are significantly different (P < 0.05). Units in percentage and related to the total number of spermatozoa (*) or in total counts (cpm 103) taken up by 50 106 spermatozoa incubated in the absence (for chromatin condensation) or presence of heparin (for chromatin stability) (**). Smaller counts values mean higher chromatin compactness.
of heparin. All samples were gently shaken, transferred to glass tubes and incubated at 37 8C. One-millilitre solutions were taken at 0 and 60 min and centrifuged (12500 g, 10 min). Three aliquots of 0.1 mL were taken from each supernatant and placed in scintillation vials. Subsequently, 9 mL of scintillation fluid was added to each sample. Counts were obtained from a liquid scintillation counter B 1215 Rachbeta II (LKD Wallac, Turku, Finland) and corrected for background and the appropriate dilution factor. Results were presented as the total counts taken up by 50 106 spermatozoa, obtained from the difference in cpm of the supernatants corresponding to the sample incubated for 60 and 0 min.
motility, motility after caffeine treatment, NAR, chromatin condensation and stability (experiment 1): Y i jk ¼ M þ Ri þ J j þ Oi j þ ei jk where Yijk is the observation; M the overall mean of the population; Ri the random effect of the repetition (i = 1, 2, . . ., 10); Jj the fixed effect of the lactose concentration in the first extender (j = 1, 2, 3); Oij the effect of the interaction within the previously described factors; eijk is the residual effect. The effect glycerol concentration as well as the equilibration time with glycerol on the sperm motility, NAR and chromatin state (experiment 2) were analyzed according to the following model:
2.6. Statistical analysis Y i jkl ¼ M þ Ri þ J j þ Pk þ Oi j þ T ik þ S jk þ OTSi jk The following model was used to test the effects of different concentrations of lactose on the sperm
þ ei jkl
Table 2 Sperm motility, NAR, chromatin condensation and stability of frozen boar semen in a first extender with 11% lactose and second extenders with the same concentration of lactose and different concentrations of glycerol, after 0 and 30 min of incubation with the second extender at 5 8C Glycerol level (%)
Motility (%) *
Motility with caffeine (%) *
NAR (%)*
0
4 6 8 10
28.6 8.3 ad 27.1 7.0 ad 40.0 9.7 bd 35.7 7.3 bd
32.9 9.8 ad 33.0 7.5 ad 45.7 7.3 bd 41.4 6.4 bd
30
4 6 8 10
24.3 4.9 ad 30.0 7.6 bd 30.7 5.3 be 28.6 3.5 ae
37.1 6.9 ae 30.9 5.3 bd 35.7 4.9 ae 35.8 5.3 ae
Glycerol exposure time (min)
Chromatin 3H-AMD uptake (cpm) ** + Heparin (for condensation)
Heparin (for stability)
25.7 4.7ad 24.0 7.2ad 15.3 4.0bd 15.0 3.7bd
21.6 2.7ad 20.8 8.6ad 26.4 7.6bd 23.8 3.2ad
30.4 7.2ad 32.9 6.4ad 38.1 5.9bd 30.6 5.2ad
31.1 7.3ae 22.3 3.7bd 25.4 5.4be 13.4 4.0cd
24.0 2.3ad 24.5 2.5ad 25.0 5.7ad 26.2 7.1ad
36.2 2.9ae 37.6 4.1ae 39.9 6.6ad 29.8 4.8bd
Values are expressed as means S.D. (n = 10). Different supercripts (a–c) denote significant statistical difference (P < 0.05) within the same incubation time. Different superscripts (d, e) denote significant statistical difference (P < 0.05) between different incubation times. Units in percentage and related to the total number of spermatozoa (*) or in total counts (cpm 103) taken up by 50 106 spermatozoa incubated in the absence (for chromatin condensation) or presence of heparin (for chromatin stability) (**). Smaller values of counts mean higher chromatin compactness.
