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RESEARCH |
1 Unit for Reproductive Medicine of Clinics for Pigs and Small Ruminants2 Clinic for Cattle3 Institute for Reproductive Biology, University of Veterinary Medicine Hannover, Bünteweg 15, D-30559 Hannover, Germany
Correspondence should be addressed to D Waberski; Email: dagmar.waberski{at}tiho-hannover.de
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Abstract |
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Introduction |
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The objective of the present study was to investigate the prevalence of chromatin instability in the fertilizing-competent sperm population in the porcine oviduct in vivo through qualitative analysis of the chromatin structure status of accessory sperm found in in vivo-derived embryos. In addition, we studied whether, and to what extent, boar sperm with unstable chromatin have the capacity to bind to the oviductal epithelia in vitro; similar binding in vivo is an essential step in establishing the oviductal sperm reservoir.
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Presence of chromatin-unstable sperm in boar ejaculates
Prevalence of chromatin instability
85.5% of the semen samples evaluated (n=173, one per boar) showed <5% of sperm with unstable chromatin. The range was 0.1–19% and the mean for all samples was 3.2% chromatin-unstable sperm.
Persistency of chromatin instability
In 15 boars showing >5% chromatin-unstable sperm in an initial semen sample, two further ejaculates collected on successive weeks were investigated. Six of these boars showed elevated percentages of chromatin-unstable sperm only in the first semen sample. However, seven showed constant elevated numbers of chromatin-unstable sperm in all three samples. The mean percentage of chromatin instability in boars with three successive samples containing >5% chromatin-unstable sperm was significantly higher (P<0.05) than in boars with either one or two samples with enhanced chromatin instability (Table 1).
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5%) percentages of sperm with unstable chromatin than in those showing low (<5%) percentages of this defect (Table 2). The percentage of sperm with unstable chromatin correlated significantly with the percentage of sperm with cytoplasmic droplets (r=0.44, P<0.01, n=128 ejaculates from 54 boars; Fig. 2). No correlation was found between the percentage of sperm with unstable chromatin and other standard sperm parameters, i.e., motility and morphological abnormalities other than attached cytoplasmic droplets (data not shown).
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Discussion |
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In conclusion, the results of this study suggest that numbers of boar sperm with unstable chromatin are reduced in the oviductal sperm reservoir in vivo, possibly because of associated changes in the plasma membrane, which prevent sperm from binding to the oviductal epithelium. Association with other sperm defects might also hinder the entrance of chromatin-unstable sperm into the oviducts. Thus, in vivo the likelihood that boar sperm with unstable chromatin will reach the eggs and fertilize them is low.
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Materials and Methods |
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Semen and oviducts
The semen was generously provided by a local artificial insemination (AI) center and delivered overnight using an express delivery service. The semen doses had been diluted at the AI center in Beltsville Thawing Solution (Johnson et al. 1988) and contained a total of 2x109 sperm cells. The slaughterhouse of the city of Hannover donated the oviducts of multiparous sows that had been sent for commercial slaughter. Only oviducts from clinically normal genital tracts were collected.
Animals
The 14 gilts for the in vivo experiment were bought from a nearby commercial farm. They were 6–8 months old, weighted at least 90 kg, and were housed in groups of two to three at the facilities of the Unit for Reproductive Medicine. The animals were treated according to the Animal Welfare Act of the German Federal Ministry, consistent with the International Guiding Principles for Biomedical Research Involving Animals as promulgated by the Society for the Study of Reproduction.
Modified fluorescence microscopic sperm chromatin structure assay (mfSCSA)
Essentially, the methods of Evenson et al. (1980), Tejada et al. (1984), Kosower et al. (1992), and Acevedo et al. (2002) were combined while including essential modifications of our own.
Media
The citrate buffer solution was composed of 98.6 mM trisodium citrate and 11.5 mM EDTA (pH 6.8). The dithiothreitol/dimethyl sulfoxide (DTT/DMSO) solution was prepared by adding 3.2 ml hydrated DMSO to 30 ml trisodium citrate buffer solution containing 10 mM DTT; the final DTT concentration was 8.6 mM. The Carnoy solution (Tejada et al. 1984) was prepared with methanol and acetic acid at a ratio of 2:1. The acridine orange staining solution was prepared by mixing 2.5 ml of 376.2 mM sodium phosphate dibasic stock solution, 40 ml of 100 mM citric acid monohydrate stock solution, and 10 ml of 3.3 mM acridine orange stock solution; all stock solutions were prepared in distilled water, kept at 4 °C, and used within a month.
