Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing–thawing

Animal Reproduction Science 99 (2007) 317–329

Effect of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing–thawing L. Fraser, J. Strze˙zek ∗ Department of Animal Biochemistry and Biotechnology, Faculty of Animal Bioengineering, Warmia and Mazury University in Olsztyn, Oczapowskiego 5, 10-718 Olsztyn, Poland Received 6 January 2006; received in revised form 7 June 2006; accepted 12 June 2006 Available online 4 August 2006

Abstract Whole ejaculate or sperm-rich fraction, collected from four sexually mature boars, was frozen in an extender containing lactose-hen egg yolk with glycerol (lactose-HEY-G) or extender containing lactose, lyophilized lipoprotein fractions isolated from ostrich egg yolk and glycerol (lactose-LPFo-G), and Orvus Es Paste, respectively. The sperm samples were also frozen in a standard boar semen extender (Kortowo-3), without the addition of cryoprotective substances. Sperm DNA integrity was assessed using a modified neutral comet assay. Sperm characteristics such as motility, plasma membrane integrity (SYBR-14/PI), mitochondrial function (rhodamine 123) and acrosome integrity were monitored. Freezing–thawing caused a significant increase (P < 0.05) in sperm DNA fragmentation, irrespective of the procedures of ejaculate collection and extender type. Sperm DNA fragmentation was significantly lower (P < 0.05) in the whole ejaculate compared with the sperm-rich fraction, indicating that spermatozoa maintained in the whole seminal plasma prior to its removal for freezing–thawing procedure were less vulnerable to cryo-induced DNA fragmentation. Furthermore, spermatozoa frozen in lactose-HEY-G or lactose-LPFo-G extender exhibited lower (P < 0.05) DNA fragmentation than those frozen in the absence of cryoprotective substances. The levels of sperm DNA damage, as expressed by comet tail length and tail moment values, were significantly higher (P < 0.05) in sperm samples frozen in the absence of cryoprotective substances. The deterioration in post-thaw sperm DNA integrity was concurrent with reduced sperm characteristics. It can be suggested that evaluation of DNA integrity, coupled with different sperm characteristics such as motility, plasma membrane integrity and mitochondrial function, may aid in determining the quality of frozen-thawed boar semen. © 2006 Elsevier B.V. All rights reserved. Keywords: Boar; Spermatozoa; Seminal plasma; Comet assay; DNA fragmentation

∗

Corresponding author. Tel.: +48 89 523 33 91; fax: +48 89 524 01 38. E-mail address: [email protected] (J. Strze˙zek).