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where Yijkl is the observation; M the overall mean of the population; Ri the random effect of the repetition (i = 1, 2, . . ., 10); Jj the fixed effect of the glycerol concentration in the second extender (j = 1, 2, 3, 4); Pk the fixed effect of the incubation time with glycerol at 5 8C (k = 1, 2); Oij, Tik, Sjk the effect of the interaction of two factors; OTSijk the effect of the interaction of three factors; eijkl is the residual effect. Data were first analyzed by Harvey’s least squares maximum likelihood analysis of variance [30]. The least squares means and standard deviations of untransformed percentages are presented in the Tables 1 and 2. For the statistical analysis, percentages values were subjected to arcsim transformation before applying the unpaired student t-test to means and standard deviations for each datum point [31]. Results from the first and second experiments were analyzed by one-way analysis of variance using SAS software [32]. A probability level of P < 0.05 was considered to be statistically significant. 3. Results 3.1. Effect of the different concentrations of lactose on motility, NAR and chromatin state The results obtained for the experiment 1 (Table 1) showed that immediately after thawing, the motility of spermatozoa frozen in the 12% lactose extender was significantly higher than spermatozoa frozen in either 11 or 14% lactose extenders. When motility was evaluated after the caffeine treatment, spermatozoa frozen in 14% lactose extender showed significantly lower motility than those frozen in 11 or 12% lactose extenders. Spermatozoa frozen in 14% lactose extender showed significantly lower motility values in presence and absence of caffeine. The percentage of spermatozoa showing normal apical ridge was significantly higher in those groups frozen in the 12 and 14% lactose extenders compared to those frozen in the 11% lactose extender. Analysis of chromatin condensation on boar spermatozoa frozen in 11% lactose extender showed significantly higher total counts compared to those spermatozoa frozen in either 12 or 14% lactose extenders. An increase in total counts, related to easier accessibility of DNA, was found after the exposure to heparin. No significant differences (P > 0.05) in chromatin stability were observed among spermatozoa frozen in extenders containing different lactose concentrations.
3.2. Effect of the different concentrations of glycerol on motility, NAR and chromatin state The results obtained for the experiment 2 (Table 2) showed significantly higher post-thaw motility in spermatozoa frozen immediately (0 min) after dilution in 8 and 10% than those frozen after dilution in 4 and 6% glycerol extenders. As previously observed in experiment 1, the caffeine treatment induced an increase in post thaw sperm motility values. The results were similar in presence or absence of caffeine. The samples frozen in 8 and 10% glycerol showed a significantly higher post-thaw motility than those frozen in 4 and 6% glycerol extenders. The integrity of the apical ridge was affected by the concentration of glycerol of the extender when samples were frozen immediately after the addition of glycerol. The percentage of spermatozoa showing normal apical ridge was higher when diluted in 4 and 6% than in 8 and 10% glycerol extenders. When comparing post-thaw motility among incubation times (0 and 30 min), it was observed that increasing incubation time reduced the post thaw motility of those spermatozoa frozen in 8 and 10% glycerol. These results were similar in caffeine treated and non-treated samples. Increasing the time of exposure to 6% glycerol did not affect motility parameters. However, at 4% glycerol, motility increased in the presence of caffeine. The post thaw NAR values either were not altered or significantly increased after 30 min of exposure to glycerol, with respect to those spermatozoa frozen immediately after dilution with glycerol. When analyzing the chromatin state it was observed that spermatozoa frozen immediately after dilution in the 8% glycerol extender showed higher counts, thus less condensed chromatin, than frozen in lower glycerol concentrations. After heparin treatment, significantly higher counts were also observed in spermatozoa frozen in 8% glycerol than in lower concentrations, indicating that the former had less stable chromatin. A further increase of the concentration to 10% caused more condensed and more stable chromatin. When the time of exposure to glycerol was 30 min no significant differences on counts related to chromatin condensation or stability were found among spermatozoa frozen in 4, 6 and 8% glycerol. These values were close to those obtained at 0 min for 8% glycerol. Furthermore, when the concentrations of glycerol were 4 and 6%, a significant change towards less stable chromatin was observed when increasing the incubation time with glycerol from 0 to 30 min. On the other hand,
