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Influence of different centrifugation protocols on equine semen preservation
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Theriogenology 74 (2010) 118–126 www.theriojournal.com
Influence of different centrifugation protocols on equine semen preservation Maarten Hoogewijs *, Tom Rijsselaere, Sarne De Vliegher, Emilie Vanhaesebrouck, Catharina De Schauwer, Jan Govaere, Mirjan Thys, Geert Hoflack, Ann Van Soom, Aart de Kruif Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 29 June 2009; received in revised form 18 January 2010; accepted 31 January 2010
Abstract Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 g for 10 min = CP1) was compared to four protocols with increasing g-force and decreased time period (600 g, 1200 g, 1800 g and 2400 g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 g and 2400 g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P < 0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P < 0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P < 0.005), whereas CP3 and CP5 yielded a lower BCF (P < 0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800 g or 2400 g for a shorter period of time (5 min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate. # 2010 Elsevier Inc. All rights reserved. Keywords: Semen; Centrifugation; Sperm loss; Stallion; Sperm quality parameters
1. Introduction Numerous studies have demonstrated the beneficial effect of diluting raw semen with an appropriate * Corresponding author. Tel.: +32 9 264 75 61; fax: +32 9 264 77 97. E-mail address: [email protected] (M. Hoogewijs). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.01.022
extender. As such, the negative influence of seminal plasma (SP) on the preservation of equine sperm can be reduced. Centrifugation of diluted equine semen and subsequent resuspension of the sperm pellet in fresh extender can even further reduce the amount of SP in stored samples. A small proportion of SP in semen improves sperm motility after cooling and storage [1].
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However, centrifugation itself may act like a double edged sword exerting both beneficial and deleterious effects on sperm motility [2]. Although the number of sperm cells can be maximized by increasing centrifugation time and force, physical damage is exerted on the sperm cells at the same time. The direct negative outcome of centrifugation on semen quality is demonstrated by the decrease in motility and velocity in contrast to uncentrifuged controls [1]. After 24 h of cooled storage the harmful effect of centrifugation is no longer present [1] which indicates that the centrifugation protocol (CP) may play an important role in the quality of the inseminate. A CP of 600 g for 10 min is commonly used for equine semen [3–6]. In literature, data on the amount of sperm loss after using this protocol are contradictory and vary from 1.9% [6] to 25% [3,4]. Nowadays, it is generally accepted that sperm losses will vary around 25% when diluted semen is centrifuged at 400-600 g for 10–15 min [7,8]. The importance of an appropriate CP has been clearly demonstrated for human sperm. The duration of centrifugation was shown to be more important than the centrifugation force for causing iatrogenic sperm membrane injuries which resulted in an increased formation of reactive oxygen species [9]. In boars, similar findings were described: a CP with a high g-force for a short time (2400 g for 3 min) was used without detrimental effects on sperm yield compared to the standard regime (800 g for 10 min) [10]. Additionally, a positive effect on semen quality after cryopreservation was detected when using high g-forces for a shorter period of time [10]. Experiments with equine semen have been performed where semen was centrifuged at 400 g for an increasing period of time. Sperm loss was approximately 20%, if samples were centrifuged for 10 min or longer, while no adverse effects on motility immediately after centrifugation were present, unless semen was centrifuged for 20 min. However, effects on preserved semen by cooling or freezing were not determined [11]. Since a high g-force for a short time was not detrimental for porcine sperm [10] and prolonged centrifugation of equine sperm negatively influenced sperm quality [11], using a short term high speed CP might be beneficial. The aim of the present study was to compare high speed CPs with the standard protocol on cooled and cryopreserved equine semen. The impact of CP on the subsequent number and quality of sperm cells was assessed daily during 3 consecutive days of cooled preservation. Additionally, the effect of different CPs on the quality of frozen-thawed equine sperm was investigated.
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2. Materials and methods 2.1. Stallions and semen collection For experiments 1 and 2, five ejaculates from each of five experienced Shetland ponies (n = 25), aged 3–6 yr, were collected between August and October 2007. For experiment 3, one ejaculate from 4 of these ponies was used. The ponies were housed in groups in a straw bedded stable and were fed good quality hay ad libitum. Prior to the unset of the experiment, the extra-gonodal sperm reserves were depleted with 3 collections every other day for 1 week. For the experiment, the stallions were collected twice a week. Semen was collected on a teaser mare using standard procedures with a custom made open ended artificial vagina based on the Colorado State University model as described by Pickett [12]. After collection, semen was filtered through a sterile gauze to remove the gel fraction and debris. The gel-free volume was noted and the sperm concentration was determined using a Bu¨rker hemocytometer after 1:9 dilution with HCl 1 M 2.2. Media Fresh-cooled semen was processed and stored in Gent Extender (Minitu¨b, Landshut, Germany), which is a skimmed milk based diluter containing 5% clarified egg yolk and gentamycin (1 mg/mL). If the semen was to be cryopreserved, a second dilution was performed in Gent Extender for Freezing (Minitu¨b, Landshut, Germany) which has the same composition as the Gent Extender plus 5% glycerol. For the fluorescent staining procedures, the samples were diluted in a HEPES buffered solution containing 0.04% BSA. 2.3. Semen processing After determination of the initial sperm concentration, the semen was diluted to a final concentration of 25 106 sperm/mL using Gent Extender. Six conical bottom centrifuge tubes of 15 mL (Cellstar1, Greiner bio-one, Germany) were filled with the diluted semen (total of 375 106 sperm cells per tube) and served as the negative control or were subjected to one of the five different CPs, respectively. After centrifugation, 90% of the supernatant was aspirated, leaving a sperm pellet with a volume of approximately 1.5 mL. The concentration in the aspirated supernatant was determined using a Neubauer hemocytometer. The obtained concentration was multiplied by the volume of
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aspirated supernatant in order to calculate the total number of sperm cells lost per tube when the supernatant was discarded. The remaining sperm pellet was resuspended in the appropriate diluter for further processing as cooled or frozen semen. 2.4. Evaluation of sperm characteristics and staining procedures Prior to analysis, an aliquot (500 mL) of semen was equilibrated to 37 8C. The morphology of sperm cells was examined on eosin-nigrosin stained smears which were prepared as described by Barth and Oko [13]. At least 200 sperm cells were evaluated and recorded per slide, individual morphological abnormalities were noted according to their location (head, midpiece or tail). Motility was evaluated with a computer-assisted sperm analyzer (CASA) (Hamilton-Thorne Ceros 12.3). For each analysis, 5 mL of diluted semen was mounted on a disposable Leja counting chamber (Orange Medical, Brussels, Belgium) and maintained at 37 8C using a minitherm stage warmer. Five randomly selected microscopic fields in the center of the slide were scanned 5 times each, obtaining 25 scans for every semen sample. The mean of the 5 scans for each microscopic field was used for the statistical analysis [14]. The software settings of the HTR 12.3, based on Loomis and Graham [15], are summarized in Table 1. Membrane integrity was evaluated using a fluorescent SYBR14-Propidium Iodide (PI) staining technique (Molecular Probes cat no.: L-7011, Leiden, The Netherlands) based on a previously described method [16]. Briefly, 225 mL HEPES-TALP was mixed with 25 mL of diluted semen and 1.25 mL SYBR14 (1:50 dilution) was added. After 5 min of incubation at 37 8C, 1.25 mL PI was added and incubated for another 5 min. Table 1 Software settings of the Hamilton Thorne Ceros 12.3 used in this study. Parameter
Value