0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.06.003

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1. Introduction Artificial insemination (AI) is widely used in pig industry, with semen preservation by cooling at 15 ◦ C since in the early 1970s (Pursel et al., 1973). Nevertheless, interest in freezing of semen is rising, mainly due to the increasing value of high quality boars. Cryopreservation of mammalian semen is the most commonly accepted method of preserving male reproductive capacity and is generally acknowledged to cause impaired fertility (Johnson et al., 2000). Over the last decade, rapid advances in reproductive molecular biology have resulted in numerous techniques to assess sperm DNA integrity (Evenson et al., 2002; Agarwal and Said, 2004). The highly compact organization of sperm DNA is thought to maintain the integrity of DNA during transmission of genetic material from male to female at the time of fertilization (Ward and Coffey, 1991). Sperm DNA integrity is important for the success of natural or assisted fertilization, including normal development of the embryo, fetus or offspring (Lopes et al., 1998). Evidence has been shown that the predictive value of routine semen analysis is limited, although some progress has been made in recent years by the introduction of standardized techniques and automation of motility estimations for semen analysis. Although these analyses may describe some aspects of the function of the testis and spermatozoa, they do not address the integrity of the male genome contained in the sperm head. Abnormalities in the male genome are, however, a clear potential reason for post-fertilization failure (Sakkas et al., 2002; Sergerie et al., 2005). The effect of freezing–thawing on DNA integrity of mammalian spermatozoa has been investigated using the comet assay. The comet assay is extensively used in somatic cells to measure genotoxic damage, especially single and double strand breaks, and was originally applied to spermatozoa (Singh et al., 1989). However, the comet assay has been modified to investigate DNA damage in cryopreserved spermatozoa of human (Donnelly et al., 2000), stallion (Linfor and Meyers, 2002), boar (Fraser and Strze˙zek, 2005) and bull (Hansen-Boe et al., 2005). In a recent study it has been shown that freezing–thawing of spermatozoa from fractionated ejaculate of boar semen can have different effects on post-thaw sperm survival (Rodr´ıguezMartinez et al., 2005). Generally, the sperm-rich fraction of boar ejaculate, which is devoid of most of its seminal plasma, is widely used for cryopreservation because of its high concentration in spermatozoa. The whole ejaculate, including the sperm-rich fraction and seminal plasma, originating in the accessory sex glands of the male reproductive system, is well endowed with antioxidants, which protect spermatozoa against oxidative insult (Strze˙zek et al., 1999). Moreover, even though the nature of DNA damage is not clear, evidence has been shown that sperm DNA quality is influenced by the presence of whole seminal plasma (Twigg et al., 1998a; Potts et al., 2000). The aim of this study was to investigate the effects of different procedures of ejaculate collection, extenders and packages on DNA integrity of boar spermatozoa following freezing–thawing. In addition, sperm characteristics were assessed by analyzing motility, plasma membrane integrity and mitochondrial function. 2. Materials and methods 2.1. Chemical agents All chemical were purchased from Sigma Chemical Company (St. Louis, MO, USA), unless otherwise stated. Orvus Es Paste was purchased from Nova Chemical Sales Inc., Scituate, MA, USA. Normal-melting and low melting point agarose (NMPA, LMPA) were purchased from Gibco-BRL, Life Technologies (Paisley, Scotland).

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2.2. Semen collection Four sexually mature Polish Large White boars, with ages ranged from 1.5 to 2 years, were used in this study. The boars were fed with a commercial porcine ration and were kept in individual pens, under standard environmental conditions. Water was available ad libitum. Whole ejaculates (including the pre-sperm, sperm-rich and post-sperm fractions) and only the sperm-rich fraction were collected at a 1-week interval from four boars, using the glovedhand technique. Following the collection of whole ejaculates from the four boars, the sperm-rich fraction was collected on the next week from the same four boars. A total number of 35 ejaculates (10 ejaculates each from three boars and five ejaculates from one boar) for the whole ejaculate and 37 ejaculates (10 ejaculates each from three boars and seven ejaculates from one boar) for the sperm-rich fraction were collected. At collections the gel portion was removed using double gauge and the ejaculates were subjected to microscopic analyses. Animal experiments were carried out in accordance with the guidelines set out by the Local Ethics Committee. 2.3. Freezing–thawing procedure Semen was collected in pre-warmed container and processed according to a standard protocol (Strze˙zek et al., 1985), with modifications. Besides whole hen egg yolk (HEY), we also used lyophilized lipoprotein fractions isolated from whole ostrich egg yolk, LPFo, which gave acceptable results (Strze˙zek et al., 2005a). Whole ejaculate and the sperm rich fractions were diluted 1:1 and 1:4, respectively, in a standard boar semen extender, Kortowo-3, (K-3), extender 1 (Strze˙zek et al., 1998) and cooled at 16 ◦ C for 3 h. Samples were centrifuged (800 × g for 10 min) and the sperm pellets (500–750 × 106 spermatozoa/ml) were re-suspended in a second extender (extender 2) containing 11% lactose (w/v) and 20% HEY (lactose-HEY extender) or 11% lactose and 5% LPFo (lactose-LPFo extender) and cooled at 5 ◦ C for 3 h. Next, 89.5 ml of lactose-HEY extender or lactose-LPFo extender were mixed with 9 ml glycerol (v/v) and 1.5 ml Orvus Es Paste (extender 3). Two parts of lactose-HEY extender or lactose-LPFo extender were mixed with one part of extender 3, respectively (lactose-HEY-G extender or lactose-LPFo-G extender). The final concentrations of glycerol and Orvus Es Paste were 3% and 0.5%, respectively. The sperm samples were also frozen in K-3 extender (control), without the addition of cryoprotective substances. The cooled semen samples were loaded into 10-ml sterilized aluminium tubes and 5-ml maxistraws (Minit¨ub, Tienfenbach, Germany) and sealed manually and with glass beads, respectively. The samples were placed onto a programmable computer freezing machine (Ice Cube 1810, SYLAB, Austria) and were cooled using an appropriate freezing protocol and then stored in liquid nitrogen. Thawing in a water bath at 50 ◦ C for 60 and 40 s for aluminium tubes and maxi-straws, respectively, was performed 2–3 days after freezing. Thawed samples were diluted immediately with K3 extender (50 × 106 spermatozoa) for sperm quality evaluation. Also, thawed samples were diluted with Ca2+ and Mg2+ -free phosphate-buffered saline, PBS (Vaccine Production, Warsaw, Poland) for DNA analysis. 2.4. Comet DNA damage assay We used a modified neutral comet assay procedure to provide quantitative measures of DNA damage in boar spermatozoa (Fraser and Strze˙zek, 2004). Briefly, mercaptoethanol-treated sperm samples (10 × 106 spermatozoa/ml) were added to 75 ␮l of 0.50% LMPA at 37 ◦ C. A 85 ␮l aliquot