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higher concentrations (10%) of glycerol resulted in more stable chromatin regardless the incubation time. 4. Discussion 4.1. Effect of cryoprotectants on frozen sperm motility and NAR Among the lactose concentrations used in this study, the best motility results were obtained when freezing in 12% lactose extenders while higher concentrations (14%) showed a negative effect. This agrees with previous studies that have reported that high levels of lactose exert a detrimental effect on motility [33–35]. It was also shown that a number of spermatozoa apparently immotile had actually the potential to move as evidenced by the caffeine treatment. The causes of this reversible loss of motility are not clear. It has been shown that the reversible loss of motility may be due to temporary metabolic failure leading to changes in ATP content and calcium uptake that are highly correlated with motility [11,36]. A reduction of intracellular water content could also be the cause of the reversible inhibition of motility since it could increase the friction in tail causing an inhibition of sliding of microtubules or other structural elements in the flagellum [37,38]. On the other hand, increasing the lactose concentration from 11 to 12 and 14% improved NAR values. This agrees with previous studies that have reported the protective effect of lactose on the acrosome membrane of boar spermatozoa [33–35], that could be due to the formation of hydrogen bonds with the phosphate groups on membrane phospholipids [38,39]. This study confirms that high levels of lactose exert a negative effect on motility but a positive effect on the proportion of normal acrosomes [33–35]. Among the conditions used, 12% lactose showed the best results for motility and NAR. Results showed that increasing concentration of glycerol improved the post thaw motility of boar spermatozoa but decreased the percentage of normal acrosomes. The best glycerol concentrations for motility were 8 and 10% whereas for acrosomal integrity (NAR) were 4 and 6%. These results corroborates previous dose dependent studies that have shown that increasing glycerol concentration in the range of 0 to 7% improved motility but negatively affected acrosome integrity [40,41]. However, they differ from Bamba and Cran [42] that have observed, in addition to the negative effect on NAR, a decrease in motility when glycerol exceeded 6%. These differences could be attributed to differences in the experimental
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procedures, since it has been shown that the concentration of glycerol required for optimum motility and NAR are determined by several factors including the cooling rate and the temperature and time of exposure to glycerol [38,43]. In general terms, increasing the glycerol exposure time to 30 min either did not alter or was beneficial for motility at lower glycerol concentration (4–6%) but was detrimental for higher concentrations (8–10%). The 30 min exposure to glycerol either improved or maintained NAR values. These results agree with previous results which showed a beneficial effect of increasing the exposure time to glycerol (0–6%) for motility and NAR [13,44]. Considering the results for glycerol, the best motility and NAR values were obtained for boar spermatozoa frozen in 4% glycerol extender (2% final concentration) and allowed to equilibrate for 30 min. 4.2. Effect of cryoprotectants on frozen sperm chromatin This study showed that a higher concentration of lactose led to increased chromatin compactness but did not show differences regarding the stability. Glycerol, however, showed different behavior. In general, those conditions that favoured higher intracellular glycerol levels, such as the use of extenders with higher glycerol concentration or a longer equilibration time, showed a less condensed and less stable chromatin. However, this effect was observed for concentrations between 4 and 8% glycerol, since higher concentrations (10% glycerol) showed more condensed and more stable chromatin. Previous studies have shown increased chromatin condensation upon freezing and thawing compared to fresh semen species [14,17,43]. The state of chromatin condensation depends on the nature and degree of interactions among its components. Protamines play an important role in these interactions, that include disulphide bonds (–SS–), interactions of thiol groups with zinc ions and non-covalent interactions [45,46]. Stronger interactions could be involved in those samples showing more condensed chromatin. On the other hand, there is increasing evidence suggesting that DNA breaks and overoxidation of thiol groups in protamines can play an important role in chromatin destabilisation after freezing and thawing [14,18]. Less stable chromatin found after heparin treatment may be related to more damaged DNA. However, it can also be due to decreased interactions among chromatin components, particularly –SS– bonds