Frames acquired Frame rate (Hz) Minimum contrast Minimum cell size (pixels) Minimum static contrast Straightness cut-off (%, STR) Average-path velocity cut-off PM (mm/s,VAP) VAP cut-off static cells (mm/s) Cell intensity Static head size Static head intensity Static elongation
30 60 60 6 25 75 50 20 100 0.55–2.04 0.45–1.70 11–99
Two hundred cells were evaluated per slide using a Leica DMR fluorescence microscope and three populations of sperm cells could be identified (green = living, red = dead and orange = moribund). Acrosomal status was determined using fluorescent Pisum Sativum Agglutinin (PSA) conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich cat no.: L 0770, Bornem, Belgium). The staining was performed in a similar way as described by Rathi et al. [17]. Briefly, 500 mL of semen was centrifuged for 10 min at 720 g and the pellet was resuspended with HEPES-TALP. The semen was centrifuged again for 10 min at 720 g, the supernatant was removed and the pellet fixed in 100 mL absolute ethyl alcohol (Vel cat no.: 1115, Haasrode, Belgium) and cooled for 30 min at 4 8C. A drop of 20 mL semen was smeared on a glass slide and 40 mL PSA-FITC (2 mg PSA-FITC diluted in 2 mL phosphate buffered saline (PBS)) was added. The glass slide was kept at 4 8C for 15 min, washed 10 times with aqua bidest and allowed to air-dry in the dark. Immediately after drying, 200 sperm cells were evaluated per slide. The acrosomal region of the acrosome intact sperm cells was labeled heavily green, while the acrosome reacted sperm retained only an equatorial band of label with little or no labeling of the anterior head region. In Experiment 3, DNA integrity was analyzed using a Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay. The TUNEL assay was performed using the In Situ Cell Death Detection Kit (Boehringer, Mannheim, Germany) to detect the presence of free 30 -OH termini in single and doublestranded sperm DNA [18]. In short, sperm samples were diluted with PVP solution (1 mg/mL in PBS) to a final concentration of 10 106 sperm/mL from which a 10 mL aliquot was smeared onto a poly-l-lysine-coated microslide. After fixation with 4% paraformaldehyde in polyvinyl pyrrolidone (PVP) solution (pH 7.4) and permeabilisation with 0.5% (v/v) Triton X-100 in PBS, the sperm cells were incubated with the TUNELmixture (fluorescein-dUTP and terminal deoxynucleotidyl transferase) for 1 h at 37 8C in the dark. Both positive (1 mg/mL DNAse I) and negative controls (nucleotide mixture in the absence of transferase) were included in each replicate. Hoechst 33342 was used to counter stain sperm DNA. The samples were examined by fluorescence microscopy (Leica DMR; magnification 400 , oil immersion). At least 200 sperm cells from each sample were analyzed randomly to evaluate the percentage of TUNEL-positive sperm cells (bright green nuclear fluorescence) (see De Pauw et al. [18] for further details).
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2.5. Centrifugation protocols After dilution of the raw semen with Gent Extender (at 37 8C), each tube, except for the uncentrifuged control, was centrifuged with a Heraeus Multifuge 3 LR (Kendro Laboratory Products, Waltham, USA) at ambient temperature using 1 of 5 CPs. Centrifugation protocol 1 was the reference protocol being 10 min at 600 g [3–6]; CPs 2, 3, 4, and 5 were 5 min at 600 g, 1200 g, 1800 g, and 2400 g, respectively. The deceleration speed was 1, which was the slowest possible deceleration in order to minimize disturbance due to turbulence. Since only one centrifuge was available, the processing of each ejaculate from the same stallion started with a different CP to eliminate influence of a prolonged contact time with the SP. So for each stallion every ejaculate was centrifuged first with a different alternating protocol. 2.6. Experimental design 2.6.1. EXPERIMENT 1: influence of the centrifugation protocol on the function of fresh and cooled sperm Fresh semen was analyzed (concentration, CASA, eosin/nigrosin staining and SYBR14-PI) immediately after collection (T0). The semen was diluted to 25 106 sperm cells/mL and allocated to six 15-mL test tubes (Cellstar1, Greiner bio-one, Germany). Five of these tubes (1–5) were subjected to one of the 5 CPs whereas tube 6 served as a negative control (no centrifugation). After centrifugation, the sperm concentration was determined in the aspirated supernatant. The sperm pellet was resuspended in fresh diluter and an aliquot of each test tube was analyzed (T1). The test tubes containing the processed semen were allowed to cool gradually to 5 8C in 50-mL test tubes filled with water (22 8C) and placed in a 5 8C refrigerator. Sperm analyses were repeated at 24 h (T2), 48 h (T3) and 72 h (T4). PSA-FITC staining was performed immediately after centrifugation and at 72 h. 2.6.2. EXPERIMENT 2: influence of the prefreezing centrifugation protocol on the function of thawed sperm The initial semen processing was performed as described in Experiment 1. After centrifugation the sperm pellet was resuspended in Gent extender for freezing to a final concentration of 50 106 sperm cells/mL. The extended semen was loaded into 0.5 mL straws (MRS1, L’Aigle Cedex, France) at ambient temperature, placed on racks and transported to the
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freezing chamber of an automated controlled rate freezer (IceCube 14S, Neupurkersdorf, Austria). The semen was first cooled from 22 8C to 4 8C in 80 min and then frozen from 4 8C to -140 8C at 60 8C/min (adapted from Vidament et al. [5]). At -140 8C the freezing racks were removed from the freezing chamber and plunged into liquid nitrogen. Frozen semen was stored for at least 1 mo before analysis. The samples were thawed by plunging into a 37 8C water bath for 30 s. Five straws from the same batch (same ejaculate, same treatment) were pooled, allowed to equilibrate for 5 min at 37 8C and analyzed once, performing the same analyses as described in Experiment 1. 2.6.3. EXPERIMENT 3: influence of the centrifugation protocol on the DNA integrity of fresh and cooled sperm From four stallions, one ejaculate was collected and diluted in Gent Extender to a final concentration of 25 106 sperm cells/mL. The diluted semen was subjected to three different CP’s (CP1, CP2, and CP5) whereas one tube served as negative (uncentrifuged) control. Immediately after centrifugation (T1) and 72 h (T4) after cooled storage, an aliquot of each tube was examined for DNA integrity using TUNEL assay. 2.7. Statistical analysis 2.7.1. EXPERIMENT 1: influence of centrifugation protocol on function of fresh and cooled sperm 1/sperm loss To evaluate whether the treatment (CP 1, 2, 3, 4, and 5) had an influence on sperm concentration in the aspirated supernatant, a Kruskal Wallis H test was conducted (overall treatment effect) as well as a Mann-Whitney U test (to test the difference between CP 1 versus the other treatments, separately) using SPSS software package (version 17.0, SPSS Inc., Chicago, IL). 2/effect of centrifugation (non CP vs. CP) Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with centrifugation (non CP vs. CP), time (T1–T4) and interaction centrifugation time included as predictor variables. Stallion (1–5) and ejaculate (5 per stallion) were included as random effects to account for clustering of ejaculate in stallion and the repeated measurements over time, within ejaculate (including an AR(1) correlation structure), respectively. The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm cells, BCF, TM and PM) were normally distributed based on the inspection of QQ-plots and Kolmogorov-Smirnov tests.
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Table 2 Fresh semen quality characteristics of 5 Shetland pony stallions (25 ejaculates). Parameter
Means SD
Min
Max
Volume (mL) Concentration (106/mL) Total number of sperm (109) Total motility (%) Progressive motility (%) Intact plasma membrane (%; Sybr14-PI staining) Intact acrosome (%; PSA-staining) Live (%; eosin/nigrosin staining) Normal sperm (%; eosin/nigrosin staining) Head defects (%) Tail defects (%) Proximal droplets (%) Distal droplets (%)
18.3 6.5 479.4 203.6 7.8 1.8 75.2 12.8 40.7 10.4 85.9 7.6 84.5 4.4 89.6 6.2 81.4 3.6 2.3 1.0 3.7 1.3 5.4 1.1 7.2 1.9
8 238.4 5.1 57.2 22.6 73.5 78.0 78.5 74.5 0.5 1.5 4.0 3.0
30 952.9 10.5 97.8 71.0 99.0 93.0 99.5 85.5 4.0 6.0 8.5 10.5
3/effect of centrifugation protocol Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with CP (1–5), time (T1–T4) and the interaction CP time included as predictor variables. Stallion (1–5) and ejaculate (5 per stallion) were included as random effects to account for clustering of ejaculate in stallion and the repeated measurements over time, within ejaculate (including an AR(1) correlation structure), respectively. The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm cells, BCF, TM, and PM) were normally distributed based on the inspection of QQ-plots and Kolmogorov-Smirnov tests.
inspection of QQ-plots and Kolmogorov-Smirnov tests.
