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Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing
Accepted Manuscript Title: Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing Authors: J. Santiago-Moreno, M.C. Esteso, C. Casta˜no, A. Toledano-D´ıaz, J.A. Delgadillo, A. L´opez-Sebasti´an PII: DOI: Reference:
S0378-4320(16)30709-6 http://dx.doi.org/doi:10.1016/j.anireprosci.2017.04.002 ANIREP 5579
To appear in:
Animal Reproduction Science
Received date: Accepted date:
30-11-2016 7-4-2017
Please cite this article as: Santiago-Moreno, J., Esteso, M.C., Casta˜no, C., Toledano-D´ıaz, A., Delgadillo, J.A., L´opez-Sebasti´an, A., Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2017.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing Running title: Selection of goat sperm
J. Santiago-Morenoa,*, M.C. Estesoa, C. Castañoa, A. Toledano-Díaza, J.A. Delgadillob A. LópezSebastiána
a
Departamento de Reproducción Animal, INIA, 28040 Madrid, Spain
bCentro
de Investigación en Reproducción Caprina, CIRCA, Departamento de Ciencias Médico-
Veterinarias, Universidad Autónoma Agraria Antonio Narro, 27054 Torreón, Coahuila, Mexico
*
Corresponding author: Tel.: +34 91 347 40 20; Fax: +34 91 347 40 14; E-mail address:
[email protected]
ABSTRACT Seminal plasma removal is routine in goat sperm cryopreservation protocols. The classical washing procedure designed to accomplish this usually leaves the pellet resulting from use of this procedure contaminated with dead sperm, debris, and cells other than sperm. This contamination negatively affects viability of sperm after cryopreservation. The present research was conducted to compare the effect on chilled and frozen-thawed goat sperm of the classical washing method to that of a selective washing method involving density gradient centrifugation (DGC). In the first experiment, sperm variables were measured in freshly
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collected sperm, and again after its washing with both methods and chilling at 5 ºC for 0, 3, 24, 48, 72 or 96 h. The DGC-washed sperm had greater (P<0.01) straight line velocity (VSL), average path velocity (VAP) and progression ratio values at all chilling times. The amplitude of lateral head displacement (ALH) was, however, less (P<0.001) in the DGC-washed sperm at all chilling times. There was a negative correlation (P<0.05) between ALH and VSL. In the second experiment involving the freezing-thawing of sperm washed by using either method, aliquots were post-wash diluted with a Tris-citric acid/glucose/egg yolk/glycerol-based medium and frozen in liquid nitrogen for 5 days. After thawing, neither the VCL, VSL nor VAP of the DGC-washed samples were affected, whereas the traditionally washed samples had less motility. In conclusion, the use of DGC was associated with enhanced sperm motility variables after chilling and freezing-thawing. This procedure would, therefore, be a useful means of removing seminal plasma from goat semen and obtaining greater quality sperm for insemination purposes.
Keywords: Sperm purification; Sperm selection; Cryopreservation; Buck
1. Introduction The seminal plasma of goats contains a phospholipase, secreted from the bulbourethral glands, which can hydrolyse the membrane phospholipids of sperm, reducing the chances of efficient sperm preservation. The presence of seminal plasma also reduces sperm survival via production of a toxin derived from the phospholipids in egg yolk extenders (Aamdal et al., 1965; Pellicer-Rubio and Combarnous, 1998; Sias et al., 2005). The removal of seminal plasma is, therefore, routine in goat sperm cryopreservation protocols. Seminal plasma is usually removed by washing sperm samples with a solution such as Krebs-Ringer phosphate glucose, 2
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followed by centrifugation (Coloma et al., 2010). The supernatant is then removed and the pellet of sperm resuspended in an extender. The removal of the seminal plasma by this classical method is associated with greater acrosome integrity (Memon et al., 1985) and sperm motility after freezing-thawing (Ritar and Salamon, 1982; 1991), and is now a routine part of many goat semen cryopreservation protocols. This washing procedure, however, does not facilitate selection of viable sperm populations, and the centrifugation pellet routinely contains abnormal, moribund and dead sperm, as well as leukocytes, epithelial cells and debris. It can also be contaminated with microorganisms (Mortimer, 1994; Santiago-Moreno et al., 2011). In addition, non-functional sperm can cause damage to the membranes of other sperm cells by producing reactive oxygen species (ROS) within the pellet (Aitken and Clarkson, 1987), and increasing the risk of the development of anti-sperm antibodies after sperm is deposited in the uterus. Selective washing techniques that remove these unwanted components along with the seminal plasma are, therefore, needed (Rodríguez-Martínez et al., 1997; Phillips et al., 2012; Galuppo et al., 2013; Guimarães et al., 2015). Density gradient centrifugation (DGC) is a selective washing technique that produces seminal plasma-free suspensions of highly motile sperm devoid of cell debris, lymphocytes, epithelial cells, abnormal and immature sperm, sperm with damaged DNA, bacteria and dust (Jayaraman et al., 2012; Jiménez-Rabadán et al., 2012). Motile and morphologically normal sperm can then be selected according to the kinetic capacities and sperm head density. During cryopreservation (chilling, freezing-thawing), sperm cells are exposed to cold shock and atmospheric oxygen. This leads to the overproduction of ROS and increases the susceptibility of the sperm cell membranes to lipid peroxidation (Bucak et al., 2008). The
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elimination of debris, dead sperm and other cells by selective washing methods such as DCG should help avoid a harmful increase in the concentration of ROS and improve the motility variables and viability of both chilled and frozen-thawed goat sperm. The aim of the present research was to compare the classic and DGC washing methods. 2. Material and methods All diluents and media were prepared in-house using reagent-grade chemicals purchased from Panreac Química S.A. (Barcelona, Spain) and Sigma Chemical Co. (St. Louis, Missouri, USA). 2.1. Animals and semen collection The experimental animals were five adult Malagueña breed bucks (Capra hircus; 1.5-3 years old at the onset of the study). The bucks were housed at the INIA Department of Animal Reproduction facility (Madrid, 40º 25´N) and fed Visan K59 (Visan Ind. Zoot., Madrid, Spain) plus barley grain, barley straw and dry alfalfa supplements. Water, mineral and vitamin blocks were available ad libitum. All animals were managed according to procedures approved by the INIA Ethics Committee, and techniques were performed in accordance with the Spanish Policy for Animal Protection (RD53/2013), which conforms to the European Union Directive 86/609 regarding the protection of animals used in scientific experiments. The same five animals were used in both experiments of the present study. For semen collection, the bucks were deeply sedated with 0.09 mg/kg of intravenous (i.v.) xylazine (Rompun, Química Farmacéutica Bayer SA, Barcelona, Spain) plus 0.8 mg/kg i.v. flunixin meglumine (Meganyl®, Syva, León, Spain). Sperm were collected by electroejaculation using a Lane Pulsator IIIZ electroejaculator (Lane Manufacturing Inc., Denver, Colorado, USA), using 0.1to 0.3 mA electrical pulses lasting 5 s with 2 s intervals between pulses. Fresh semen