Washing of semen and preparation of slides
Four milliliters of semen were mixed with 2 ml citrate buffer solution and centrifuged at 2100 g for 10 min. The supernatant was discarded and 2 ml citrate buffer added. The samples were mixed thoroughly and centrifuged once again at 2100 g for 10 min. The supernatant was removed leaving a small amount for resuspension of the sperm pellet. A 10 µl droplet of resuspended sperm was placed on a Superfrost Plus slide (Carl Roth) and smeared using another slide. The slides were air dried for at least 20 min and kept refrigerated until further processing.
Disulfide reduction (chromatin decondensation)
This step took place under an extractor at a temperature between 18 and 23 °C. The slides were placed horizontally on test tube racks, taking care that they were as level as possible. Then, each slide was completely covered with 2 ml DTT/DMSO solution. They were left to react for 30 min. Then, each slide was rinsed with citrate buffer using a wash bottle and placed in a Hellendahl vertical glass stain jar, previously filled with 30 ml buffer. The slides were left in the jar for 10 min, then taken out, wiped once on the sides and back with absorbent paper, and placed vertically against the test tube racks to air dry for 20 min.
Acid denaturation
This step took place at room temperature under an extractor while preventing direct contact of the samples with light. Sixty milliliters of Carnoy solution were prepared in a vertical Hellendahl glass stain jar while the slides were being air dried. The jar was covered with an aluminum foil. The air-dried slides were placed in the jar and left to react for 100 min; then they were taken out, wiped once on the sides and back with absorbent paper, and placed vertically against the test tube racks to air dry for at least 10 min.
Staining with acridine orange
The slides were protected from direct contact with light during this step, which was carried out strictly at 4 °C. A pre-cooled, vertical Hellendahl glass stain jar was placed in a pre-cooled water bath. The acridine orange staining solution was prepared in the stain jar. The slides were placed in the jar and left for 20 min. Then slides were taken out, wiped once on the sides and back with absorbent paper, and placed in a pre-cooled jar filled with 30 ml citrate buffer. They were left in the buffer solution for 10 min. Then they were taken out, wiped again, and placed vertically against the test tube racks to air dry for at least 20 min. Once the slides were dry, they were stored in a slide storage box at 4 °C until evaluation.
An aliquot of a frozen control sample of sperm was processed with each staining batch to assure that the factors related to each work day, such as room temperature and humidity, would not influence the results. Temperature, humidity, and any abnormal circumstances were noted to determine their influence on the slides' quality.
Fluorescence microscopy and digital image analysis
The Zeiss Axioscope fluorescence microscope (Jena, Germany) was set to 200x, phase 2, 450–490 filter, FT 510, LP 520. The AnalySIS 3.0 (Soft Imaging System, Münster, Germany) computer software was used for the evaluation. The software was set to recognize as sperm particles bigger than 700 pixels, to prevent the detection of small dust particles as sperm.
The classification of sperm is based on the fluorescence acquired by the DNA after acridine orange staining. Acridine orange intercalates between the stacked bases of double-stranded (unfragmented, stable) DNA (ds-DNA) and fluoresces green at 530 nm. In the case of single-stranded (fragmented) DNA (ss-DNA), stacked arrays of acridine orange bind to the phosphate backbone of the nucleotide and fluoresces red at 640 nm.
The exposure time for the digital camera Olympus DP 50 (Olympus, Hamburg, Germany) was set at 
40 ms, the light sensitivity to ISO 2000, and the correction of the color scale was set to 0.9, 0.9, and 1.57 for red, green, and blue respectively. The fields were selected for evaluation using the ‘live-window’ feature of the software and photographed at a resolution of 2776x2074 pixels.