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Fig. 1. Photograph of a spermatozoon with damaged DNA assessed with the neutral comet assay. Scale bar = 40 ␮m.

of this mixture was transferred onto frosted slides (pre-coated the day before with 0.75% NMPA in PBS, Ca2+ - and Mg2+ -free), covered with a coverslip and left on ice to allow the agarose to solidify. The coverslips was gently removed and the third layer (75 ␮l of 0.50% LMPA) was spread, covered and left on ice to solidify. The slides were immersed in freshly prepared cold lysing solution (2.5 mol/l NaCl, 100 mmol/l EDTA tetrasodium salt, 10 mmol/l Tris-base, pH 10, 1% sodium lauroyl sarcosine and 1% Triton X-100) at 4 ◦ C for 2 h. Thereafter the cells were subjected to enzymatic treatment with RNase A (2.5 mol NaCl, 5 mmol Tris, and 0.05% sodium lauroyl sarcosine, 20 ␮g/cm3 , pH 7.4) for 4 h and then with Proteinase K in a similar buffer composition, without RNase A. The slides were placed in electrophoreses unit, covered with the electrophoresis buffer (300 mmol/l sodium acetate, 100 mmol/l Tris, pH 9) for 15 min, and then electrophoresed (12 V, 120 mA, 1 h), during which fragmented DNA migrated from the nucleus towards the anode. Immediately before scoring, the slides were air-dried and stained with ethidium bromide (20 ␮g/ml). Slides were prepared in duplicate for each sample. 2.4.1. Comet scoring and analysis The ethidium bromide-stained slides were examined at 400× magnification under an epifluorescence microscope (Olympus BX 41) equipped with a 50 W high-pressure mercury lamp (HBO 50 Osram, Germany), using appropriate optical filters (excitation filter 530–550 nm and barrier filter BA 590). Fluorescence-stained comet images were captured with a CCD camera (Exwave HAD, Sony, Tokyo, Japan) attached to the fluorescent microscope and linked to a comet assay software programme, Komet Image Analysis System, Version 5.0 (Kinetic Imaging Ltd., Liverpool, UK). Spermatozoa with non-fragmented DNA (undamaged) do not form a “comet” and were considered negative, whereas sperm cells with fragmented DNA (positive) display increased migration of the DNA from the nucleus towards the anode, and had the appearance of a “comet” with brightly fluorescent head and tail formed by the DNA (Fig. 1). Samples were run in duplicate and a maximum of 50 cells were analyzed for the comet assay parameters, which were used to characterize DNA damage in fresh and frozen-thawed boar spermatozoa. The comet assay parameters analyzed were tail length (␮m) and tail moment (tail length multiplied by the fraction of DNA in the tail and is expressed as an arbitrary unit). 2.5. Evaluation of sperm characteristics 2.5.1. Motility and plasma membrane integrity The percentage of total motile spermatozoa was examined visually. Six microliters of sperm sample were paced on pre-warmed microscope slide, covered with a glass cover slide and examined under a light microscope (200×) equipped with an attached heated stage (37 ◦ C).