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samples were transported to an adjacent laboratory immediately after collection. The diluents and all other materials coming into contact with semen were maintained at 37 ºC. All materials and equipment used to collect, handle and process the semen were either new or sterilised prior to use by autoclaving or ultraviolet irradiation. When semen collection was complete, the effects of the xylazine were reversed by the administration of 0.7 mg/kg yohimbine hydrochloride (Sigma, St. Louis, Missouri, USA) (half i.m/half i.v-injected). 2.2. Experimental design 2.2.1. Experiment 1: Influence of washing method on the quality of chilled sperm A total of 28 sperm samples (5 or 6 from each animal) were collected over the period March to July 2014. Each sample was divided into three aliquots; one remained unwashed (control), one was washed by the classic washing (CW) method, and the third subjected to DGC washing following the method of Santiago-Moreno et al. (2014). The samples were then diluted with Tris-citric acid-glucose (TCG) medium plus 6% egg yolk (v/v; TCG-e.y) to 400 x 106 sperm/mL in a 15 ml centrifuge tube, set in a beaker with 30 ml of water at room temperature and transferred to a refrigerator at 5 ºC, for 3, 24, 48, 72 or 96 h. Both before and after treatment, sperm motility variables were measured using a Computer-Aided Sperm Analysis system (CASA; see below), and sperm viability and acrosome status by fluorescence microscopy was assessed. 2.2.2. Experiment 2: Influence of washing method on frozen-thawed sperm quality Ten sperm samples (two from each animal) collected in October 2012, were divided into two aliquots and motility variables (measured by CASA; see below), sperm viability and
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acrosome status (measured by fluorescence microscopy) analysed before washing. One aliquot was then washed following the CW method and the other by the DGC method. The sperm suspensions were then diluted with TCG medium plus 6% egg yolk (v/v) and glycerol 684 mM (5% v/v; TCG-e.y-gly) to 400 x 106 sperm/mL in a 15 ml centrifuge tube, set in a beaker containing 30 ml of water at room temperature, and subsequently transferred to a refrigerator at 5 ºC followed by freezing in straws (see below; about 100 x 10 6 sperm in each straw). After 5 days the straws were thawed in a water bath at 37 ºC for 30 s, the contents poured into a glass tube, and the CASA- and fluorescence microscopy-determined sperm variables measured. One aliquot of the frozen-thawed sample was then incubated at 38.5 ºC in a 5% CO2 atmosphere for 2 h and the sperm variables again assessed. 2.3. Sperm analysis The volume of each ejaculate was measured using a micropipette (Gilson, France). Total fresh sperm concentrations were calculated using a Neubauer chamber (Marienfeld, LaudaKönigshofen, Germany). For both ejaculated and post-wash sperm, motility was objectively assayed using a CASA system coupled to a phase contrast microscope (Nikon Eclipse model 50i; negative contrast; Izasa S.A., Barcelona, Spain) and using Sperm Class Analyzer® v.4.0. software (Microptic S.L., Barcelona, Spain). Sperm samples were diluted over the range 1:60 to 1:100 (% v/v) as required in a Tris-citric acid-glucose washing medium (Coloma et al., 2010; 345 mOsm, pH 6.8) and loaded onto a warmed (37 ºC) 20 µm Leja® 8-chamber slide (Leja Products B.V., Nieuw-Vennep, The Netherlands). The percentage of motile sperm and sperm with progressive motility (MP) were recorded. Sperm movement characteristics - curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF) - were also analysed by CASA. Three
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progression ratios, expressed as percentages, were calculated from the three velocity measurements
described
above:
linearity
(LIN=VSL/VCL
x
100),
straightness
(STR=VSL/VAP x 100), and wobble (WOB = VAP/VCL x 100). A minimum of three fields and 500 sperm tracks were evaluated at 100x for each sample chamber (image acquisition rate 25 frames/s). Sperm viability and acrosomal status were also analysed by fluorescence microscopy (counting 200 cells), using a Nikon Eclipse E200 epifluorescence microscope (DFL epifluorescence, C-SHG1 super high pressure mercury power supply; Nikon Instruments Inc., New York, USA). These variables were simultaneously evaluated using a fluorochrome combination of propidium iodide (PI) and fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin (PNA-FITC), as previously described (Soler et al., 2005). 2.4. Sperm washing 2.4.1. Classic washing method The CW solution used for this experiment was composed of Tris 313.7 mM, citric acid 104.7 mM and glucose 30.3 mM (TCG medium; 345 mOsm/kg, pH 6.8). Sperm samples were diluted 1:9 (v/v) with the washing solution at 37 ºC, and centrifuged at 900 g for 20 min. The supernatant was then removed and the sperm resuspended at room temperature (23 ºC) in TCG-e.y chilling medium (Experiment 1) or TCG-e.y-gly freezing medium (Experiment 2). The final sperm concentration in the medium was 400 x 106 sperm/mL. 2.4.2. Selective washing method: density gradient centrifugation Bovipure™ (Nidacon, Mölndal, Sweden), an iso-osmotic salt solution containing colloidal silica particles coated with silane, was developed for the DGC selection of bull and can also be used for sheep and goat sperm assessments. The bottom layer medium was prepared by adding 8 mL Bovipure™ to 2 mL Bovidilute™ (Nidacon, Mölndal, Sweden); the top layer
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medium was prepared by adding 4 mL Bovipure™ to 6 mL Bovidilute™. Both bottom and top layer media were similar to Capripure Bottom Layer™ medium and Capripure Top Layer™ medium, respectively, which were previously used in the laboratory where the present experiments were conducted for sperm selection of wild goats (Santiago-Moreno et al., 2014). Bottom layer medium (0.5 mL for every 333 x 106 sperm/mL to be added) was placed in a 15 mL centrifuge tube (Sterilin®, Stone, UK), and then carefully overlain with the same volume of top layer medium (Santiago-Moreno et al., 2014). The sperm sample was carefully placed at the top of the gradient and centrifuged at 300 g for 20 min. The supernatant was then removed. The pellet was resuspended in the chilling medium (TCG-e.y; Experiment 1) or in the freezing medium (TCG-e.y-gly; Experiment 2). The final sperm concentration in the medium was 400 x 106 sperm/mL. 2.5. Freezing of sperm After 5 min at room temperature (see above), the diluted sperm samples were transferred to a refrigerator at 5 ºC. Cooling to this temperature took about 1 hand samples were then maintained at this temperature for a further 2 h. At this point, aliquots of samples were loaded into 0.25 ml French straws (IMV®; L’Aigle, France) and frozen by placing them in the nitrogen vapour 5 cm above the surface of an open liquid nitrogen tank for 10 min before plunging them into the liquid nitrogen. 2.6. Statistical analysis The results are presented as means ± SE. The sperm variable values that had non-normal distributions, as determined by the Shapiro-Wilks test, were arcsine-transformed before analysis. The influence of the washing method (CW or DGC) on the sperm variables after refrigeration at different times (Experiment 1) was analysed by repeated measured ANOVA.
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The influence of washing method on the sperm variables after freezing-thawing (Experiment 2) was analysed by ANOVA, following the statistical model: xij = m + Ai + eij, where xij = the value of the measured sperm variable, m = the overall mean, A i = the effect of washing method (i = 1-3; no wash, CW and DGC), and eij = the residual (j = 1-10). In addition, comparisons between fresh and frozen-thawed sperm variables and between frozen-thawed samples prepared by the different washing methods were made using the paired t test. Pearson linear correlations were determined to examine the association between ALH and the sperm velocity variables VSL, VCL and VAP. All calculations were performed using Statistica software for Windows v.12 (StatSoft Inc. Tulsa, OK, USA). 3. Results 3.1. Experiment 1: Influence of washing method on chilled sperm The percentage of motile sperm at different chilling times was affected by the type of washing treatment (P<0.001). The percentage value for motile sperm in the DGC samples remained nearly constant over time, with only a slight reduction seen at 96 h. This value was, however, less (P<0.05) than in both the control and CW samples between 0 and 24 h. In both the control and CW samples, the percentage of motile sperm were not significantly different between 0 and 3 h, but progressively decreased after 3 h to reach - at 48 h - values similar to those recorded for the DGC samples (Fig. 1). The percentage of sperm with progressive motility tended (P = 0.06) to be greater in the DGC samples than in the CW or control samples. Values for this variable, however, decreased (P<0.01) in all groups from maximum values at 0 h to minimum values to 96 h of chilling (Fig. 1).