2.7.2. EXPERIMENT 2: influence of pre-freezing centrifugation protocol on function of thawed sperm Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with CP (1–5), ejaculate (1–5) and the interaction CP ejaculate included as predictor variables. Stallion (1–5) was included as random effects to account for clustering of ejaculate in stallion. The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm cells, BCF, TM, and PM) were normally distributed based on the
3.1. EXPERIMENT 1: influence of the centrifugation protocol on the function of fresh and cooled sperm
2.7.3. EXPERIMENT 3: influence of centrifugation protocol on DNA integrity of fresh and cooled sperm Since the percentages of DNA intact sperm cells were not normally distributed, a time effect (T1 vs. T4) was analyzed using Wilcoxon signed ranks test. Effect of treatment was determined using Kruskal-Wallis test at T1 and T4. Analyses were done using SPSS software package (version 17.0, SPSS Inc., Chicago, IL). 3. Results
The general sperm characteristics over all the ejaculates from the 5 stallions immediately after collection are presented in Table 2. There was an overall effect of CP on sperm concentration in the recovered supernatant (P < 0.001). The supernatants of CP 2 and CP 3 contained higher
Table 3 Concentration of sperm (mean SD) recovered from the supernatant and percentage lost sperm after centrifugation of diluted semen (25 106/mL) using five different centrifugation protocols (n = 25). Centrifugation protocol N8
Time
Speed
1 2 3 4 5
10 min 5 min 5 min 5 min 5 min
600 g 600 g 1200 g 1800 g 2400 g
a,b,c,d
Values with same superscript are statistically different (P < 0.05).
Concentration of sperm recovered from the supernatant ( 106/mL)
% Sperm Loss
6.1 0.04a,b,c,d 11.2 0.04a 6.4 0.04b 2.1 0.02c 0.6 0.007d
22.0 40.4 23.0 7.4 2.1
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Fig. 1. Effect of centrifugation on percentage membrane intact sperm (A), beat cross frequency (B), total motility (C) and progressive motility (D) during chilled storage for 72 h.
concentrations of sperm cells compared to CP 1 (P < 0.001) (Table 3). In contrast, CP 4 and CP 5 resulted in lower concentrations of sperm cells in the supernatant compared to CP1 (P < 0.001). The relative losses of sperm cells when aspirating 90% of the supernatant compared to the initial number for CP 1, 2, 3, 4, and 5 were 22.0%, 40.4%, 23.0%, 7.4%, and 2.1%, respectively. When comparing the uncentrifuged samples to all the centrifuged samples, we found a time effect; percentage membrane intact (SYBR14-PI) sperm cells,
percentage acrosome intact sperm cells (results not shown), BCF, TM and PM decreased over time (P < 0.001) (Fig. 1). No overall CP effect was present for the percentage of membrane intact sperm cells, percentage acrosome intact sperm cells, TM and PM. Except for the percentage acrosome intact sperm cells, the interaction time CP (non CP vs. CP) was significant for these parameters (P < 0.05 for BCF and P < 0.001 for SYBR-PI, TM and PM). Beat cross frequency was lower for uncentrifuged compared with centrifuged sperm cells (P < 0.001).The decrease in
Fig. 2. Effect of different centrifugation protocols on percentage membrane intact sperm (A), beat cross frequency (B), total motility (C) and progressive motility (D) during chilled storage for 72 h.
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Fig. 3. Effect of different centrifugation protocols on beat cross frequency of frozen-thawed stallion sperm, error bars are SD (columns with the same character are statistically different from the reference protocol (600 g for 10 min), for a P < 0.005 and for b and c P < 0.05).
BCF over time for the uncentrifuged samples was faster compared to the centrifuged samples (P < 0.001). All the examined parameters decreased over time in all treatments (P < 0.001). When comparing CP 1 to the other CPs, no effect was found on the percentage of membrane intact sperm cells, percentage acrosome intact sperm cells and PM (Fig. 2). A significant effect on TM (P < 0.05) was noticed where CP 1 was significantly higher than CP 2 (P < 0.005). However, CP 1 was not significantly different from CP 3, 4, and 5. Centrifugation protocol 1 resulted in higher BCF than CP 2 (P < 0.05) and CP 4 (P < 0.005). The interaction time CP was not significant for the analyzed parameters. 3.2. EXPERIMENT 2: influence of the pre-freezing centrifugation protocol on the function of thawed sperm There was a significant effect of ejaculate for all evaluated parameters (% acrosome intact sperm, P < 0.05; other parameters, P < 0.001). Centrifugation protocol 2 resulted in higher BCF (P < 0.005), while CP 3 and 5 resulted in lower BCF of sperm, compared to CP 1 (P < 0.05). There was no difference in BCF for CP 1 and CP 4 (Fig. 3). The other examined sperm parameters were not influenced by the CP. 3.3. EXPERIMENT 3: influence of the centrifugation protocol on the DNA integrity of fresh and cooled sperm The percentage of DNA intact sperm cells was lower at T4 (72 h) compared to T1 (0 h) (P < 0.01) (Fig. 4). There was no effect of treatment on DNA integrity, neither at T1 nor at T4 (P = 0.90 and P = 0.74, respectively).
Fig. 4. Effect of different centrifugation protocols on DNA integrity of equine sperm during chilled storage.
4. Discussion In this study, we demonstrated that the loss of sperm after centrifugation can be minimized by using an appropriate protocol without damaging the sperm. Centrifugation protocol had a limited effect on in vitro sperm characteristics in our study. We specifically showed that the use of the standard protocol (CP 1) led to an average sperm loss of 22%, resulting in a considerable reduction in the number of AI doses obtained. If the centrifugation time was decreased while the exerted g-force was increased up to 1800 g or even 2400 g, sperm losses were reduced to 7.4% and 2.1%, respectively, without any apparent changes in the in vitro sperm quality. If semen was cooled and stored after centrifugation, only a minimal effect of the used CP was noticed on total motility as evaluated by CASA. Surprisingly, the motility was reduced in the group where the lowest centrifugation force was used for the shortest time (600 g for 5 min). The sperm pellet probably was very soft which allowed the most motile sperm cells to swim up immediately after centrifugation had ceased. As a consequence, these highly motile sperm may be discarded during aspiration of the supernatant. This was confirmed in a preliminary experiment (n = 4) by analyzing the supernatant of semen samples centrifuged according to CP1, 2 and 5 (data not shown). The average TM and PM in the supernatant was 41.3% and 23.5%, 54.8% and 31.8%, and 8.3% and 4.0% for CP1, 2, and 5, respectively, suggesting that the motility of the discarded sperm cells in the supernatant is much higher when low centrifugation forces are used. Therefore, application of a low g-force for a short time leads to important losses of top quality sperm cells and must definitely be avoided.