Threshold values for each fluorescence channel were set for the classification of sperm. For this, representative red and green sperm in a field were selected and their intensity profile was evaluated. The threshold values referred to the quantity of green and red colors found in green- and red-appearing sperm. After the threshold values were chosen, the sperm were classified either automatically or manually as either chromatin stable (ds-DNA, green fluorescence) or chromatin unstable (ss-DNA, red fluorescence). The same threshold values were used for all pictures taken from a given slide. Manual classification was used where few sperm per field were available or unspecific fluorescence was present from oviductal or embryonic cells. A minimum of four fields and at least 500 sperm cells per slide were analyzed. Regions with unequal staining, i.e., the edges and the center of the slide were not considered for evaluation.
Reliability mfSCSA data
The original microscopic AOT described by Tejada et al. (1984) was found to give variable results due to indistinct colors, rapid fading, and heterogenous slide staining (Evenson et al. 2002, Chohan et al. 2006). Considering the SCSA as gold standard (Chohan et al. 2006), two experiments were performed to show that the mfSCSA, as used in the present study, gave results comparable with the SCSA data. In the first experiment, the results of mfSCSA were compared with the results obtained from flow cytometric SCSA using split semen samples of 40 boar ejaculates snap frozen in liquid nitrogen. The ability to freeze raw or extended semen in liquid nitrogen without affecting SCSA parameters has been shown previously (Evenson & Jost 1994, Evenson et al. 2002) and was confirmed in our own experiments (data not shown). The flow cytometric SCSA was carried out following the procedure described by Evenson & Jost (1994) using the equipment, chemicals, and data analysis software in same laboratory as described by Schmid et al. (2003). In the second experiment, 30 slides obtained from the semen samples from 30 different boars were processed with the mfSCSA and each slide was evaluated on two different days. The first evaluation was performed at the day of processing and the second evaluation between 6 and 11 months later. During the time between evaluations, the slides had been kept in the dark at 4 °C. Classification of green (chromatin-unstable) and red (chromatin-unstable) sperm were compared between the first and second days of analysis.
Presence of chromatin-unstable sperm in boar ejaculates
For the selection of boars with different percentages of chromatin-unstable sperm, single diluted semen samples from 173 boars were analyzed using the mfSCSA. In 54 boars, sperm morphology (as per Hancock 1956) and motility were additionally evaluated. In 15 boars with elevated percentages of chromatin-unstable sperm in the first semen sample, a further two ejaculates were collected and investigated in successive weeks.
Chromatin status in sperm bound to oviductal epithelium in vitro
Oviduct preparation
Fallopian tubes were obtained at the local abattoir from sows at different cycle stages. The oviducts were transported to the laboratory in ice-cold PBS. The protocol essentially follows the oviductal explant assay as described by Petrunkina et al. (2001a) with few modifications. Briefly, oviducts were placed in a Petri dish containing ice-cold PBS, stripped from all surrounding tissue, and then opened longitudinally. Oviducts were then fixed with needles on a Petri dish filled with paraffin wax. To maximize the number of sperm bound to oviductal epithelium, long pieces (7–9 cm long, 0.6–0.8 cm wide) of isthmic longitudinal folds of the oviductal epithelia were dissected out under a stereomicroscope. For each assay, two such oviductal strips, each from a different sow, were then put in a small Petri dish containing 2.5 ml modified TALP medium (Petrunkina et al. 2001a). The oviductal strips were refrigerated while the sperm were prepared.
Sperm preparation
This experiment compared semen from two boars with <5% chromatin-unstable sperm (boar 1: n=4 ejaculates and boar 2: n=4 ejaculates) with semen from two further boars with >5% chromatin-unstable sperm in all ejaculates (boar 3: n=3 ejaculates and boar 4: n=3 ejaculates). Sperm samples were evaluated for motility and morphology using the criteria of Krause (1965). Sperm data are given in Table 3. Ten milliliters of diluted semen containing 25x106 sperm/ml were subjected to centrifugation at 170 g for 10 min. Four milliliters of the resuspended sperm pellet were subjected to discontinuous (70/35) Percoll gradient centrifugation (Harrison et al. 1993). The sperm pellet was resuspended with 500 µl modified Androhep without EDTA (Petrunkina et al. 2001a) and placed in a 1.5 ml Eppendorf vial.