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The VSL (P<0.01) and VAP (P<0.001) values were greater for the DGC samples over the entire chilling period. The VCL was not affected by the washing type, with values decreasing as chilling time increased in all groups (Fig. 2). The LIN, STR and WOB values were all greater (P<0.001) in the DGC samples at all times (Fig. 3) The ALH remained less (P<0.001) in the DGC samples than in the control and CW samples (Fig. 4). There was a significant negative correlation (P<0.05; r = -0.59) between ALH and VSL. The BCF was not affected by washing treatment nor by chilling time: 9.1 ± 0.1 Hz in non-washed samples (controls), 9.2 ± 0.1 Hz in CW samples, and 9.0 ± 0.1 Hz in DGC samples. The washing treatment affected (P<0.05) the percentage of live sperm as chilling time advanced. Live sperm counts were less in the initial DGC samples but remained relatively constant as chilling time advanced. In the control samples the percentage of live sperm was greater at 0 h than in either the CW or DGC samples, but decreased (P<0.05) with chilling time to reach - at 96 h - values similar to those recorded for the DGC samples (Fig. 5). The percentage of sperm with intact acrosomes was not affected by the washing procedure (Fig. 5). 3.2. Experiment 2: Influence of washing method on frozen-thawed sperm variables Data in Table 1 indicates the effect of the DGC washing method on sperm variables before freezing and after thawing at 0 h and after 2 h of incubation at 38.5 ºC in a 5% CO2 atmosphere. Both VCL (P = 0.06) and VSL (P = 0.09) trended towards being greater in the pre-freezing DCG than CW samples. In the DGC samples, VCL, VSL and VAP were not affected by freezing-thawing; however, these variables were less desirable (P<0.05) in the CW samples. The differences regarding VCL, VSL and VAP values after freezing-thawing were no longer evident following 2 hours of incubation. There were no differences between the washing
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methods in terms of post-thaw motile sperm, percentage MP, percentage LIN, percentage STR, BCF, ALH, percentage live sperm, or the percentage of sperm with an intact acrosome. 4. Discussion For the majority of sperm motility variables, using DGC resulted in more desirable values for sperm quality than either not washing or using the classical CW procedure. It would, therefore, appear that use of DGC is a useful way of removing seminal plasma and selecting high quality sperm in goats. Subsequent to washing (0 h), DGC treatment was associated with an enhanced VSL and progression ratio values, but lesser total motile sperm and sperm viability values. The latter has also been reported when using Capripure® DGC to prepare ibex (Capra pyrenaica) sperm (Santiago-Moreno et al., 2014). This may be a consequence of centrifugation (which has a detrimental effect on sperm; Aitken and Clarkson, 1988) with the centrifugation forces involved affecting the motility and membrane integrity of the sperm of small ruminants (Gil et al., 1999; Ritar, 1993). The advantages of DGC, however, are evident when storing chilled sperm. The percentage of live sperm and total motile sperm remained relatively constant during the chilled storage period - unlike that for the CW samples - suggesting that the use of the DGC procedure allows for the removal of sperm with sub-optimal functionality along with most apoptotic sperm (Ricci et al., 2009). The more desirable VSL values and progression ratios recorded for the chilled DCG samples suggests that motile sperm that were present when this procedure was used had greater mitochondrial membrane potentials (Donnelly et al., 2000; Marchetti et al., 2002). Use of the DGC results in selection of sperm with characteristics of movement that allow these cells to have greater movement velocities. In the present studies, ALH values remained less in chilled DGC samples than similarly treated CW samples. Moreover, there was a negative correlation between ALH and VSL. These results suggest that a lateral head displacement of >3 µm leads to less efficient progressive movement of goat sperm. These findings are not consistent with
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those reported for other species (e.g., humans), in which selection procedures, where use of the swim-up and Percoll, methods resulted in increased VSL and ALH (Liu et al., 1991; Swanson et al., 1995). The ALH is an important motility variable, and adequate values are required if barriers, such as that imposed by cervical mucus, are to be overcome, and the oocyte barriers to fertilization (e.g., the cumulus cell layers and the zona pellucida) are to be penetrated (Aitken et al., 1985). The ALH increases greatly in hyperactive sperm undergoing capacitation, but freshly ejaculated semen usually contains only a small fraction of hyperactive sperm. This may explain the negative correlation between ALH and VSL observed in the present experiments. The use of the DGC, thus, appears to facilitate selection of non-capacitated goat sperm. Neither washing procedure affected the percentage of intact acrosomes either before or after chilling, suggesting centrifugation may have no harmful effect on the acrosome. Inconsistent with this finding, Maxwell et al. (2007) reported that, when using PureSperm® DGC with bull sperm, the proportion of sperm with an intact acrosome was greater, although the mechanism underlying this is unclear. Discontinuous colloidal silica gradients may affect the capacitation dynamics and responsiveness of mouse and boar sperm to capacitation stimuli (Furimsky et al., 2005; Henning et al., 2015), and the effect of DGC in this respect in goats needs to be further examined. Previous reports have indicated that sperm selection techniques are more beneficial when working with low quality sperm (Valcarcel et al., 1996; Morató et al., 2013). The present results, however, suggest the used of DGC may be beneficial regardless of sperm quality. In Experiment 1, the sperm velocity variables in the fresh samples were greater than in Experiment 2; substantial improvements in progression ratios in the DGC-selected sperm were observed during the entire chilling period compared to no washing or use of the classical CW procedure. In Experiment 2, a lesser initial sperm quality was recorded, but both VSL and VAP trended towards increasing soon after DGC treatment. The differences between the
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results of Experiment 1 and 2 in terms of the influence of washing method on prechilling/freezing sperm variables might be related to differences in the initial sperm quality caused by the advancing age of the animals between the times Experiments 1 and 2 (some 2 years) were conducted, or the season of semen collection (Roca et al., 1992; Arrebola et al., 2010). Although both use of CW and DGC are effective in the removal of seminal plasma, the CW method has the major drawback that immotile, dead and abnormal sperm all remain present, as does any debris, along with any unwanted cell (epithelial cells, inflammatory cells and bacteria etc.). Dead sperm exert a well-known toxic effect on viable sperm cells. Further, CW-washed sperm had greater DNA fragmentation than DGC-selected sperm (Jackson et al., 2010). The ROS produced by leukocytes and male germ cells, which may be present in ejaculates, also induce lipid peroxidation and other damage to sperm cells (Henkel, 2011). Because freezingthawing also increases the production of free radicals, frozen-thawed sperm variables should be enhanced when sperm selection techniques such as DGC are used. Indeed, the present findings indicate that the use of DGC has advantages over the CW procedure when freezing sperm; after thawing, the values of many sperm motility variables were greater. There are inconsistencies of results in the present study and those reported for bull sperm when using both single and double layer centrifugation techniques with iodixanol. In the study with bulls, sperm quality after cryopreservation was similar to that of untreated samples (Gloria et al., 2016). Differences in the species and the methods used may explain these inconsistences. Indeed, the use of DGC prior to cryopreservation is associated with enhanced frozen-thawed sperm quality in stallions (Hoogewijs et al., 2011) and boars (Martinez-Alborcia et al., 2013). In the present study, however, no benefits of DCG were observed with respect to membrane integrity, unlike that reported in previous studies with stallion sperm involving two-layer iodixanol density centrifugation (Heutelbeck et al., 2015).
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In conclusion, use of both CW and DGC can be used to remove seminal plasma from goat sperm samples. Sperm selection by use of DGC is highly recommended when chilling and freezing goat semen because of the greater sperm of quality when this procedure is used.
Acknowledgements This research was funded by MINECO grant AGL2014-52081-R. The authors thank B. Labrador, L. Maribel Cedillo and J. Anabel Loya for assistance with data management.
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Martinez-Alborcia, M.J., Morrell, J.M., Gil, M.A., Barranco, I., Maside, C., Alkmin, D.V., Parrilla, I., Martinez, E.A., Roca, J., 2013. Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa. Anim. Reprod. Sci. 140,173–179. Maxwell, W.M.C, Parrilla, I., Caballero, I., Garcia, E., Roca, J., Martinez, E.A.., Vazquez, J.M., Rath, D., 2007. Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSpem ® density gradient centrifugation. Reprod. Dom. Anim. 42, 489-494. Memon, M.A., Bretzlaff, K.N., Ott, R.S., 1985. Effect of washing on motility and acrosome morphology of frozen-thawed goat spermatozoa. Am. J. Vet. Res. 46, 473-475. Morató, R., de Souza Soares, J.M., Orero, G., Mogas, T., 2013. Pre-selection by double layer density gradient centrifugation improve the fertilising capacity of frozen-thawed, capacity stallion sperm. Anim. Reprod. Sci. 139, 62-68. Mortimer, D., 1994. Sperm washing. In: Practical Laboratory Andrology, Oxford University Press Inc. New York, pp. 267-286. Pellicer-Rubio, M.T., Combarnous, Y., 1998. Deterioration of goat spermatozoa in skimmed milk-based extenders as a result of oleic acid released by the bulbourethral lipase BUSgp60. J. Reprod. Fertil. 112:95-105. Phillips, T.C., Dhaliwal, G.K., Verstegen-Onclin, K.M., Verstegen, J.P., 2012. Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen. Theriogenology 77, 39-45. Ricci, G., Perticarari, S., Boscolo, R., Montico, M., Guaschino, S., Presani, G., 2009. Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique. Fertil. Steril. 91, 632-638.