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Additionally, we found a significant effect on BCF for sperm samples cooled and stored after centrifugation. The BCF of the sperm subjected to CPs 2 and 4 was reduced compared to the standard protocol. Moreover, the quality of the frozen-thawed samples after being subjected to one of the tested CPs did not differ significantly for any of the tested quality parameters except for BCF. Protocols 2 and 4 yielded the highest BCF values after thawing, which was in contrast with our observations for cooled preservation. However, the actual impact of BCF on fertility is difficult to predict since few data are available in literature [19]. BCF is a parameter indicative for spermatic vigor. Changes in BCF under capacitating conditions in vitro are thought to be related to sperm hyperactivation that occurs in vivo and might favor the penetration of oocytes [19]. The results of the experiment 3 showed a decrease of DNA integrity over time, although centrifugation did not influence DNA integrity. These findings are contradictory with the study of Love and coworkers [20] where increased DNA damage immediately after centrifugation was reported. These authors showed that complete removal of SP after centrifugation and subsequent resuspension of the sperm pellet in fresh diluter resulted in better sperm quality (motility and DNA) compared to samples in which SP was still present after simple dilution of the semen. Therefore the impact of the centrifugation on DNA integrity needed to be further examined. In our study, no difference in DNA damage was present immediately after centrifugation which might be explained by the method used to evaluate DNA integrity. The study of Love et al. [20] evaluated DNA integrity using the sperm chromatin structure assay (SCSA). In comparison to the TUNEL assay, with the SCSA a larger number of sperm cells is analyzed by a flow cytometer based on the colour of the metachromatic dye acridine orange [21]. Therefore it might be advisable to measure the direct impact of different CPs with flow cytometry (SCSA or TUNEL) to get a more accurate impression of the possible side effect of centrifugation on DNA integrity. Cushioned centrifugation techniques have previously been described as an answer to reduce sperm damage while maximizing the sperm harvest. A variety of dense solutions acting as a cushion have been described [22]. The frequently used iodixanol was first reported for stallion sperm cells in 1997 [23]. Very high recovery rates have been described [3,4] using such a commercially available cushion, when centrifuging at 1000 g for 20 min. However, the most critical step is carefully removing the cushion material after centrifugation. Indeed, it is critical to aspirate as much cushion
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fluid as possible without aspirating sperm cells from the adjacent band to avoid possible deleterious effects of the iodixanol solution [24]. The use of very small amounts (30 mL) of cushion without removal was proven to be harmless. However, these small volumes require specially designed, custom-made nipple centrifugation tubes which are not readily available [24]. Because of these important drawbacks, cushioned centrifugation is not commonly used in practice. Therefore, the impact of CP on sperm quality remains an important subject to examine. In summary, sperm losses can be reduced substantially by centrifuging stallion semen for 5 min at 1800 or 2400 g without a cushion and without impairing the in vitro sperm function. This leads to an increase in the number of AI doses produced per ejaculate. It can easily result in an additional two AI doses from an average ejaculate of 10 billion sperm. Nevertheless, the effects of these CPs on DNA damage requires further investigation as well as the impact on in vivo fertility. References [1] Jasko DJ, Moran DM, Farlin ME, Squires EL. Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Therio 1991;35:1059–67. [2] Martin JC, Klug E, Gu¨nzel A-R. Centrifugation of stallion semen and its storage in large volume straws. J Reprod Fert Suppl 1979;27:47–51. [3] Ecot P, Decuadro-Hansen G, Delhomme G, Vidament M. Evaluation of a cushioned centrifugation technique for processing equine semen for freezing. Anim Reprod Sci 2005;89:245–8. [4] Knop K, Hoffmann N, Rath D, Sieme H. Effects of cushioned centrifugation technique on sperm recovery and sperm quality in stallions with good and poor semen freezability. Anim Reprod Sci 2005;89:294–7. [5] Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E. Centrifugation and addition of glycerol at 22 degrees C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa. Therio 2000;54:907–19. [6] Weiss S, Janett F, Burger D, Ha¨ssig M, Thun R. The influence of centrifugation on quality and freezability of stallion semen. Schweiz Arch Tierheilk 2004;146:285–93. [7] Loomis PR. Advanced methods for handling and preparation of stallion semen. Vet Clin North Am Equine Pract 2006;22:663–76. [8] Aurich C. Recent advances in cooled-semen technology. Anim Reprod Sci 2008;107:268–75. [9] Shekarriz M, DeWire DM, Thomas Jr AJ, Agarwal A. A method of human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur Urol 1995;28:31–5. [10] Carvajal G, Cuello C, Ruiz M, Va´zquez JM, Martı´nez EA, Roca J. Effects of centrifugation before freezing on boar sperm cryosurvival. J Androl 2004;25:389–96. [11] Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C. Factors affecting motion characteristics of frozenthawed stallion spermatozoa. Equine Vet J 1996;28:47–53.
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[12] Picket BW. Collection and evaluation of stallion semen for artificial insemination. In: McKinnon AO, Voss JL, editors. Equine Reproduction. Williams & Wilkins; 1993. p. 705–14. [13] Barth AD, Oko RJ. Abnormal morphology of bovine spermatozoa. Ames, Iowa: Iowa State University Press; 1989. [14] Rijsselaere T, Van Soom A, Maes D, de Kruif A. Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer. Therio 2003;60:1553–68. [15] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105: 119–28. [16] Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276–84. [17] Rathi R, Colenbrander B, Stout TA, Bevers MM, Gadella BM. Progesterone induces acrosome reaction in stallion spermatozoa via a protein tyrosine kinase dependent pathway. Mol Reprod Dev 2003;64:120–8. [18] De Pauw IMC, Van Soom A, Mintiens K, Verberckmoes S, de Kruif A. In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions. Therio 2003;59:1093–107.
[19] Gil MC, Garcı´a-Herreros M, Baro´n FJ, Aparicio IM, Santos AJ, Garcı´a-Marı´n LJ. Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen. Therio 2009;71:254–63. [20] Love CC, Brinsko SP, Rigby SL, Thompson JA, Blanchard TL, Varner DD. Relationship of seminal plasma level and extender type to sperm motility and DNA integrity. Therio 2005;63: 1584–91. [21] Evans DP, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Therio 2006;65: 979–91. [22] Cochran JD, Amann RP, Froman DP, Pickett BW. Effects of centrifugation, glycerol level, cooling to 5 8C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Therio 1984;22:25–38. [23] Revell SG, Pettit MT, Ford TC. Use of centrifugation over iodixanol to reduce damage when processing stallion sperm for freezing. J Reprod Fertil 1997, Abstr series no. 19, p38. [24] Waite JA, Love CC, Brinsko SP, Teague SR, Salazar Jr JL, Mancil SS, Varner DD. Factors impacting equine sperm recovery rate and quality following cushioned centrifugation. Therio 2008;70:704–14.