Co-incubation of sperm and oviductal strips
The Eppendorf vial with resuspended sperm and the Petri dish containing the pair of oviductal strips in 3.5 ml TALP medium were equilibrated at 39 °C for 5 min in a humidified atmosphere containing 5% CO2. Five hundred microliters of resuspended semen with a concentration of 35x107 sperm/ml were added to the Petri dish containing the oviductal strips. The samples were co-incubated for 30 min at 39 °C in a humidified atmosphere containing 5% CO2 to allow maximum sperm binding (Waberski et al. 2006). At the end of co-incubation, free-swimming spermatozoa were removed by extensive washings with TALP. The washings consisted of ‘grasping’ the strips with medium dissecting forceps, moving the strips vigorously around the Petri dish, and transferring them in another Petri dish containing 4 ml TALP. Three washes in all were performed. The oviductal strips with the bound spermatozoa were then transferred to 1 ml TALP in a 1.5 ml Eppendorf vial.
Sperm release
Virtually all sperm were released from oviductal strips by snap freezing in liquid nitrogen for 10 min. After thawing at 39 °C for 5 min, the oviductal strips were carefully removed. A few oviductal cells left in the suspension of released sperm were largely removed by centrifugation at 170 g for 10 min. The released sperm (suspension in TALP) were submitted to the mfSCSA as described above.
Chromatin status in accessory sperm from in vivo-derived embryos
Semen
Semen from two boars with >5% chromatin-unstable sperm (boars 5 and 6) and one boar with <5% chromatin-unstable sperm (boar 7) in at least three successive ejaculates was used. Sperm samples were evaluated for motility and morphology using the criteria of Krause (1965). Aliquots of semen were snap frozen in liquid nitrogen for the mfSCSA. Sperm data are given in Table 4.
Estrus detection, insemination, and recovery of embryos/oocytes
This experiment was performed following the Hannover Gilt Model (HGM) guidelines (Ardón et al. 2003) with few modifications. The HGM includes the use of spontaneous ovulating gilts in their second estrus after stalling, estrus detection, and only one insemination in the presence of boars, and detection of ovulation via transabdominal ultrasound observation of the ovaries every 12 h. In the present study, gilts were inseminated repeatedly with the purpose of maximizing the number of sperm recovered. The first insemination took place 12–18 h after first detection of estrus. Inseminations were repeated every 12 h, as long as the gilt showed the standing reflex. Reproductive tracts of the gilts were recovered after slaughter, 3–5 days after ovulation. The reproductive tracts were kept at 37 °C and immediately transported to the laboratory. The embryos and oocytes were flushed from the reproductive tracts and their morphology was evaluated as described by Ardón et al. (2003). As seen in earlier studies (Ardón et al. 2003, 2005), no sperm bound to the outer zona pellucida were present.
Evaluation of the chromatin structure in accessory sperm
The sperm chromatin structure was evaluated in the accessory sperm present in the zona pellucida of embryos. Accessory sperm are those found trapped in the zona pellucida; they interacted and partially penetrated the zona pellucida due to incomplete zona block in the pig (Hunter 1997). Embryos were mounted on Superfrost Plus slides using a micropipette. Slides were air dried and then refrigerated until treated. During this procedure, the zona pellucida bursts and the sperm disperse on the slide. The sperm on the slides were subjected to the mfSCSA as described above. The semen used for insemination, which had been snap frozen in liquid nitrogen, was subjected to the mfSCSA at the same time as the accessory sperm.
Statistical analysis
Data are presented as arithmetic means with standard deviations calculated using the Excel software. All other statistical analyses were performed using SAS software (SAS Institute Inc., Cary, NC, USA). The correlation of two evaluations of the same slide was analyzed with the Spearman correlation (procedure CORR); the similarity between both evaluations was analyzed using the Kruskal–Wallis test (procedure NPAR1WAY) and the Student's t-test for paired samples. The following analyses were performed using the Wilcoxon test: effects of boars on sperm parameters, effects of sperm preparations (diluted, Percoll washed, oviduct bound, and accessory sperm) on the percentage of chromatin instability, effect of the embryo morphology on chromatin instability in accessory sperm. The Spearman correlation was also used to evaluate the correlation between chromatin instability and standard sperm parameters. The Spearman correlation and the Wilcoxon test were chosen because the data were not normally distributed. The criterion for significance was P<0.05.
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Acknowledgements |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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Received 19 July 2007
First decision 4 September 2007
Revised manuscript received 6 December 2007
Accepted 3 January 2008
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References |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsAcknowledgementsReferences |
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