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Ritar, A.J., 1993. Control of ovulation, storage of semen and artificial insemination of fibreproducing goats in Australia: a review. Aust. J. Exp. Agric. 33, 807-820. Ritar, A.J., Salamon, S., 1982. Effects of seminal plasma and of its removal and of egg yolk in the diluent on the survival of fresh and frozen-thawed spermatozoa of the Angora goat. Aust. J. Biol. Sci. 35, 305-312. Ritar, A.J., Salamon, S., 1991. Effects of month of collection, method of processing, concentration of egg yolk and duration of frozen storage on viability of Angora goat spermatozoa. Small Rumin. Res. 4, 29-37. Roca, J., Martinez, E., Vazquez, J.M., Coy, P., 1992. Characteristics and seasonal variations in the semen of Murciano-Granaina goats in the Mediterranean area. Anim. Reprod. Sci. 29, 255-262. Rodriguez-Martinez, H., Larsson, B., Pertoft, H., 1997. Evaluation of sperm damage and techniques for sperm clean-up. Reprod. Fertil. Dev. 9, 297-308. Santiago-Moreno, J., Carvajal, A., Astorga, R.J., Coloma, M.A., Toledano-Díaz, A., GómezGuillamón, F., Salas-Vega, R., López-Sebastián, A., 2011. Potential impact of diseases transmissible by sperm on the establishment of Iberian Ibex (Capra pyrenaica) genome resource banks. Eur. J. Wildl. Res. 57, 211-216. Santiago-Moreno, J., Esteso, M.C., Castaño, C., Toledano-Díaz, A., Rodríguez, E., LópezSebastián, A., 2014. Sperm selection by Capripure® density-gradient versus dextran swim-up procedure in wild mountain ruminants. Anim. Reprod. Sci. 149, 178-186. Sias, B., Ferrato, F., Pellicer-Rubio, M.T., Forgerit, Y., Guillouet, P., Leboeuf, B., Carriere, F., 2005. Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma. Biochim. Biophys. Acta 1686,169-180.
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Soler, A.J., Esteso, M.C., Fernández-Santos, M.R., Garde, J.J., 2005. Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5ºC in the epididymis for several days. Theriogenology 64, 1503-1517. Swanson, M.L., Collins, J.M., Freiman, S.F., Dubin, N.H., 1995. Effect of Percoll wash on sperm motion parameters and subsequent fertility in intrauterine insemination cycles. J. Assist. Reprod. Genet. 12, 48-54. Valcarcel, A., de las Heras, M.A., Moses, D.F., Perez, L.J., Baldassarre, H., 1996. Comparison between Sephadex G-10 and Percoll for preparation of normospermic, asthenospermic and frozen/thawed ram semen. Anim. Reprod. Sci. 41, 215-224.
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Figure legends Fig. 1. Percentage of motile and progressively motile (MP) sperm in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.05) between DGC and both CONTROL and CW.
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Fig. 2. Curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP), in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.01 and P<0.001 for VSL and VAP, respectively) between DGC and both CONTROL and CW.
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Fig. 3. Linearity (LIN), straightness (STR) and wobble (WOB) in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.001) between DGC and both CONTROL and CW.
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Fig. 4. Amplitude of lateral head displacement (ALH) in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.001) between DGC and both CONTROL and CW.
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Fig. 5. Percentage of live sperm and sperm with intact acrosomes in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.05) between DGC and both CONTROL and CW. The arrow represents the time of decrease (P<0.05) in the percentage of live sperm, for CW and CONTROL regarding 0 H.
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Table 1 Values of sperm variables for fresh and frozen-thawed sperm, pre-washed either by the classical (CW) or density gradient centrifugation (DGC) methods, and for similar frozenthawed samples after 2 h of incubation at 38.5ºC in a 5% CO2 atmosphere (means±SE). Sperm variables
Original ejaculate pre-freezing
CW pre- DGC freezing pre-freezing
CW frozenthawed
DGC frozenthawed
Motile sperm (%) MP (%)
59.7±6.2a
54.7±7.0a
59.0±6.2a
26.8±6.8b
30.1±5.4a
32.6±6.8a
36.0±5.3a
15.7±5.1b
VCL (µm/s) VSL (µm/s) VAP (µm/s) LIN (%)
91.9±7.9a
98.3±15.7a
117.8±16.6a 69.6±8.4b
103.6±8.8a 40.8±4.8c
41.3±6.8c
54.0±8.0a
68.6±14.0a
84.9±14.9a
42.9±8.1b
81.2±7.6a
16.7±3.2c
17.7±5.8c
69.2±8.5a
82.6±16.1a
102.8±17.1a 51.6±8.8b
93.0±8.4a
22.6±3.7c
24.3±7.1c
56.6±5.3abc 65.2±6.1abc 66.0±8.4abc
57.8±4.9ac 77.9±1.1ab 40.3±5.1d
38.2±5.4d
STR (%)
75.8±3.2a
80.8±2.5a
80.4±3.4a
76.8±6.8a
30.7±4.9b
CW frozenthawed after 2 h incubation 12.4±3.3c
DGC frozenthawed after 2 h incubation 14.1±6.1c
19.5±3.1b
2.4±1.1c
4.6±3.3c
87.1±0.9a
71.7±3.0bc 69.2±4.3c
WOB 73.3±4.1b 79.6±5.2ab 81.6±7.5ab 70.8±4.4b 89.4±1.0a 54.7±4.7c (%) BCF 9.0±0.2 9.3±0.3 8.2±0.7 9.2±0.5 8.8±0.4 8.0±1.1 (Hz) ALH 2.7±0.2 2.6±0.2 2.3±0.3 2.5±0.1 2.3±0.2 1.8±0.3 (µm) Live 65.8±7.2a 52.9±5.4a 59.5±7.1a 26.1±5.1b 22.5±2.9b 19.6±4.2bc sperm (%) Intact 88.0±5.9a 77.6±5.0a 80.4±6.7a 58.3±5.6b 53.9±5.7b 54.8±5.4b acrosome (%) Values with different letters (a,b,c) in the same row are significantly different (P<0.05)
53.7±5.3c 6.9±1.2 1.7±0.3 14.0±2.3c
46.9±8.1b
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S0378-4320(16)30709-6 http://dx.doi.org/doi:10.1016/j.anireprosci.2017.04.002 ANIREP 5579
To appear in:
Animal Reproduction Science
Received date: Accepted date:
30-11-2016 7-4-2017
Please cite this article as: Santiago-Moreno, J., Esteso, M.C., Casta˜no, C., Toledano-D´ıaz, A., Delgadillo, J.A., L´opez-Sebasti´an, A., Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing.Animal Reproduction Science http://dx.doi.org/10.1016/j.anireprosci.2017.04.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Seminal plasma removal by density-gradient centrifugation is superior for goat sperm preservation compared with classical sperm washing Running title: Selection of goat sperm
J. Santiago-Morenoa,*, M.C. Estesoa, C. Castañoa, A. Toledano-Díaza, J.A. Delgadillob A. LópezSebastiána
a
Departamento de Reproducción Animal, INIA, 28040 Madrid, Spain
bCentro
de Investigación en Reproducción Caprina, CIRCA, Departamento de Ciencias Médico-
Veterinarias, Universidad Autónoma Agraria Antonio Narro, 27054 Torreón, Coahuila, Mexico
*
Corresponding author: Tel.: +34 91 347 40 20; Fax: +34 91 347 40 14; E-mail address:
[email protected]
ABSTRACT Seminal plasma removal is routine in goat sperm cryopreservation protocols. The classical washing procedure designed to accomplish this usually leaves the pellet resulting from use of this procedure contaminated with dead sperm, debris, and cells other than sperm. This contamination negatively affects viability of sperm after cryopreservation. The present research was conducted to compare the effect on chilled and frozen-thawed goat sperm of the classical washing method to that of a selective washing method involving density gradient centrifugation (DGC). In the first experiment, sperm variables were measured in freshly
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collected sperm, and again after its washing with both methods and chilling at 5 ºC for 0, 3, 24, 48, 72 or 96 h. The DGC-washed sperm had greater (P<0.01) straight line velocity (VSL), average path velocity (VAP) and progression ratio values at all chilling times. The amplitude of lateral head displacement (ALH) was, however, less (P<0.001) in the DGC-washed sperm at all chilling times. There was a negative correlation (P<0.05) between ALH and VSL. In the second experiment involving the freezing-thawing of sperm washed by using either method, aliquots were post-wash diluted with a Tris-citric acid/glucose/egg yolk/glycerol-based medium and frozen in liquid nitrogen for 5 days. After thawing, neither the VCL, VSL nor VAP of the DGC-washed samples were affected, whereas the traditionally washed samples had less motility. In conclusion, the use of DGC was associated with enhanced sperm motility variables after chilling and freezing-thawing. This procedure would, therefore, be a useful means of removing seminal plasma from goat semen and obtaining greater quality sperm for insemination purposes.