Theriogenology 74 (2010) 118–126 www.theriojournal.com
Influence of different centrifugation protocols on equine semen preservation Maarten Hoogewijs *, Tom Rijsselaere, Sarne De Vliegher, Emilie Vanhaesebrouck, Catharina De Schauwer, Jan Govaere, Mirjan Thys, Geert Hoflack, Ann Van Soom, Aart de Kruif Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, 9820 Merelbeke, Belgium Received 29 June 2009; received in revised form 18 January 2010; accepted 31 January 2010
Abstract Three experiments were conducted to evaluate the impact of centrifugation on cooled and frozen preservation of equine semen. A standard centrifugation protocol (600 g for 10 min = CP1) was compared to four protocols with increasing g-force and decreased time period (600 g, 1200 g, 1800 g and 2400 g for 5 min for CP2, 3, 4, and 5, respectively) and to an uncentrifuged negative control. In experiment 1, the influence of the different CPs on sperm loss was evaluated by calculating the total number of sperm cells in 90% of the supernatant. Moreover, the effect on semen quality following centrifugation was assessed by monitoring several sperm parameters (membrane integrity using SYBR14-PI, acrosomal status using PSA-FITC, percentage total motility (TM), percentage progressive motility (PM) and beat cross frequency (BCF) obtained with computer assisted sperm analysis (CASA)) immediately after centrifugation and daily during chilled storage for 3 d. The use of CP1 resulted in a sperm loss of 22%. Increasing the centrifugation force to 1800 g and 2400 g for 5 min led to significantly lower sperm losses (7.4% and 2.1%, respectively; P < 0.05). Compared to the uncentrifuged samples, centrifugation of semen resulted in a better sperm quality after chilled storage. There were minimal differences between the CPs although total motility was lower for CP2 than for the other treatments (P < 0.005). In experiment 2, the centrifuged samples were cryopreserved using a standard freezing protocol and analyzed immediately upon thawing. Samples centrifuged according to CP2 resulted in a higher BCF (P < 0.005), whereas CP3 and CP5 yielded a lower BCF (P < 0.05) when compared to CP1. There were no post thaw differences between CP1 and CP4. In experiment 3, DNA integrity of the different samples was analyzed using TUNEL. Although DNA integrity decreased over time, CP had no impact. In conclusion, the loss of sperm cells in the supernatant after centrifugation can be substantially reduced by increasing the g-force up to 1800 g or 2400 g for a shorter period of time (5 min) compared to the standard protocol without apparent changes in semen quality, resulting in a considerable increase in the number of insemination doses per ejaculate. # 2010 Elsevier Inc. All rights reserved. Keywords: Semen; Centrifugation; Sperm loss; Stallion; Sperm quality parameters
1. Introduction Numerous studies have demonstrated the beneficial effect of diluting raw semen with an appropriate * Corresponding author. Tel.: +32 9 264 75 61; fax: +32 9 264 77 97. E-mail address: [email protected] (M. Hoogewijs). 0093-691X/$ – see front matter # 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.01.022
extender. As such, the negative influence of seminal plasma (SP) on the preservation of equine sperm can be reduced. Centrifugation of diluted equine semen and subsequent resuspension of the sperm pellet in fresh extender can even further reduce the amount of SP in stored samples. A small proportion of SP in semen improves sperm motility after cooling and storage [1].
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However, centrifugation itself may act like a double edged sword exerting both beneficial and deleterious effects on sperm motility [2]. Although the number of sperm cells can be maximized by increasing centrifugation time and force, physical damage is exerted on the sperm cells at the same time. The direct negative outcome of centrifugation on semen quality is demonstrated by the decrease in motility and velocity in contrast to uncentrifuged controls [1]. After 24 h of cooled storage the harmful effect of centrifugation is no longer present [1] which indicates that the centrifugation protocol (CP) may play an important role in the quality of the inseminate. A CP of 600 g for 10 min is commonly used for equine semen [3–6]. In literature, data on the amount of sperm loss after using this protocol are contradictory and vary from 1.9% [6] to 25% [3,4]. Nowadays, it is generally accepted that sperm losses will vary around 25% when diluted semen is centrifuged at 400-600 g for 10–15 min [7,8]. The importance of an appropriate CP has been clearly demonstrated for human sperm. The duration of centrifugation was shown to be more important than the centrifugation force for causing iatrogenic sperm membrane injuries which resulted in an increased formation of reactive oxygen species [9]. In boars, similar findings were described: a CP with a high g-force for a short time (2400 g for 3 min) was used without detrimental effects on sperm yield compared to the standard regime (800 g for 10 min) [10]. Additionally, a positive effect on semen quality after cryopreservation was detected when using high g-forces for a shorter period of time [10]. Experiments with equine semen have been performed where semen was centrifuged at 400 g for an increasing period of time. Sperm loss was approximately 20%, if samples were centrifuged for 10 min or longer, while no adverse effects on motility immediately after centrifugation were present, unless semen was centrifuged for 20 min. However, effects on preserved semen by cooling or freezing were not determined [11]. Since a high g-force for a short time was not detrimental for porcine sperm [10] and prolonged centrifugation of equine sperm negatively influenced sperm quality [11], using a short term high speed CP might be beneficial. The aim of the present study was to compare high speed CPs with the standard protocol on cooled and cryopreserved equine semen. The impact of CP on the subsequent number and quality of sperm cells was assessed daily during 3 consecutive days of cooled preservation. Additionally, the effect of different CPs on the quality of frozen-thawed equine sperm was investigated.
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2. Materials and methods 2.1. Stallions and semen collection For experiments 1 and 2, five ejaculates from each of five experienced Shetland ponies (n = 25), aged 3–6 yr, were collected between August and October 2007. For experiment 3, one ejaculate from 4 of these ponies was used. The ponies were housed in groups in a straw bedded stable and were fed good quality hay ad libitum. Prior to the unset of the experiment, the extra-gonodal sperm reserves were depleted with 3 collections every other day for 1 week. For the experiment, the stallions were collected twice a week. Semen was collected on a teaser mare using standard procedures with a custom made open ended artificial vagina based on the Colorado State University model as described by Pickett [12]. After collection, semen was filtered through a sterile gauze to remove the gel fraction and debris. The gel-free volume was noted and the sperm concentration was determined using a Bu¨rker hemocytometer after 1:9 dilution with HCl 1 M 2.2. Media Fresh-cooled semen was processed and stored in Gent Extender (Minitu¨b, Landshut, Germany), which is a skimmed milk based diluter containing 5% clarified egg yolk and gentamycin (1 mg/mL). If the semen was to be cryopreserved, a second dilution was performed in Gent Extender for Freezing (Minitu¨b, Landshut, Germany) which has the same composition as the Gent Extender plus 5% glycerol. For the fluorescent staining procedures, the samples were diluted in a HEPES buffered solution containing 0.04% BSA. 2.3. Semen processing After determination of the initial sperm concentration, the semen was diluted to a final concentration of 25 106 sperm/mL using Gent Extender. Six conical bottom centrifuge tubes of 15 mL (Cellstar1, Greiner bio-one, Germany) were filled with the diluted semen (total of 375 106 sperm cells per tube) and served as the negative control or were subjected to one of the five different CPs, respectively. After centrifugation, 90% of the supernatant was aspirated, leaving a sperm pellet with a volume of approximately 1.5 mL. The concentration in the aspirated supernatant was determined using a Neubauer hemocytometer. The obtained concentration was multiplied by the volume of