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Sperm plasma membrane integrity was assessed dual fluorescent probes, SYBR-14 and propidium iodide, PI (Live/Dead Sperm Viability Kit; Molecular Probes, Eugene, OR, USA), as described by Garner and Johnson (1995). Stained sperm slides were evaluated by means of fluorescence microscope (Olympus CH 30, Tokyo, Japan) equipped with blue and green excitations for SYBR-14 and PI, respectively. The SYBR-14 stained the DNA of membrane-damaged sperm cells, resulting in a bright green fluorescence, while PI only stained the nuclei of membranedamaged spermatozoa red. Two slides were evaluated per sample and 200 spermatozoa were counted per slide. The percentage of spermatozoa with normal acrosomal ridges (NAR) was estimated using Giemsa staining method described by Watson (1975). Spermatozoa which did not show a clear and uniform acrosomal ridge were considered to have altered acrosomes. Approximately 200 spermatozoa were counted at a random order under a light microscope equipped with oil-immersion lens at a magnification 1000×. 2.5.2. Mitochondrial activity The percentage of live spermatozoa with functional mitochondria was assessed using a combination of fluorescent stains, rhodamine 123 (Molecular Probes, Eugene, OR, USA) and PI, as described in a previous study (Fraser et al., 2002). Rhodamine 123 was prepared from a stock solution of 5 mg/ml anhydrous dimethyl sulfoxide (DMSO) and was stored in aliquots of 30 ␮l until required. Sperm cells unstained with PI and stained green with R123 at the mid-piece region were considered viable spermatozoa with functional mitochondrial. A minimum of 100 cells per slide was examined in random fields of each aliquot, using an epifluorescence microscope (Olympus CH 30, Tokyo, Japan). 2.6. Statistical analysis All results are expressed as mean ± S.E.M. The duplicate samples were averaged per treatment to be used as single data for statistical analysis. Normality was assessed by the Shapiro Wilk Wtest. The percentile data of sperm DNA integrity was subjected to arcsine transformation, while the variables of both comet tail length and tail moment were log-transformed to achieve normally distributed data prior to one-way analysis of variance (ANOVA). Data were subjected to a mixed factorial design with repeated measurements ANOVA using the general linear model (GLM) procedure from Statistica software package, Version 5 (StatSoft Incorporation, Tulsa OK, USA). The characteristics of the model included ejaculate collection procedure (whole ejaculate, sperm-rich fraction), extender (lactose-HEY-G, lactose-LPFo, K-3) and package (aluminium tubes, plastic straws). Arcsine-transformed and the log-transformed data were transformed back to the original values to be shown in figures. Multiple comparisons were made using the Neuman–Keuls post hoc test. Treatments were considered significant at P < 0.05. 3. Results 3.1. Sperm DNA assessment The procedure of ejaculate collection (whole ejaculate, sperm-rich fraction) and extender type (lactose-HEY-G, lactose-LPFo, K-3) significantly affected (P < 0.001) post-thaw DNA integrity (Table 1). A significant effect (P < 0.05) in post-thaw sperm DNA integrity was obtained for all the first-order interactions: ejaculate collection × extender; ejaculate collec-

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Table 1 ANOVA sources in variation in sperm DNA parameters Source

Ejaculate collection (Ej) Extender (E) Package (P) Ej × E Ej × P E×P Ej × E × P

d.f.