Keywords: Sperm purification; Sperm selection; Cryopreservation; Buck
1. Introduction The seminal plasma of goats contains a phospholipase, secreted from the bulbourethral glands, which can hydrolyse the membrane phospholipids of sperm, reducing the chances of efficient sperm preservation. The presence of seminal plasma also reduces sperm survival via production of a toxin derived from the phospholipids in egg yolk extenders (Aamdal et al., 1965; Pellicer-Rubio and Combarnous, 1998; Sias et al., 2005). The removal of seminal plasma is, therefore, routine in goat sperm cryopreservation protocols. Seminal plasma is usually removed by washing sperm samples with a solution such as Krebs-Ringer phosphate glucose, 2
3
followed by centrifugation (Coloma et al., 2010). The supernatant is then removed and the pellet of sperm resuspended in an extender. The removal of the seminal plasma by this classical method is associated with greater acrosome integrity (Memon et al., 1985) and sperm motility after freezing-thawing (Ritar and Salamon, 1982; 1991), and is now a routine part of many goat semen cryopreservation protocols. This washing procedure, however, does not facilitate selection of viable sperm populations, and the centrifugation pellet routinely contains abnormal, moribund and dead sperm, as well as leukocytes, epithelial cells and debris. It can also be contaminated with microorganisms (Mortimer, 1994; Santiago-Moreno et al., 2011). In addition, non-functional sperm can cause damage to the membranes of other sperm cells by producing reactive oxygen species (ROS) within the pellet (Aitken and Clarkson, 1987), and increasing the risk of the development of anti-sperm antibodies after sperm is deposited in the uterus. Selective washing techniques that remove these unwanted components along with the seminal plasma are, therefore, needed (Rodríguez-Martínez et al., 1997; Phillips et al., 2012; Galuppo et al., 2013; Guimarães et al., 2015). Density gradient centrifugation (DGC) is a selective washing technique that produces seminal plasma-free suspensions of highly motile sperm devoid of cell debris, lymphocytes, epithelial cells, abnormal and immature sperm, sperm with damaged DNA, bacteria and dust (Jayaraman et al., 2012; Jiménez-Rabadán et al., 2012). Motile and morphologically normal sperm can then be selected according to the kinetic capacities and sperm head density. During cryopreservation (chilling, freezing-thawing), sperm cells are exposed to cold shock and atmospheric oxygen. This leads to the overproduction of ROS and increases the susceptibility of the sperm cell membranes to lipid peroxidation (Bucak et al., 2008). The
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elimination of debris, dead sperm and other cells by selective washing methods such as DCG should help avoid a harmful increase in the concentration of ROS and improve the motility variables and viability of both chilled and frozen-thawed goat sperm. The aim of the present research was to compare the classic and DGC washing methods. 2. Material and methods All diluents and media were prepared in-house using reagent-grade chemicals purchased from Panreac Química S.A. (Barcelona, Spain) and Sigma Chemical Co. (St. Louis, Missouri, USA). 2.1. Animals and semen collection The experimental animals were five adult Malagueña breed bucks (Capra hircus; 1.5-3 years old at the onset of the study). The bucks were housed at the INIA Department of Animal Reproduction facility (Madrid, 40º 25´N) and fed Visan K59 (Visan Ind. Zoot., Madrid, Spain) plus barley grain, barley straw and dry alfalfa supplements. Water, mineral and vitamin blocks were available ad libitum. All animals were managed according to procedures approved by the INIA Ethics Committee, and techniques were performed in accordance with the Spanish Policy for Animal Protection (RD53/2013), which conforms to the European Union Directive 86/609 regarding the protection of animals used in scientific experiments. The same five animals were used in both experiments of the present study. For semen collection, the bucks were deeply sedated with 0.09 mg/kg of intravenous (i.v.) xylazine (Rompun, Química Farmacéutica Bayer SA, Barcelona, Spain) plus 0.8 mg/kg i.v. flunixin meglumine (Meganyl®, Syva, León, Spain). Sperm were collected by electroejaculation using a Lane Pulsator IIIZ electroejaculator (Lane Manufacturing Inc., Denver, Colorado, USA), using 0.1to 0.3 mA electrical pulses lasting 5 s with 2 s intervals between pulses. Fresh semen
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samples were transported to an adjacent laboratory immediately after collection. The diluents and all other materials coming into contact with semen were maintained at 37 ºC. All materials and equipment used to collect, handle and process the semen were either new or sterilised prior to use by autoclaving or ultraviolet irradiation. When semen collection was complete, the effects of the xylazine were reversed by the administration of 0.7 mg/kg yohimbine hydrochloride (Sigma, St. Louis, Missouri, USA) (half i.m/half i.v-injected). 2.2. Experimental design 2.2.1. Experiment 1: Influence of washing method on the quality of chilled sperm A total of 28 sperm samples (5 or 6 from each animal) were collected over the period March to July 2014. Each sample was divided into three aliquots; one remained unwashed (control), one was washed by the classic washing (CW) method, and the third subjected to DGC washing following the method of Santiago-Moreno et al. (2014). The samples were then diluted with Tris-citric acid-glucose (TCG) medium plus 6% egg yolk (v/v; TCG-e.y) to 400 x 106 sperm/mL in a 15 ml centrifuge tube, set in a beaker with 30 ml of water at room temperature and transferred to a refrigerator at 5 ºC, for 3, 24, 48, 72 or 96 h. Both before and after treatment, sperm motility variables were measured using a Computer-Aided Sperm Analysis system (CASA; see below), and sperm viability and acrosome status by fluorescence microscopy was assessed. 2.2.2. Experiment 2: Influence of washing method on frozen-thawed sperm quality Ten sperm samples (two from each animal) collected in October 2012, were divided into two aliquots and motility variables (measured by CASA; see below), sperm viability and