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aspirated supernatant in order to calculate the total number of sperm cells lost per tube when the supernatant was discarded. The remaining sperm pellet was resuspended in the appropriate diluter for further processing as cooled or frozen semen. 2.4. Evaluation of sperm characteristics and staining procedures Prior to analysis, an aliquot (500 mL) of semen was equilibrated to 37 8C. The morphology of sperm cells was examined on eosin-nigrosin stained smears which were prepared as described by Barth and Oko [13]. At least 200 sperm cells were evaluated and recorded per slide, individual morphological abnormalities were noted according to their location (head, midpiece or tail). Motility was evaluated with a computer-assisted sperm analyzer (CASA) (Hamilton-Thorne Ceros 12.3). For each analysis, 5 mL of diluted semen was mounted on a disposable Leja counting chamber (Orange Medical, Brussels, Belgium) and maintained at 37 8C using a minitherm stage warmer. Five randomly selected microscopic fields in the center of the slide were scanned 5 times each, obtaining 25 scans for every semen sample. The mean of the 5 scans for each microscopic field was used for the statistical analysis [14]. The software settings of the HTR 12.3, based on Loomis and Graham [15], are summarized in Table 1. Membrane integrity was evaluated using a fluorescent SYBR14-Propidium Iodide (PI) staining technique (Molecular Probes cat no.: L-7011, Leiden, The Netherlands) based on a previously described method [16]. Briefly, 225 mL HEPES-TALP was mixed with 25 mL of diluted semen and 1.25 mL SYBR14 (1:50 dilution) was added. After 5 min of incubation at 37 8C, 1.25 mL PI was added and incubated for another 5 min. Table 1 Software settings of the Hamilton Thorne Ceros 12.3 used in this study. Parameter
Value
Frames acquired Frame rate (Hz) Minimum contrast Minimum cell size (pixels) Minimum static contrast Straightness cut-off (%, STR) Average-path velocity cut-off PM (mm/s,VAP) VAP cut-off static cells (mm/s) Cell intensity Static head size Static head intensity Static elongation
30 60 60 6 25 75 50 20 100 0.55–2.04 0.45–1.70 11–99
Two hundred cells were evaluated per slide using a Leica DMR fluorescence microscope and three populations of sperm cells could be identified (green = living, red = dead and orange = moribund). Acrosomal status was determined using fluorescent Pisum Sativum Agglutinin (PSA) conjugated with fluorescein isothiocyanate (FITC) (Sigma-Aldrich cat no.: L 0770, Bornem, Belgium). The staining was performed in a similar way as described by Rathi et al. [17]. Briefly, 500 mL of semen was centrifuged for 10 min at 720 g and the pellet was resuspended with HEPES-TALP. The semen was centrifuged again for 10 min at 720 g, the supernatant was removed and the pellet fixed in 100 mL absolute ethyl alcohol (Vel cat no.: 1115, Haasrode, Belgium) and cooled for 30 min at 4 8C. A drop of 20 mL semen was smeared on a glass slide and 40 mL PSA-FITC (2 mg PSA-FITC diluted in 2 mL phosphate buffered saline (PBS)) was added. The glass slide was kept at 4 8C for 15 min, washed 10 times with aqua bidest and allowed to air-dry in the dark. Immediately after drying, 200 sperm cells were evaluated per slide. The acrosomal region of the acrosome intact sperm cells was labeled heavily green, while the acrosome reacted sperm retained only an equatorial band of label with little or no labeling of the anterior head region. In Experiment 3, DNA integrity was analyzed using a Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) assay. The TUNEL assay was performed using the In Situ Cell Death Detection Kit (Boehringer, Mannheim, Germany) to detect the presence of free 30 -OH termini in single and doublestranded sperm DNA [18]. In short, sperm samples were diluted with PVP solution (1 mg/mL in PBS) to a final concentration of 10 106 sperm/mL from which a 10 mL aliquot was smeared onto a poly-l-lysine-coated microslide. After fixation with 4% paraformaldehyde in polyvinyl pyrrolidone (PVP) solution (pH 7.4) and permeabilisation with 0.5% (v/v) Triton X-100 in PBS, the sperm cells were incubated with the TUNELmixture (fluorescein-dUTP and terminal deoxynucleotidyl transferase) for 1 h at 37 8C in the dark. Both positive (1 mg/mL DNAse I) and negative controls (nucleotide mixture in the absence of transferase) were included in each replicate. Hoechst 33342 was used to counter stain sperm DNA. The samples were examined by fluorescence microscopy (Leica DMR; magnification 400 , oil immersion). At least 200 sperm cells from each sample were analyzed randomly to evaluate the percentage of TUNEL-positive sperm cells (bright green nuclear fluorescence) (see De Pauw et al. [18] for further details).
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2.5. Centrifugation protocols After dilution of the raw semen with Gent Extender (at 37 8C), each tube, except for the uncentrifuged control, was centrifuged with a Heraeus Multifuge 3 LR (Kendro Laboratory Products, Waltham, USA) at ambient temperature using 1 of 5 CPs. Centrifugation protocol 1 was the reference protocol being 10 min at 600 g [3–6]; CPs 2, 3, 4, and 5 were 5 min at 600 g, 1200 g, 1800 g, and 2400 g, respectively. The deceleration speed was 1, which was the slowest possible deceleration in order to minimize disturbance due to turbulence. Since only one centrifuge was available, the processing of each ejaculate from the same stallion started with a different CP to eliminate influence of a prolonged contact time with the SP. So for each stallion every ejaculate was centrifuged first with a different alternating protocol. 2.6. Experimental design 2.6.1. EXPERIMENT 1: influence of the centrifugation protocol on the function of fresh and cooled sperm Fresh semen was analyzed (concentration, CASA, eosin/nigrosin staining and SYBR14-PI) immediately after collection (T0). The semen was diluted to 25 106 sperm cells/mL and allocated to six 15-mL test tubes (Cellstar1, Greiner bio-one, Germany). Five of these tubes (1–5) were subjected to one of the 5 CPs whereas tube 6 served as a negative control (no centrifugation). After centrifugation, the sperm concentration was determined in the aspirated supernatant. The sperm pellet was resuspended in fresh diluter and an aliquot of each test tube was analyzed (T1). The test tubes containing the processed semen were allowed to cool gradually to 5 8C in 50-mL test tubes filled with water (22 8C) and placed in a 5 8C refrigerator. Sperm analyses were repeated at 24 h (T2), 48 h (T3) and 72 h (T4). PSA-FITC staining was performed immediately after centrifugation and at 72 h. 2.6.2. EXPERIMENT 2: influence of the prefreezing centrifugation protocol on the function of thawed sperm The initial semen processing was performed as described in Experiment 1. After centrifugation the sperm pellet was resuspended in Gent extender for freezing to a final concentration of 50 106 sperm cells/mL. The extended semen was loaded into 0.5 mL straws (MRS1, L’Aigle Cedex, France) at ambient temperature, placed on racks and transported to the
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freezing chamber of an automated controlled rate freezer (IceCube 14S, Neupurkersdorf, Austria). The semen was first cooled from 22 8C to 4 8C in 80 min and then frozen from 4 8C to -140 8C at 60 8C/min (adapted from Vidament et al. [5]). At -140 8C the freezing racks were removed from the freezing chamber and plunged into liquid nitrogen. Frozen semen was stored for at least 1 mo before analysis. The samples were thawed by plunging into a 37 8C water bath for 30 s. Five straws from the same batch (same ejaculate, same treatment) were pooled, allowed to equilibrate for 5 min at 37 8C and analyzed once, performing the same analyses as described in Experiment 1. 2.6.3. EXPERIMENT 3: influence of the centrifugation protocol on the DNA integrity of fresh and cooled sperm From four stallions, one ejaculate was collected and diluted in Gent Extender to a final concentration of 25 106 sperm cells/mL. The diluted semen was subjected to three different CP’s (CP1, CP2, and CP5) whereas one tube served as negative (uncentrifuged) control. Immediately after centrifugation (T1) and 72 h (T4) after cooled storage, an aliquot of each tube was examined for DNA integrity using TUNEL assay. 2.7. Statistical analysis 2.7.1. EXPERIMENT 1: influence of centrifugation protocol on function of fresh and cooled sperm 1/sperm loss To evaluate whether the treatment (CP 1, 2, 3, 4, and 5) had an influence on sperm concentration in the aspirated supernatant, a Kruskal Wallis H test was conducted (overall treatment effect) as well as a Mann-Whitney U test (to test the difference between CP 1 versus the other treatments, separately) using SPSS software package (version 17.0, SPSS Inc., Chicago, IL). 2/effect of centrifugation (non CP vs. CP) Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with centrifugation (non CP vs. CP), time (T1–T4) and interaction centrifugation time included as predictor variables. Stallion (1–5) and ejaculate (5 per stallion) were included as random effects to account for clustering of ejaculate in stallion and the repeated measurements over time, within ejaculate (including an AR(1) correlation structure), respectively. The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm cells, BCF, TM and PM) were normally distributed based on the inspection of QQ-plots and Kolmogorov-Smirnov tests.