1 2 1 2 1 2 2

Sperm DNA integrity

Tail length DNA

Tail moment DNA

F

P-value

F

P-value

F

P-value

25.03 191.67 0.52 15.29 7.41 18.94 103.11

<0.001 <0.001 >0.476 <0.001 <0.001 <0.001 <0.001

0.49 22.29 1.23 47.38 2.64 25.44 105.66

>0.485 <0.001 >0.276 <0.001 >0.114 <0.001 <0.001

1.04 18.27 3.75 27.25 0.07 27.25 105.20

>0.315 <0.001 >0.061 <0.001 >0.792 <0.001 <0.001

A three-way ANOVA (2 × 3 × 2) with repeated measures was used to analyze the interactions of the main effects: semen collection (whole ejaculate, sperm-rich fraction), extender type (lactose-HEY-G, lactose-LPFo-G, K-3) and packaging materials (aluminium tubes, plastic straws). d.f.: degree of freedom; F: Fisher test.

tion × package; extender × package (Table 1). Similarly, the second-order interaction ejaculate collection × extender × package was significant (P < 0.001). Post-thaw sperm DNA fragmentation in the whole ejaculate or the sperm-rich fraction of boar semen following freezing–thawing in 10-ml aluminium tubes or 5-ml plastic straws is shown in Fig. 2A. Freezing–thawing of boar semen caused a significant increase (P < 0.05) in sperm DNA fragmentation, regardless of the extender type and packaging materials. It was observed that post-thaw sperm DNA fragmentation was significantly lower (P < 0.05) in the whole ejaculate compared with the sperm-rich fraction. Post-thaw sperm DNA fragmentation in whole ejaculate or sperm-rich fraction for sperm samples frozen in lactose-HEY-G or lactose-LPFo-G extender was significantly lower (P < 0.05) than those frozen in K-3 extender, without cryoprotective substances. We did not find any significant differences (P > 0.05) in post-thaw DNA fragmentation between sperm samples frozen in lactose-HEY-G and lactose-LPFo-G extenders. The extent of sperm DNA damage, as measured by the comet tail length and tail moment, was affected (P < 0.001) by the extender type following freezing–thawing (Table 1). Furthermore, the interactions exerting a significant influence (P < 0.001) on these analyzed comet assay parameters were ejaculate collection × extender and extender × package (Table 1). A very highly significant the second-order interaction ejaculate collection × extender × package was found (P < 0.001) for both post-thaw comet tail length and tail moment. Variations in comet tail length and tail moment of sperm DNA were less marked (P < 0.05) in lactose-HEY-G or lactose-LPFo-G extender compared with K-3 extender following freezing–thawing, irrespective of the ejaculate collection procedures and packaging materials (Fig. 2B and C). There was a trend towards increased post-thaw tail length and tail moment in K-3 extender, indicating that the level of DNA damage was more extensive in those samples frozen in the absence of cryoprotective substances. Furthermore, no significant differences (P > 0.05) in comet tail length or tail moment between sperm samples frozen in lactose-HEY-G and lactoseLPFo-G extender were observed, regardless of the ejaculate collection procedures. 3.2. Sperm motility, plasma membrane integrity and mitochondrial function The characteristics of boar spermatozoa prior to extension and freezing are illustrated in Table 2. In this study there were no significant differences in sperm charactertics between boars prior to freezing–thawing.

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Fig. 2. Sperm DNA damage in whole ejaculate (n = 35) or sperm-rich fraction (n = 37) of boar semen following freezing–thawing in aluminium tubes or plastic straws. (A) Sperm DNA fragmentation. (B) Comet tail length of sperm DNA. (C) Comet tail moment of sperm DNA. Values are expressed as mean ± S.E.M. A Neuman–Keuls post hoc test was used for multiple comparisons. Values with different letters (a, b, c, d) differed significantly (P < 0.05).

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Table 2 Characteristics of spermatozoa from whole ejaculate or sperm-rich fraction of boar semen prior to freezing–thawing Sperm variable

Semen collection type Whole ejaculate

Motility (%) Concentration (×106 /ml) Plasma membrane integrity (SYBR14/PI) (%) Mitochondrial function (R123/PI) (%) Normal acrosome ridges (%)

72.7 394.2 86.1 89.5 92.5

± ± ± ± ±

Sperm-rich fraction

1.0 15.6 1.1 1.3 0.5

73.8 856.3 88.9 89.4 88.1

± ± ± ± ±

0.5 30.8 0.4 0.6 1.4

Values are expressed as mean ± S.E.M.