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acrosome status (measured by fluorescence microscopy) analysed before washing. One aliquot was then washed following the CW method and the other by the DGC method. The sperm suspensions were then diluted with TCG medium plus 6% egg yolk (v/v) and glycerol 684 mM (5% v/v; TCG-e.y-gly) to 400 x 106 sperm/mL in a 15 ml centrifuge tube, set in a beaker containing 30 ml of water at room temperature, and subsequently transferred to a refrigerator at 5 ºC followed by freezing in straws (see below; about 100 x 10 6 sperm in each straw). After 5 days the straws were thawed in a water bath at 37 ºC for 30 s, the contents poured into a glass tube, and the CASA- and fluorescence microscopy-determined sperm variables measured. One aliquot of the frozen-thawed sample was then incubated at 38.5 ºC in a 5% CO2 atmosphere for 2 h and the sperm variables again assessed. 2.3. Sperm analysis The volume of each ejaculate was measured using a micropipette (Gilson, France). Total fresh sperm concentrations were calculated using a Neubauer chamber (Marienfeld, LaudaKönigshofen, Germany). For both ejaculated and post-wash sperm, motility was objectively assayed using a CASA system coupled to a phase contrast microscope (Nikon Eclipse model 50i; negative contrast; Izasa S.A., Barcelona, Spain) and using Sperm Class Analyzer® v.4.0. software (Microptic S.L., Barcelona, Spain). Sperm samples were diluted over the range 1:60 to 1:100 (% v/v) as required in a Tris-citric acid-glucose washing medium (Coloma et al., 2010; 345 mOsm, pH 6.8) and loaded onto a warmed (37 ºC) 20 µm Leja® 8-chamber slide (Leja Products B.V., Nieuw-Vennep, The Netherlands). The percentage of motile sperm and sperm with progressive motility (MP) were recorded. Sperm movement characteristics - curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), amplitude of lateral head displacement (ALH), beat-cross frequency (BCF) - were also analysed by CASA. Three
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progression ratios, expressed as percentages, were calculated from the three velocity measurements
described
above:
linearity
(LIN=VSL/VCL
x
100),
straightness
(STR=VSL/VAP x 100), and wobble (WOB = VAP/VCL x 100). A minimum of three fields and 500 sperm tracks were evaluated at 100x for each sample chamber (image acquisition rate 25 frames/s). Sperm viability and acrosomal status were also analysed by fluorescence microscopy (counting 200 cells), using a Nikon Eclipse E200 epifluorescence microscope (DFL epifluorescence, C-SHG1 super high pressure mercury power supply; Nikon Instruments Inc., New York, USA). These variables were simultaneously evaluated using a fluorochrome combination of propidium iodide (PI) and fluorescein isothiocyanate-conjugated peanut (Arachis hypogaea) agglutinin (PNA-FITC), as previously described (Soler et al., 2005). 2.4. Sperm washing 2.4.1. Classic washing method The CW solution used for this experiment was composed of Tris 313.7 mM, citric acid 104.7 mM and glucose 30.3 mM (TCG medium; 345 mOsm/kg, pH 6.8). Sperm samples were diluted 1:9 (v/v) with the washing solution at 37 ºC, and centrifuged at 900 g for 20 min. The supernatant was then removed and the sperm resuspended at room temperature (23 ºC) in TCG-e.y chilling medium (Experiment 1) or TCG-e.y-gly freezing medium (Experiment 2). The final sperm concentration in the medium was 400 x 106 sperm/mL. 2.4.2. Selective washing method: density gradient centrifugation Bovipure™ (Nidacon, Mölndal, Sweden), an iso-osmotic salt solution containing colloidal silica particles coated with silane, was developed for the DGC selection of bull and can also be used for sheep and goat sperm assessments. The bottom layer medium was prepared by adding 8 mL Bovipure™ to 2 mL Bovidilute™ (Nidacon, Mölndal, Sweden); the top layer
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medium was prepared by adding 4 mL Bovipure™ to 6 mL Bovidilute™. Both bottom and top layer media were similar to Capripure Bottom Layer™ medium and Capripure Top Layer™ medium, respectively, which were previously used in the laboratory where the present experiments were conducted for sperm selection of wild goats (Santiago-Moreno et al., 2014). Bottom layer medium (0.5 mL for every 333 x 106 sperm/mL to be added) was placed in a 15 mL centrifuge tube (Sterilin®, Stone, UK), and then carefully overlain with the same volume of top layer medium (Santiago-Moreno et al., 2014). The sperm sample was carefully placed at the top of the gradient and centrifuged at 300 g for 20 min. The supernatant was then removed. The pellet was resuspended in the chilling medium (TCG-e.y; Experiment 1) or in the freezing medium (TCG-e.y-gly; Experiment 2). The final sperm concentration in the medium was 400 x 106 sperm/mL. 2.5. Freezing of sperm After 5 min at room temperature (see above), the diluted sperm samples were transferred to a refrigerator at 5 ºC. Cooling to this temperature took about 1 hand samples were then maintained at this temperature for a further 2 h. At this point, aliquots of samples were loaded into 0.25 ml French straws (IMV®; L’Aigle, France) and frozen by placing them in the nitrogen vapour 5 cm above the surface of an open liquid nitrogen tank for 10 min before plunging them into the liquid nitrogen. 2.6. Statistical analysis The results are presented as means ± SE. The sperm variable values that had non-normal distributions, as determined by the Shapiro-Wilks test, were arcsine-transformed before analysis. The influence of the washing method (CW or DGC) on the sperm variables after refrigeration at different times (Experiment 1) was analysed by repeated measured ANOVA.
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The influence of washing method on the sperm variables after freezing-thawing (Experiment 2) was analysed by ANOVA, following the statistical model: xij = m + Ai + eij, where xij = the value of the measured sperm variable, m = the overall mean, A i = the effect of washing method (i = 1-3; no wash, CW and DGC), and eij = the residual (j = 1-10). In addition, comparisons between fresh and frozen-thawed sperm variables and between frozen-thawed samples prepared by the different washing methods were made using the paired t test. Pearson linear correlations were determined to examine the association between ALH and the sperm velocity variables VSL, VCL and VAP. All calculations were performed using Statistica software for Windows v.12 (StatSoft Inc. Tulsa, OK, USA). 3. Results 3.1. Experiment 1: Influence of washing method on chilled sperm The percentage of motile sperm at different chilling times was affected by the type of washing treatment (P<0.001). The percentage value for motile sperm in the DGC samples remained nearly constant over time, with only a slight reduction seen at 96 h. This value was, however, less (P<0.05) than in both the control and CW samples between 0 and 24 h. In both the control and CW samples, the percentage of motile sperm were not significantly different between 0 and 3 h, but progressively decreased after 3 h to reach - at 48 h - values similar to those recorded for the DGC samples (Fig. 1). The percentage of sperm with progressive motility tended (P = 0.06) to be greater in the DGC samples than in the CW or control samples. Values for this variable, however, decreased (P<0.01) in all groups from maximum values at 0 h to minimum values to 96 h of chilling (Fig. 1).