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Table 2 Fresh semen quality characteristics of 5 Shetland pony stallions (25 ejaculates). Parameter
Means SD
Min
Max
Volume (mL) Concentration (106/mL) Total number of sperm (109) Total motility (%) Progressive motility (%) Intact plasma membrane (%; Sybr14-PI staining) Intact acrosome (%; PSA-staining) Live (%; eosin/nigrosin staining) Normal sperm (%; eosin/nigrosin staining) Head defects (%) Tail defects (%) Proximal droplets (%) Distal droplets (%)
18.3 6.5 479.4 203.6 7.8 1.8 75.2 12.8 40.7 10.4 85.9 7.6 84.5 4.4 89.6 6.2 81.4 3.6 2.3 1.0 3.7 1.3 5.4 1.1 7.2 1.9
8 238.4 5.1 57.2 22.6 73.5 78.0 78.5 74.5 0.5 1.5 4.0 3.0
30 952.9 10.5 97.8 71.0 99.0 93.0 99.5 85.5 4.0 6.0 8.5 10.5
3/effect of centrifugation protocol Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with CP (1–5), time (T1–T4) and the interaction CP time included as predictor variables. Stallion (1–5) and ejaculate (5 per stallion) were included as random effects to account for clustering of ejaculate in stallion and the repeated measurements over time, within ejaculate (including an AR(1) correlation structure), respectively. The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm cells, BCF, TM, and PM) were normally distributed based on the inspection of QQ-plots and Kolmogorov-Smirnov tests.
inspection of QQ-plots and Kolmogorov-Smirnov tests.
2.7.2. EXPERIMENT 2: influence of pre-freezing centrifugation protocol on function of thawed sperm Mixed models were fitted in SAS 9.1.3. (PROC MIXED) with CP (1–5), ejaculate (1–5) and the interaction CP ejaculate included as predictor variables. Stallion (1–5) was included as random effects to account for clustering of ejaculate in stallion. The outcome variables (percentage membrane intact sperm cells, percentage acrosome intact sperm cells, BCF, TM, and PM) were normally distributed based on the
3.1. EXPERIMENT 1: influence of the centrifugation protocol on the function of fresh and cooled sperm
2.7.3. EXPERIMENT 3: influence of centrifugation protocol on DNA integrity of fresh and cooled sperm Since the percentages of DNA intact sperm cells were not normally distributed, a time effect (T1 vs. T4) was analyzed using Wilcoxon signed ranks test. Effect of treatment was determined using Kruskal-Wallis test at T1 and T4. Analyses were done using SPSS software package (version 17.0, SPSS Inc., Chicago, IL). 3. Results
The general sperm characteristics over all the ejaculates from the 5 stallions immediately after collection are presented in Table 2. There was an overall effect of CP on sperm concentration in the recovered supernatant (P < 0.001). The supernatants of CP 2 and CP 3 contained higher
Table 3 Concentration of sperm (mean SD) recovered from the supernatant and percentage lost sperm after centrifugation of diluted semen (25 106/mL) using five different centrifugation protocols (n = 25). Centrifugation protocol N8
Time
Speed
1 2 3 4 5
10 min 5 min 5 min 5 min 5 min
600 g 600 g 1200 g 1800 g 2400 g
a,b,c,d
Values with same superscript are statistically different (P < 0.05).
Concentration of sperm recovered from the supernatant ( 106/mL)
% Sperm Loss
6.1 0.04a,b,c,d 11.2 0.04a 6.4 0.04b 2.1 0.02c 0.6 0.007d
22.0 40.4 23.0 7.4 2.1
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Fig. 1. Effect of centrifugation on percentage membrane intact sperm (A), beat cross frequency (B), total motility (C) and progressive motility (D) during chilled storage for 72 h.
concentrations of sperm cells compared to CP 1 (P < 0.001) (Table 3). In contrast, CP 4 and CP 5 resulted in lower concentrations of sperm cells in the supernatant compared to CP1 (P < 0.001). The relative losses of sperm cells when aspirating 90% of the supernatant compared to the initial number for CP 1, 2, 3, 4, and 5 were 22.0%, 40.4%, 23.0%, 7.4%, and 2.1%, respectively. When comparing the uncentrifuged samples to all the centrifuged samples, we found a time effect; percentage membrane intact (SYBR14-PI) sperm cells,
percentage acrosome intact sperm cells (results not shown), BCF, TM and PM decreased over time (P < 0.001) (Fig. 1). No overall CP effect was present for the percentage of membrane intact sperm cells, percentage acrosome intact sperm cells, TM and PM. Except for the percentage acrosome intact sperm cells, the interaction time CP (non CP vs. CP) was significant for these parameters (P < 0.05 for BCF and P < 0.001 for SYBR-PI, TM and PM). Beat cross frequency was lower for uncentrifuged compared with centrifuged sperm cells (P < 0.001).The decrease in
Fig. 2. Effect of different centrifugation protocols on percentage membrane intact sperm (A), beat cross frequency (B), total motility (C) and progressive motility (D) during chilled storage for 72 h.
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Fig. 3. Effect of different centrifugation protocols on beat cross frequency of frozen-thawed stallion sperm, error bars are SD (columns with the same character are statistically different from the reference protocol (600 g for 10 min), for a P < 0.005 and for b and c P < 0.05).
BCF over time for the uncentrifuged samples was faster compared to the centrifuged samples (P < 0.001). All the examined parameters decreased over time in all treatments (P < 0.001). When comparing CP 1 to the other CPs, no effect was found on the percentage of membrane intact sperm cells, percentage acrosome intact sperm cells and PM (Fig. 2). A significant effect on TM (P < 0.05) was noticed where CP 1 was significantly higher than CP 2 (P < 0.005). However, CP 1 was not significantly different from CP 3, 4, and 5. Centrifugation protocol 1 resulted in higher BCF than CP 2 (P < 0.05) and CP 4 (P < 0.005). The interaction time CP was not significant for the analyzed parameters. 3.2. EXPERIMENT 2: influence of the pre-freezing centrifugation protocol on the function of thawed sperm There was a significant effect of ejaculate for all evaluated parameters (% acrosome intact sperm, P < 0.05; other parameters, P < 0.001). Centrifugation protocol 2 resulted in higher BCF (P < 0.005), while CP 3 and 5 resulted in lower BCF of sperm, compared to CP 1 (P < 0.05). There was no difference in BCF for CP 1 and CP 4 (Fig. 3). The other examined sperm parameters were not influenced by the CP. 3.3. EXPERIMENT 3: influence of the centrifugation protocol on the DNA integrity of fresh and cooled sperm The percentage of DNA intact sperm cells was lower at T4 (72 h) compared to T1 (0 h) (P < 0.01) (Fig. 4). There was no effect of treatment on DNA integrity, neither at T1 nor at T4 (P = 0.90 and P = 0.74, respectively).
Fig. 4. Effect of different centrifugation protocols on DNA integrity of equine sperm during chilled storage.
4. Discussion In this study, we demonstrated that the loss of sperm after centrifugation can be minimized by using an appropriate protocol without damaging the sperm. Centrifugation protocol had a limited effect on in vitro sperm characteristics in our study. We specifically showed that the use of the standard protocol (CP 1) led to an average sperm loss of 22%, resulting in a considerable reduction in the number of AI doses obtained. If the centrifugation time was decreased while the exerted g-force was increased up to 1800 g or even 2400 g, sperm losses were reduced to 7.4% and 2.1%, respectively, without any apparent changes in the in vitro sperm quality. If semen was cooled and stored after centrifugation, only a minimal effect of the used CP was noticed on total motility as evaluated by CASA. Surprisingly, the motility was reduced in the group where the lowest centrifugation force was used for the shortest time (600 g for 5 min). The sperm pellet probably was very soft which allowed the most motile sperm cells to swim up immediately after centrifugation had ceased. As a consequence, these highly motile sperm may be discarded during aspiration of the supernatant. This was confirmed in a preliminary experiment (n = 4) by analyzing the supernatant of semen samples centrifuged according to CP1, 2 and 5 (data not shown). The average TM and PM in the supernatant was 41.3% and 23.5%, 54.8% and 31.8%, and 8.3% and 4.0% for CP1, 2, and 5, respectively, suggesting that the motility of the discarded sperm cells in the supernatant is much higher when low centrifugation forces are used. Therefore, application of a low g-force for a short time leads to important losses of top quality sperm cells and must definitely be avoided.