The effect of ejaculate collection procedure and package affected post-thaw sperm motility (P < 0.001), plasma membrane integrity (P < 0.002) and mitochondrial function (P < 0.001), as shown in Table 3. The only interaction exerting a significant defect on sperm motility and mitochondrial function was ejaculate collection × package (P < 0.001). Also, there were no significant variations (P > 0.05) in ejaculate collection, extender and packaging materials when assessed for normal acrosome integrity following freezing–thawing. Post-thaw motility, plasma membrane integrity and mitochondrial function of boar spermatozoa frozen in 10-ml aluminium tubes or 5-ml plastic straws are shown in Table 4. It was observed that reduced post-thaw sperm motility was reflected in impaired mitochondrial function, independent on the ejaculate collection procedures, extender type and packaging materials. Furthermore, post-thaw sperm motility was higher (P < 0.05) in samples of the sperm-rich fraction when frozen in lactose-HEY-G or lactose-LPFo-G extender compared with those of the whole ejaculate. This was accompanied by higher percentage of spermatozoa exhibiting rhodamine 123 stain, indicating those viable spermatozoa within the total population with functional mitochondria. These changes were more attenuated in samples frozen in aluminium tubes. In both lactose-HEY-G and lactoseLPFo-G extenders sperm plasma membrane integrity in whole ejaculate was markedly lower (P < 0.05) in straws compared with aluminium tubes following freezing–thawing (Table 4). The proportions of spermatozoa with normal acrosomal ridges were not differed (P > 0.05) between samples frozen in lactose-HEY-G and lactose-LPFo-G extenders. Freezing–thawing had a deleterious effect on characteristics of spermatozoa frozen in the absence of cryoprotective substances (Table 4).

Table 3 ANOVA sources of variation in sperm charactertics Source

Ejaculate collection (Ej) Extender (E) Package (P) Ej × P

d.f.

1 1 1 1

Motility

Plasma membrane integrity

Mitochondrial function

F

P-value

F

P-value

F

P-value

136.18 1.05 77.01 10.96

<0.001 >0.313 <0.001 <0.002

10.80 2.60 85.54 0.07

<0.002 >0.116 <0.001 >0.832

44.59 0.76 27.52 13.07

<0.001 >0.391 <0.001 <0.001

A three-way ANOVA (2 × 2 × 2) with repeated measures was used to analyze the interactions of the main effects: semen collection (whole ejaculate, sperm-rich fraction), extender type (lactose-HEY-G, lactose-LPFo-G) and packaging materials (aluminium tubes, plastic straws). d.f.: degree of freedom; F: Fisher test.

Sperm viables (%)

Whole ejaculates

Sperm-rich fraction

Aluminium tubes L-HEY Motility Plasma membrane integrity (SYBR-14/PI) Mitochondrial function (R123/PI) Normal acrosome ridges