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The VSL (P<0.01) and VAP (P<0.001) values were greater for the DGC samples over the entire chilling period. The VCL was not affected by the washing type, with values decreasing as chilling time increased in all groups (Fig. 2). The LIN, STR and WOB values were all greater (P<0.001) in the DGC samples at all times (Fig. 3) The ALH remained less (P<0.001) in the DGC samples than in the control and CW samples (Fig. 4). There was a significant negative correlation (P<0.05; r = -0.59) between ALH and VSL. The BCF was not affected by washing treatment nor by chilling time: 9.1 ± 0.1 Hz in non-washed samples (controls), 9.2 ± 0.1 Hz in CW samples, and 9.0 ± 0.1 Hz in DGC samples. The washing treatment affected (P<0.05) the percentage of live sperm as chilling time advanced. Live sperm counts were less in the initial DGC samples but remained relatively constant as chilling time advanced. In the control samples the percentage of live sperm was greater at 0 h than in either the CW or DGC samples, but decreased (P<0.05) with chilling time to reach - at 96 h - values similar to those recorded for the DGC samples (Fig. 5). The percentage of sperm with intact acrosomes was not affected by the washing procedure (Fig. 5). 3.2. Experiment 2: Influence of washing method on frozen-thawed sperm variables Data in Table 1 indicates the effect of the DGC washing method on sperm variables before freezing and after thawing at 0 h and after 2 h of incubation at 38.5 ºC in a 5% CO2 atmosphere. Both VCL (P = 0.06) and VSL (P = 0.09) trended towards being greater in the pre-freezing DCG than CW samples. In the DGC samples, VCL, VSL and VAP were not affected by freezing-thawing; however, these variables were less desirable (P<0.05) in the CW samples. The differences regarding VCL, VSL and VAP values after freezing-thawing were no longer evident following 2 hours of incubation. There were no differences between the washing
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methods in terms of post-thaw motile sperm, percentage MP, percentage LIN, percentage STR, BCF, ALH, percentage live sperm, or the percentage of sperm with an intact acrosome. 4. Discussion For the majority of sperm motility variables, using DGC resulted in more desirable values for sperm quality than either not washing or using the classical CW procedure. It would, therefore, appear that use of DGC is a useful way of removing seminal plasma and selecting high quality sperm in goats. Subsequent to washing (0 h), DGC treatment was associated with an enhanced VSL and progression ratio values, but lesser total motile sperm and sperm viability values. The latter has also been reported when using Capripure® DGC to prepare ibex (Capra pyrenaica) sperm (Santiago-Moreno et al., 2014). This may be a consequence of centrifugation (which has a detrimental effect on sperm; Aitken and Clarkson, 1988) with the centrifugation forces involved affecting the motility and membrane integrity of the sperm of small ruminants (Gil et al., 1999; Ritar, 1993). The advantages of DGC, however, are evident when storing chilled sperm. The percentage of live sperm and total motile sperm remained relatively constant during the chilled storage period - unlike that for the CW samples - suggesting that the use of the DGC procedure allows for the removal of sperm with sub-optimal functionality along with most apoptotic sperm (Ricci et al., 2009). The more desirable VSL values and progression ratios recorded for the chilled DCG samples suggests that motile sperm that were present when this procedure was used had greater mitochondrial membrane potentials (Donnelly et al., 2000; Marchetti et al., 2002). Use of the DGC results in selection of sperm with characteristics of movement that allow these cells to have greater movement velocities. In the present studies, ALH values remained less in chilled DGC samples than similarly treated CW samples. Moreover, there was a negative correlation between ALH and VSL. These results suggest that a lateral head displacement of >3 µm leads to less efficient progressive movement of goat sperm. These findings are not consistent with
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those reported for other species (e.g., humans), in which selection procedures, where use of the swim-up and Percoll, methods resulted in increased VSL and ALH (Liu et al., 1991; Swanson et al., 1995). The ALH is an important motility variable, and adequate values are required if barriers, such as that imposed by cervical mucus, are to be overcome, and the oocyte barriers to fertilization (e.g., the cumulus cell layers and the zona pellucida) are to be penetrated (Aitken et al., 1985). The ALH increases greatly in hyperactive sperm undergoing capacitation, but freshly ejaculated semen usually contains only a small fraction of hyperactive sperm. This may explain the negative correlation between ALH and VSL observed in the present experiments. The use of the DGC, thus, appears to facilitate selection of non-capacitated goat sperm. Neither washing procedure affected the percentage of intact acrosomes either before or after chilling, suggesting centrifugation may have no harmful effect on the acrosome. Inconsistent with this finding, Maxwell et al. (2007) reported that, when using PureSperm® DGC with bull sperm, the proportion of sperm with an intact acrosome was greater, although the mechanism underlying this is unclear. Discontinuous colloidal silica gradients may affect the capacitation dynamics and responsiveness of mouse and boar sperm to capacitation stimuli (Furimsky et al., 2005; Henning et al., 2015), and the effect of DGC in this respect in goats needs to be further examined. Previous reports have indicated that sperm selection techniques are more beneficial when working with low quality sperm (Valcarcel et al., 1996; Morató et al., 2013). The present results, however, suggest the used of DGC may be beneficial regardless of sperm quality. In Experiment 1, the sperm velocity variables in the fresh samples were greater than in Experiment 2; substantial improvements in progression ratios in the DGC-selected sperm were observed during the entire chilling period compared to no washing or use of the classical CW procedure. In Experiment 2, a lesser initial sperm quality was recorded, but both VSL and VAP trended towards increasing soon after DGC treatment. The differences between the
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results of Experiment 1 and 2 in terms of the influence of washing method on prechilling/freezing sperm variables might be related to differences in the initial sperm quality caused by the advancing age of the animals between the times Experiments 1 and 2 (some 2 years) were conducted, or the season of semen collection (Roca et al., 1992; Arrebola et al., 2010). Although both use of CW and DGC are effective in the removal of seminal plasma, the CW method has the major drawback that immotile, dead and abnormal sperm all remain present, as does any debris, along with any unwanted cell (epithelial cells, inflammatory cells and bacteria etc.). Dead sperm exert a well-known toxic effect on viable sperm cells. Further, CW-washed sperm had greater DNA fragmentation than DGC-selected sperm (Jackson et al., 2010). The ROS produced by leukocytes and male germ cells, which may be present in ejaculates, also induce lipid peroxidation and other damage to sperm cells (Henkel, 2011). Because freezingthawing also increases the production of free radicals, frozen-thawed sperm variables should be enhanced when sperm selection techniques such as DGC are used. Indeed, the present findings indicate that the use of DGC has advantages over the CW procedure when freezing sperm; after thawing, the values of many sperm motility variables were greater. There are inconsistencies of results in the present study and those reported for bull sperm when using both single and double layer centrifugation techniques with iodixanol. In the study with bulls, sperm quality after cryopreservation was similar to that of untreated samples (Gloria et al., 2016). Differences in the species and the methods used may explain these inconsistences. Indeed, the use of DGC prior to cryopreservation is associated with enhanced frozen-thawed sperm quality in stallions (Hoogewijs et al., 2011) and boars (Martinez-Alborcia et al., 2013). In the present study, however, no benefits of DCG were observed with respect to membrane integrity, unlike that reported in previous studies with stallion sperm involving two-layer iodixanol density centrifugation (Heutelbeck et al., 2015).
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In conclusion, use of both CW and DGC can be used to remove seminal plasma from goat sperm samples. Sperm selection by use of DGC is highly recommended when chilling and freezing goat semen because of the greater sperm of quality when this procedure is used.
Acknowledgements This research was funded by MINECO grant AGL2014-52081-R. The authors thank B. Labrador, L. Maribel Cedillo and J. Anabel Loya for assistance with data management.
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Heutelbeck, A., Oldenhof, H., Rohn, K., Martinsson, G., Morrel, J.M., Sieme, H., 2015. Use of density centrifugation for delayed cryopreservation of stallion sperm: perform sperm selection directly after collection or after storage? Reprod. Dom. Anim. 50, 76-83. Hoogewijs, M., Morrell, J., Van Soom, A., Govaere, J., Johannisson, A., Piepers, S., De Schauwer ,C., De Kruif, A., De Vliegher, S., 2011. Sperm selection using single layer centrifugation prior to cryopreservation can increase thawed sperm quality in stallions. Equine Vet. J. 40:35–41. Jackson, R.E., Bormann, C.L., Hassun, P.A., Rocha, A.M., Motta, E.L.A., Serafini, P.C., Smith, G.D., 2010. Effects of semen storage and separation techniques on sperm DNA fragmentation. Fertil. Steril. 94, 2626-2630. Jayaraman, V., Upadhya, D., Narayan, P.K., Adiga, S.K., 2012. The sperm processing by swimup and density gradient is effective in the elimination of the sperm with DMA damage. J. Assist. Reprod. Genet. 29, 557-563. Jiménez-Rabadán, P., Morrell, J.M., Johannisson, A., Ramón, M., García-Álvarez, O., MarotoMorales, A., Álvaro-García, P.J., Pérez-Guzmán, M.D., Fernández-Santos, M.R., Garde, J.J., Soler, A.J., 2012. Single layer centrifugation (SLC) improves sperm quality of cryopreserved Blanca-Celtibérica buck semen. Anim. Reprod. Sci. 136, 47-54. Liu, D.Y., Clarke, G. N., Baker, H.W.G., 1991. Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rates in vitro. J. Androl. 12, 231-239. Marchetti, C., Obert, G., Deffosez, A., Formstecher, P., Marchetti, P., 2002. Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm. Hum. Reprod. 17, 1257–1265.