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Additionally, we found a significant effect on BCF for sperm samples cooled and stored after centrifugation. The BCF of the sperm subjected to CPs 2 and 4 was reduced compared to the standard protocol. Moreover, the quality of the frozen-thawed samples after being subjected to one of the tested CPs did not differ significantly for any of the tested quality parameters except for BCF. Protocols 2 and 4 yielded the highest BCF values after thawing, which was in contrast with our observations for cooled preservation. However, the actual impact of BCF on fertility is difficult to predict since few data are available in literature [19]. BCF is a parameter indicative for spermatic vigor. Changes in BCF under capacitating conditions in vitro are thought to be related to sperm hyperactivation that occurs in vivo and might favor the penetration of oocytes [19]. The results of the experiment 3 showed a decrease of DNA integrity over time, although centrifugation did not influence DNA integrity. These findings are contradictory with the study of Love and coworkers [20] where increased DNA damage immediately after centrifugation was reported. These authors showed that complete removal of SP after centrifugation and subsequent resuspension of the sperm pellet in fresh diluter resulted in better sperm quality (motility and DNA) compared to samples in which SP was still present after simple dilution of the semen. Therefore the impact of the centrifugation on DNA integrity needed to be further examined. In our study, no difference in DNA damage was present immediately after centrifugation which might be explained by the method used to evaluate DNA integrity. The study of Love et al. [20] evaluated DNA integrity using the sperm chromatin structure assay (SCSA). In comparison to the TUNEL assay, with the SCSA a larger number of sperm cells is analyzed by a flow cytometer based on the colour of the metachromatic dye acridine orange [21]. Therefore it might be advisable to measure the direct impact of different CPs with flow cytometry (SCSA or TUNEL) to get a more accurate impression of the possible side effect of centrifugation on DNA integrity. Cushioned centrifugation techniques have previously been described as an answer to reduce sperm damage while maximizing the sperm harvest. A variety of dense solutions acting as a cushion have been described [22]. The frequently used iodixanol was first reported for stallion sperm cells in 1997 [23]. Very high recovery rates have been described [3,4] using such a commercially available cushion, when centrifuging at 1000 g for 20 min. However, the most critical step is carefully removing the cushion material after centrifugation. Indeed, it is critical to aspirate as much cushion
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fluid as possible without aspirating sperm cells from the adjacent band to avoid possible deleterious effects of the iodixanol solution [24]. The use of very small amounts (30 mL) of cushion without removal was proven to be harmless. However, these small volumes require specially designed, custom-made nipple centrifugation tubes which are not readily available [24]. Because of these important drawbacks, cushioned centrifugation is not commonly used in practice. Therefore, the impact of CP on sperm quality remains an important subject to examine. In summary, sperm losses can be reduced substantially by centrifuging stallion semen for 5 min at 1800 or 2400 g without a cushion and without impairing the in vitro sperm function. This leads to an increase in the number of AI doses produced per ejaculate. It can easily result in an additional two AI doses from an average ejaculate of 10 billion sperm. Nevertheless, the effects of these CPs on DNA damage requires further investigation as well as the impact on in vivo fertility. References [1] Jasko DJ, Moran DM, Farlin ME, Squires EL. Effect of seminal plasma dilution or removal on spermatozoal motion characteristics of cooled stallion semen. Therio 1991;35:1059–67. [2] Martin JC, Klug E, Gu¨nzel A-R. Centrifugation of stallion semen and its storage in large volume straws. J Reprod Fert Suppl 1979;27:47–51. [3] Ecot P, Decuadro-Hansen G, Delhomme G, Vidament M. Evaluation of a cushioned centrifugation technique for processing equine semen for freezing. Anim Reprod Sci 2005;89:245–8. [4] Knop K, Hoffmann N, Rath D, Sieme H. Effects of cushioned centrifugation technique on sperm recovery and sperm quality in stallions with good and poor semen freezability. Anim Reprod Sci 2005;89:294–7. [5] Vidament M, Ecot P, Noue P, Bourgeois C, Magistrini M, Palmer E. Centrifugation and addition of glycerol at 22 degrees C instead of 4 degrees C improve post-thaw motility and fertility of stallion spermatozoa. Therio 2000;54:907–19. [6] Weiss S, Janett F, Burger D, Ha¨ssig M, Thun R. The influence of centrifugation on quality and freezability of stallion semen. Schweiz Arch Tierheilk 2004;146:285–93. [7] Loomis PR. Advanced methods for handling and preparation of stallion semen. Vet Clin North Am Equine Pract 2006;22:663–76. [8] Aurich C. Recent advances in cooled-semen technology. Anim Reprod Sci 2008;107:268–75. [9] Shekarriz M, DeWire DM, Thomas Jr AJ, Agarwal A. A method of human semen centrifugation to minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur Urol 1995;28:31–5. [10] Carvajal G, Cuello C, Ruiz M, Va´zquez JM, Martı´nez EA, Roca J. Effects of centrifugation before freezing on boar sperm cryosurvival. J Androl 2004;25:389–96. [11] Heitland AV, Jasko DJ, Squires EL, Graham JK, Pickett BW, Hamilton C. Factors affecting motion characteristics of frozenthawed stallion spermatozoa. Equine Vet J 1996;28:47–53.
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[12] Picket BW. Collection and evaluation of stallion semen for artificial insemination. In: McKinnon AO, Voss JL, editors. Equine Reproduction. Williams & Wilkins; 1993. p. 705–14. [13] Barth AD, Oko RJ. Abnormal morphology of bovine spermatozoa. Ames, Iowa: Iowa State University Press; 1989. [14] Rijsselaere T, Van Soom A, Maes D, de Kruif A. Effect of technical settings on canine semen motility parameters measured by the Hamilton-Thorne analyzer. Therio 2003;60:1553–68. [15] Loomis PR, Graham JK. Commercial semen freezing: individual male variation in cryosurvival and the response of stallion sperm to customized freezing protocols. Anim Reprod Sci 2008;105: 119–28. [16] Garner DL, Johnson LA. Viability assessment of mammalian sperm using SYBR-14 and propidium iodide. Biol Reprod 1995;53:276–84. [17] Rathi R, Colenbrander B, Stout TA, Bevers MM, Gadella BM. Progesterone induces acrosome reaction in stallion spermatozoa via a protein tyrosine kinase dependent pathway. Mol Reprod Dev 2003;64:120–8. [18] De Pauw IMC, Van Soom A, Mintiens K, Verberckmoes S, de Kruif A. In vitro survival of bovine spermatozoa stored at room temperature under epididymal conditions. Therio 2003;59:1093–107.
[19] Gil MC, Garcı´a-Herreros M, Baro´n FJ, Aparicio IM, Santos AJ, Garcı´a-Marı´n LJ. Morphometry of porcine spermatozoa and its functional significance in relation with the motility parameters in fresh semen. Therio 2009;71:254–63. [20] Love CC, Brinsko SP, Rigby SL, Thompson JA, Blanchard TL, Varner DD. Relationship of seminal plasma level and extender type to sperm motility and DNA integrity. Therio 2005;63: 1584–91. [21] Evans DP, Wixon R. Clinical aspects of sperm DNA fragmentation detection and male infertility. Therio 2006;65: 979–91. [22] Cochran JD, Amann RP, Froman DP, Pickett BW. Effects of centrifugation, glycerol level, cooling to 5 8C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Therio 1984;22:25–38. [23] Revell SG, Pettit MT, Ford TC. Use of centrifugation over iodixanol to reduce damage when processing stallion sperm for freezing. J Reprod Fertil 1997, Abstr series no. 19, p38. [24] Waite JA, Love CC, Brinsko SP, Teague SR, Salazar Jr JL, Mancil SS, Varner DD. Factors impacting equine sperm recovery rate and quality following cushioned centrifugation. Therio 2008;70:704–14.