Plastic straws L-LPFo

K-3

L-HEY

Aluminium tubes L-LPFo

Plastic straws

K-3

L-HEY

L-LPFo

K-3

L-HEY

L-LPFo

K-3

32.3 ± 1.1 a 52.6 ± 1.4 a

30.7 ± 1.2 a 52.2 ± 1.3 a

2.7 ± 0.9 b 8.8 ± 0.8 b

26.7 ± 0.9 c 46.1 ± 0.9 c

27.9 ± 0.8 c 45.5 ± 1.1 c

2.2 ± 0.6 b 8.2 ± 0.9 b

42.6 ± 1.5 d 57.8 ± 1.4 a

39.6 ± 1.5 d 54.2 ± 1.1 a

3.1 ± 0.8 b 8.2 ± 0.9 b

32.6 ± 1.1 a 48.8 ± 0.9 c

31.9 ± 0.7 a 48.3 ± 1.2 c

2.4 ± 0.5 b 7.2 ± 1.2 b

44.3 ± 0.9 ad

45.4 ± 1.1 ad

7.8 ± 0.9 b

42.9 ± 0.9 a

43.8 ± 1.0 ad

6.2 ± 0.9 b

55.7 ± 1.1 c

51.7 ± 1.2 c

5.3 ± 1.1 b

46.7 ± 1.1 d

45.6 ± 1.2 ad

4.9 ± 1.1 b

49.1 ± 1.2 a

49.7 ± 1.3 a

5.2 ± 1.1 b

52.6 ± 1.6 a

51.6 ± 1.3 a

7.2 ± 1.1 b

52.9 ± 1.2 a

50.7 ± 1.9 a

9.8 ± 1.7 b

50.6 ± 1.2 a

49.4 ± 1.3 a

8.2 ± 0.9 b

Values are expressed as the mean (±S.E.M.) of 35 samples (n = 35) and 37 samples (n = 37) for the whole ejaculate and sperm-rich fraction, respectively. A Neuman–Keuls post hoc test was used for multiple comparisons. Values within the same row with different letters (a, b, c, d) differed significantly (P < 0.05). L-HEY: extender with lactose/whole hen egg yolk and glycerol; L-LPFo: extender with lactose/lyophilized lipoprotein fractions of ostrich egg yolk and glycerol; K-3: Kortowo 3 extender without cryoprotective substances.

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Table 4 Characteristics of post-thaw boar spermatozoa following freezing–thawing in aluminum tubes or plastic straws

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4. Discussion Accordingly, the intent of this study was to show that freezing–thawing procedure had different effects on DNA integrity of boar spermatozoa, depending on the sperm source and extenders. Our data indicate that spermatozoa of the whole ejaculate were less vulnerable to cryo-induced DNA fragmentation compared with those of the sperm-rich fraction. We suggest that this difference in DNA fragmentation might be related to the antioxidant properties of the whole seminal plasma, which was used at the initial stage of the freezing–thawing procedure. Moreover, it has been reported that boar seminal plasma contains low molecular weight antioxidants, and proteins with antiperoxidant properties (Strze˙zek et al., 1999). These authors have confirmed that there is a high correlation between total protein content and antioxidant properties of the seminal plasma. Furthermore, these authors have suggested an important role of the vesicular gland secretion, which probably have a protective effect on boar spermatozoa against peroxidative damages. Accumulating evidence has been shown that increased level of oxidative stress induced by reactive oxygen species (ROS) during freezing–thawing studies can enhance sperm DNA fragmentation (Baumber et al., 2003; Chatterjee and Gagnon, 2001). In the present study we show that spermatozoa maintained in their own seminal plasma prior to its removal for extension intended for freezing–thawing were less vulnerable to DNA fragmentation, raising the possibility that factors within the seminal plasma afforded partial protection to sperm DNA integrity. It has been shown that proteins of boar seminal plasma, originating in the epididymis and accessory sex glands, can bind specifically to sperm plasma membrane and modulate sperm function (Strze˙zek et al., 2005b). Furthermore, in our laboratory it has been demonstrated that lyophilized lipoprotein fractions isolated from ostrich egg yolk interact with seminal plasma protein complex (es), resulting in enhanced antiperoxidant activity (Lecewicz et al., 2005). With this in mind, it is likely that the mutual interactions of plasma membrane proteins with components of egg yolk lipoprotein may form an environment with high antiperoxidant activity for spermatozoa of the whole ejaculate during freezing–thawing. The exact mechanism of action is not fully known, but it appears that enhanced antiperoxidant activity may provide protection against ROS-induced sperm DNA damage in the whole ejaculate. Even though the biological effect of seminal plasma on sperm DNA integrity has not been well understood, a previous study showed that freezing of human spermatozoa in seminal plasma improved post-thaw DNA integrity (Donnelly et al., 2001). According to Potts et al. (2000), the addition of seminal plasma to incubation media reduced DNA damage to human spermatozoa. Taken together, these studies reassured the beneficial effect of whole seminal plasma on DNA integrity of human spermatozoa. In contrast, Love et al. (2005) reported that stallion seminal plasma had a detrimental effect on sperm DNA integrity during 24 and 48 h of storage. However, the authors postulated that the discrepancy between the different outcomes regarding the effects of human and stallion seminal plasma on sperm DNA integrity might be related to the high sodium concentrations occurring in equine semen compared with other species. Little is known about the nature of the protective effect of antioxidants on sperm DNA integrity, particularly after freezing–thawing of boar semen. It is worth noting that the presence of cryoprotective substances helps to reduce the sperm susceptibility to cryo-induced DNA fragmentation. Furthermore, it should be stressed that throughout the entire experiments sperm samples frozen-thawed in lactose-LPFo-G extender gave similar results to those frozen-thawed in lactose-HEY-G extender. The comet assay parameters, tail length and tail moment, provide additional evidence about the level of DNA damage sustained by frozen-thawed sperm cells, and therefore increase the sensitivity of the comet assay in detecting low levels of DNA damage. In this study the comet