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Martinez-Alborcia, M.J., Morrell, J.M., Gil, M.A., Barranco, I., Maside, C., Alkmin, D.V., Parrilla, I., Martinez, E.A., Roca, J., 2013. Suitability and effectiveness of single layer centrifugation using Androcoll-P in the cryopreservation protocol for boar spermatozoa. Anim. Reprod. Sci. 140,173–179. Maxwell, W.M.C, Parrilla, I., Caballero, I., Garcia, E., Roca, J., Martinez, E.A.., Vazquez, J.M., Rath, D., 2007. Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSpem ® density gradient centrifugation. Reprod. Dom. Anim. 42, 489-494. Memon, M.A., Bretzlaff, K.N., Ott, R.S., 1985. Effect of washing on motility and acrosome morphology of frozen-thawed goat spermatozoa. Am. J. Vet. Res. 46, 473-475. Morató, R., de Souza Soares, J.M., Orero, G., Mogas, T., 2013. Pre-selection by double layer density gradient centrifugation improve the fertilising capacity of frozen-thawed, capacity stallion sperm. Anim. Reprod. Sci. 139, 62-68. Mortimer, D., 1994. Sperm washing. In: Practical Laboratory Andrology, Oxford University Press Inc. New York, pp. 267-286. Pellicer-Rubio, M.T., Combarnous, Y., 1998. Deterioration of goat spermatozoa in skimmed milk-based extenders as a result of oleic acid released by the bulbourethral lipase BUSgp60. J. Reprod. Fertil. 112:95-105. Phillips, T.C., Dhaliwal, G.K., Verstegen-Onclin, K.M., Verstegen, J.P., 2012. Efficacy of four density gradient separation media to remove erythrocytes and nonviable sperm from canine semen. Theriogenology 77, 39-45. Ricci, G., Perticarari, S., Boscolo, R., Montico, M., Guaschino, S., Presani, G., 2009. Semen preparation methods and sperm apoptosis: swim-up versus gradient-density centrifugation technique. Fertil. Steril. 91, 632-638.
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Ritar, A.J., 1993. Control of ovulation, storage of semen and artificial insemination of fibreproducing goats in Australia: a review. Aust. J. Exp. Agric. 33, 807-820. Ritar, A.J., Salamon, S., 1982. Effects of seminal plasma and of its removal and of egg yolk in the diluent on the survival of fresh and frozen-thawed spermatozoa of the Angora goat. Aust. J. Biol. Sci. 35, 305-312. Ritar, A.J., Salamon, S., 1991. Effects of month of collection, method of processing, concentration of egg yolk and duration of frozen storage on viability of Angora goat spermatozoa. Small Rumin. Res. 4, 29-37. Roca, J., Martinez, E., Vazquez, J.M., Coy, P., 1992. Characteristics and seasonal variations in the semen of Murciano-Granaina goats in the Mediterranean area. Anim. Reprod. Sci. 29, 255-262. Rodriguez-Martinez, H., Larsson, B., Pertoft, H., 1997. Evaluation of sperm damage and techniques for sperm clean-up. Reprod. Fertil. Dev. 9, 297-308. Santiago-Moreno, J., Carvajal, A., Astorga, R.J., Coloma, M.A., Toledano-Díaz, A., GómezGuillamón, F., Salas-Vega, R., López-Sebastián, A., 2011. Potential impact of diseases transmissible by sperm on the establishment of Iberian Ibex (Capra pyrenaica) genome resource banks. Eur. J. Wildl. Res. 57, 211-216. Santiago-Moreno, J., Esteso, M.C., Castaño, C., Toledano-Díaz, A., Rodríguez, E., LópezSebastián, A., 2014. Sperm selection by Capripure® density-gradient versus dextran swim-up procedure in wild mountain ruminants. Anim. Reprod. Sci. 149, 178-186. Sias, B., Ferrato, F., Pellicer-Rubio, M.T., Forgerit, Y., Guillouet, P., Leboeuf, B., Carriere, F., 2005. Cloning and seasonal secretion of the pancreatic lipase-related protein 2 present in goat seminal plasma. Biochim. Biophys. Acta 1686,169-180.
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Soler, A.J., Esteso, M.C., Fernández-Santos, M.R., Garde, J.J., 2005. Characteristics of Iberian red deer (Cervus elaphus hispanicus) spermatozoa cryopreserved after storage at 5ºC in the epididymis for several days. Theriogenology 64, 1503-1517. Swanson, M.L., Collins, J.M., Freiman, S.F., Dubin, N.H., 1995. Effect of Percoll wash on sperm motion parameters and subsequent fertility in intrauterine insemination cycles. J. Assist. Reprod. Genet. 12, 48-54. Valcarcel, A., de las Heras, M.A., Moses, D.F., Perez, L.J., Baldassarre, H., 1996. Comparison between Sephadex G-10 and Percoll for preparation of normospermic, asthenospermic and frozen/thawed ram semen. Anim. Reprod. Sci. 41, 215-224.
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Figure legends Fig. 1. Percentage of motile and progressively motile (MP) sperm in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.05) between DGC and both CONTROL and CW.
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Fig. 2. Curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP), in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.01 and P<0.001 for VSL and VAP, respectively) between DGC and both CONTROL and CW.
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Fig. 3. Linearity (LIN), straightness (STR) and wobble (WOB) in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.001) between DGC and both CONTROL and CW.
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Fig. 4. Amplitude of lateral head displacement (ALH) in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.001) between DGC and both CONTROL and CW.
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Fig. 5. Percentage of live sperm and sperm with intact acrosomes in non-washed (CONTROL), density gradient centrifugation (DGC)-washed, and classically washed (CW) sperm samples after 0, 3, 24, 48, 72 and 96 h of chilling at 5 ºC. Asterisks indicate differences (P<0.05) between DGC and both CONTROL and CW. The arrow represents the time of decrease (P<0.05) in the percentage of live sperm, for CW and CONTROL regarding 0 H.
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Table 1 Values of sperm variables for fresh and frozen-thawed sperm, pre-washed either by the classical (CW) or density gradient centrifugation (DGC) methods, and for similar frozenthawed samples after 2 h of incubation at 38.5ºC in a 5% CO2 atmosphere (means±SE). Sperm variables
Original ejaculate pre-freezing
CW pre- DGC freezing pre-freezing
CW frozenthawed
DGC frozenthawed
Motile sperm (%) MP (%)
59.7±6.2a
54.7±7.0a
59.0±6.2a
26.8±6.8b
30.1±5.4a
32.6±6.8a
36.0±5.3a
15.7±5.1b
VCL (µm/s) VSL (µm/s) VAP (µm/s) LIN (%)
91.9±7.9a
98.3±15.7a
117.8±16.6a 69.6±8.4b
103.6±8.8a 40.8±4.8c
41.3±6.8c
54.0±8.0a
68.6±14.0a
84.9±14.9a
42.9±8.1b
81.2±7.6a
16.7±3.2c
17.7±5.8c
69.2±8.5a
82.6±16.1a
102.8±17.1a 51.6±8.8b
93.0±8.4a
22.6±3.7c
24.3±7.1c
56.6±5.3abc 65.2±6.1abc 66.0±8.4abc
57.8±4.9ac 77.9±1.1ab 40.3±5.1d
38.2±5.4d
STR (%)
75.8±3.2a
80.8±2.5a
80.4±3.4a
76.8±6.8a
30.7±4.9b
CW frozenthawed after 2 h incubation 12.4±3.3c
DGC frozenthawed after 2 h incubation 14.1±6.1c
19.5±3.1b
2.4±1.1c
4.6±3.3c
87.1±0.9a
71.7±3.0bc 69.2±4.3c
WOB 73.3±4.1b 79.6±5.2ab 81.6±7.5ab 70.8±4.4b 89.4±1.0a 54.7±4.7c (%) BCF 9.0±0.2 9.3±0.3 8.2±0.7 9.2±0.5 8.8±0.4 8.0±1.1 (Hz) ALH 2.7±0.2 2.6±0.2 2.3±0.3 2.5±0.1 2.3±0.2 1.8±0.3 (µm) Live 65.8±7.2a 52.9±5.4a 59.5±7.1a 26.1±5.1b 22.5±2.9b 19.6±4.2bc sperm (%) Intact 88.0±5.9a 77.6±5.0a 80.4±6.7a 58.3±5.6b 53.9±5.7b 54.8±5.4b acrosome (%) Values with different letters (a,b,c) in the same row are significantly different (P<0.05)
53.7±5.3c 6.9±1.2 1.7±0.3 14.0±2.3c
46.9±8.1b
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