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or interactions of different nature than those mediated by zinc, since heparin acts as strong polyanion that bind (chelate) zinc cations [47]. A possible though speculative explanation of these results lies on the water binding capacity of lactose and glycerol, which could alter the physicochemical interactions among proteins, water and ions and, therefore, may influence chromatin condensation. Lactose is a non-penetrating agent that binds water in the extracellular space. A partial loss of intracellular water may favour stronger interactions among protamines and increased chromatin condensation. Glycerol is a penetrating agent that can bind and retain water within the cell, which could favour lower chromatin condensation. 4.3. Overall effects of cryoprotectants on frozen boar spermatozoa The results obtained in this study showed that the nature and concentration of cryoprotectants in the extenders caused changes in motility and NAR, as expected, but also on the chromatin state of frozen boar spermatozoa. Taking together all parameters, it was shown that increasing the concentration of lactose in the extenders from 11 to 12% caused an increase in motility, NAR and chromatin compactness, while increasing it further to 14% was detrimental only for motility. Chromatin stability was not affected by the lactose concentration. Increasing the glycerol concentration in the second extender from 4 to 6% and freezing spermatozoa immediately after dilution did not show any changes but increasing it further to 8% led to higher motility and lower NAR as well as lower chromatin condensation and stability. When a 30 min incubation time was used after dilution with the same extenders, spermatozoa showed higher NAR and lower chromatin condensation and stability. This incubation time was detrimental for motility when freezing with the 8% but favourable with the 4% glycerol extender. Increasing the glycerol concentration from 8 to 10% showed spermatozoa with no significant changes in motility but increased chromatin condensation and stability. The spermatozoa frozen in 10% glycerol showed low values of NAR that did not improve by longer incubation time. While the impact of the changes in motility and NAR on fertility can be foreseen, the significance of the chromatin changes is difficult to assess. Sperm chromatin compactness is necessary for DNA inactivation and protection [48]. On the other hand, hyperstability of chromatin could delay paternal nuclear
formation during fertilization [49,50] that can induce early embryonic death or lower embryonic development frequently observed after artificial insemination with frozen boar semen [51]. Further in vitro and in vivo studies would provide a better understanding of the significance of these findings. Acknowledgements We wish to thank to the Department of Statistics (CSIC) for their assistance, to Prof. J. Strzez´ek for his critical advice and manuscript reading and to the late Prof. S. Martı´n Rillo for his support. BDC was supported by a fellowship from the Ministerio de Educacio´n y Ciencia (Spain) under the Programa de Cooperacio´n Cientı´fica con Iberoame´rica. References [1] Johnson LA, Aalbers JG, Willems CMT, Sybesma W. Use of boar semen for artificial insemination. Fertility capacity of fresh and frozen spermatozoa in sows on 36 farms. J Anim Sci 1981;52:1130–6. [2] Holt WV, North RD. Effects of temperature and restoration of osmotic equilibrium during thawing on the induction of plasma membrane damage in cryopreserved ram spermatozoa. Biol Reprod 1994;51:414–24. [3] Meryman HT. Osmotic stress as a mechanism of freezing injury. Cryobiology 1971;8:489–500. [4] Muldrew K, McGann LE. The osmotic rupture hypothesis of intracellular freezing injury. Biophys J 1994;66:532–41. [5] Caiza De La Cueva FI, Rigau T, Rosa P, Piedrafita J, Ro´driguez GE. Resistance to hyperosmotic stress in boar spermatozoa: the role of the ionic pumps and the relatioship with cryosurvival. Anim Reprod Sci 1997;48:301–15. [6] Lui Z, Foote RT, Brockett CC. Survival of bull sperm frozen at different rates in media varying in osmolality. Cryobiology 1998;37:219–30. [7] Lui Z, Foote RT. Osmotic effects on volume and motility of bull sperm exposed to membrane permeable and nonpermeable agents. Cryobiology 1998;37:207–18. [8] Abdelhakeam AA, Graham EF, Va´zquez IA. Studies on the absence of glycerol in unfrozen and frozen ram semen. Cryobiology 1991;28:43–9. [9] Fiser PS, Fairfull RW. Combined effects of glycerol concentration, cooling velocity, and osmolality of skim milk diluents on cryopreservation of ram spermatozoa. Theriogenology 1986; 25:473–84. [10] Zeng WX, Shimada M, Isobe N, Terada T. Survival of boar spermatozoa frozen in diluents of varying osmolality. Theriogenology 2001;56:447–58. [11] Fraser L, Gorszczaruk K, Strzezek J. Relationship between motility and membrane integrity of boar spermatozoa in media varying in osmolality. Reprod Dom Anim 2001;36:325–9. [12] Almid T, Johnson LA. Effect of glycerol concentration, equilibration time and temperature of glycerol addition on post-thaw viability of boar spermatozoa frozen in straws. J Anim Sci 1988;66:2899–905.
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