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assay parameters did not detect differences in post-thaw sperm DNA damage between the whole ejaculate and sperm-rich fraction and might be due to the inherent heterogeneity of the populations of sperm cells within the individual or ejaculate. However, there was extensive DNA damage to spermatozoa frozen in the absence of cryoprotective substances, indicating increased vulnerability to cryo-induced DNA damage. Although the implications of using DNA damaged spermatozoa are fully known, there is a cause for concern. Studies have been shown that the comet assay correlates sperm DNA damage with implantation success after intracytoplasmic sperm injection, ICSI (Morris et al., 2002) and with embryo quality (Tomsu et al., 2002). Moreover, evidence has been shown that human sperm containing high loads of DNA damage, detected by the comet assay, are related to embryo failure to develop after ICSI (Twigg et al., 1998b; Morris et al., 2002). These authors reaffirmed that DNA damaged spermatozoa would fertilize less efficiently and might result in embryo failure. However, oocytes and embryo can repair DNA damage and there is a threshold beyond which sperm DNA cannot be repaired (Zini et al., 2005). In the current study evaluation of post-thaw sperm DNA integrity is presented simultaneously with sperm characteristics, such as motility, plasma membrane integrity and mitochondrial function of spermatozoa, to show that there were differential effects in response to induced oxidative stress after freezing–thawing. Our results indicate that the extent of damage caused by freezing–thawing to sperm motility was concurrent with impaired mitochondrial function. Another observation from this study was that some sperm populations with high post-thaw sperm DNA fragmentation exhibited high motility and mitochondrial activity. Similar findings were reported by Morris et al. (2002), which showed that high sperm motility was concomitant with high DNA damage in the human sperm populations. In a previous study it was demonstrated that spermatozoa exposed to oxidative conditions could possess damaged DNA yet have intact motility and improved capacity for sperm-oocyte fusion (Aitken et al., 1998). These findings are in contrast to other studies which showed that sperm samples with low motility carried high loads of DNA damage (Irvine et al., 2000; Zini et al., 2001). We propose that freezing–thawing had different effects on post-thaw sperm charactertics and DNA integrity, indicating that different measures of sperm function can respond independently to environmental factors, such as oxidative stress. Furthermore, another potential source of variation of boar spermatozoa to cryo-induced injury may be the consequence of individual characteristics. Studies have been shown that there are individual differences between boars in the ability of spermatozoa to withstand cryopreservation and have indicated that inter-male variability in sperm freezability may be genetically inherited (Holt, 2000; Thurston et al., 2002). The results of the current study implicate that routine semen parameters may not always reflect the quality of sperm DNA and reaffirm previous findings, indicating that multiple characteristics are required on individual spermatozoa to assess their physiological state (Prathalingam et al., 2006). 5. Conclusions Our study represents the first comparative investigation of sperm DNA integrity in the whole ejaculate and sperm-rich fraction of boar semen. The results have revealed a low cryo-induced DNA fragmentation to spermatozoa of the whole ejaculate, which originate from an environment with high antioxidant capacity. These findings reinforce the importance of seminal plasma components that are involved in the DNA protection of boar spermatozoa and indicate that spermatozoa harvested from the whole ejaculate have reduced vulnerability to cryo-induced oxidative stress during freezing–thawing. However, further studies are warranted to define the mechanism

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