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Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae)
Theriogenology 66 (2006) 775–784 www.journals.elsevierhealth.com/periodicals/the
Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae) Joseph Saragusty a,b, Haim Gacitua b, Roni King c, Amir Arav b,* a
Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76000, Israel b Institute of Animal Science, Agriculture Research Organization, The Volcani Center, P.O. Box 6, Bet-Dagan 50250, Israel c Israel Nature and Parks Authority, 3 Am Ve’Olamo Street, Jerusalem 95463, Israel Received 18 August 2005; received in revised form 16 January 2006; accepted 20 January 2006
Abstract Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67 11.67% (mean S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49 3.24%, intact acrosome was detected in 63.74 2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups—highly active and progressively motile spermatozoa with SMI of 62.75 0.38 and low activity and poorly progressive with SMI of 46.16 1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable. # 2006 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Spermatozoa; Genome resource bank (GRB); Epididymal sperm; Gazelle
1. Introduction Gazella gazella or Mountain gazelle, and Gazella dorcas or Desert gazelle, are two of several closely related species found in the Middle East. Both are listed as vulnerable in the World Conservation Union’s * Corresponding author. Tel.: +972 8 9484423; fax: +972 8 9475075. E-mail address: [email protected] (A. Arav). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.055
(IUCN) red list of threatened species and the Red Book of Vertebrates in Israel [54], with a decline in their world wide population of more than 20% (Desert gazelle) or 30% (Mountain gazelle) and up to 50% decline in the population of the Mountain gazelle in northern Israel in the last decade [31,38,39]. The main causes for the population decline include habitat loss or degradation, over-hunting and predation [38–40,44]. A severe outbreak of foot and mouth disease among the Mountain gazelles in natural reserves in the north of
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Israel resulted in the death of approximately 1500–2000 animals, constituting over 50% of the population there [55,56]. Such an outbreak demonstrates how vulnerable small populations can be. A tiny population of a highly endangered subspecies of the Mountain gazelle has been described recently and was named Acacia gazelle (Gazella gazella acaiae) [43]. This subspecies was found to be closer genetically to the Gazella gazella cora found in Saudi Arabia than to the Israeli Gazella gazella gazella (Mokady and Geffen, manuscript in preparation). During the last decades, even though the females are reproducing, the population is declining due to low survival rate of the fawns. According to the last survey, conducted in May– June 2005, only 12 individuals were counted. All efforts taken thus far to change this dire demographic trend proved fruitless. Recognizing these risks and the potential of captive and ex situ breeding, the World Conservation Union (IUCN) has approved in 2002 new guidelines for the conservation and maintenance of existing genetic diversity and viable populations of all taxa [28]. Captive breeding programs for various gazelle species exist for more than 3 decades now. To maintain biodiversity and to limit the negative consequences of small and fragmented populations, such as inbreeding depression [51] and susceptibility to disease outbreaks [55], there is an imperative need for the development of techniques to preserve the gametes of these endangered species. With the advances in reproduction technologies—artificial insemination (AI) and cryopreservation, and with the recognition for the need to protect many species from the danger of extinction, the establishment of genome resource banks (GRB) has been gaining increasing acceptance in recent years [19,20,34,35,46,57,58]. The number of studies carried out on semen preservation in the gazelle species is very limited and, to the best of our knowledge, none have been published on the G. gazella semen. To-date, no live offspring has been produced following insemination with cryopreserved semen from gazelle species [15] despite several attempts [25]. The limited research done on the preservation of other gazelle species and related antelopes can be helpful but, in cryobiology, species variations is a well recognized issue [24,36] and the need to develop specific techniques to preserve the gametes of each of the gazelle species is therefore due. Epididymal sperm collection and preservation is a well documented collection method [6,14,27,29,33,36, 41,48,49]. Probably the main advantage of this method is that it enables us to collect sperm post-mortem and,
if stored, it can be used to extend the reproductive ‘‘life span’’ of that individual. When dealing with endangered species, this may enable us to preserve the spermatozoa of wild and genetically valuable captive males who die in an accident or otherwise. The spermatozoa accumulated in the cauda epididymis is already mature and fertile [12] making it a useful source. Several methods were described as to how to extract the sperm out of the cauda epididymis. These include squeezing the cauda epididymis [33], making cuts in the cauda epididymis [23,33,41], cutting and squeezing [49], extrusion by air pressure [27,32] and flushing the vas deferens [41]. Flushing the vas deference, when compared with the cutting method [41], was showed to be superior, yet it seems to be less suitable for field work. In directional freezing technique, utilizing the multithermal gradient device (MTG1, IMT Ltd., Nes Ziona, Israel), damage to the cells, even when freezing in large volumes, can be minimized [2–4]. Using this technology, several studies have demonstrated the viability and fertility of frozen-thawed semen in a variety of species [4,13,52]. Large volume freezing has numerous additional advantages, making it an attractive alternative to freezing in straws. Risks of contamination, mix ups or loss of straws can all be avoided and storage, handling costs and space can all be reduced. A single insemination dose in a single large tube makes it easy for use in artificial insemination and easy to mark and control. Identifying samples stored in straws exposes them to high temperatures as soon as they are taken out of the liquid nitrogen, incurring further damage to the cells. For a large volume sample taken out of liquid nitrogen to reach a temperature of 100 8C takes over 2 min at room temperature (unpublished observation), giving the handler ample time to identify it and decided if to take it out or return it back to the liquid nitrogen. The aim of this study was to characterize and evaluate the feasibility of cryopreserving post-mortem epididymal sperm. Achieving this goal may provide us with the means to preserve genetic material from the last few individuals left from G. gazella acaiae and hopefully assist in preventing its extinction. 2. Materials and methods 2.1. Semen collection, processing and evaluation Testicles were obtained post-mortem from each of six sexually mature gazelles that were killed or severely injured (and were later euthanized) in car accidents in
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Israel. These testicles were submerged in saline and transported in ambient temperature to the laboratory within 24 h of death. Using a sterile scalpel, the skin and parietal tunic were removed, leaving the cauda epididymides exposed. These were isolated and placed in pre-warmed (30 8C) sperm-TALP medium containing NaCl 4.18% (v/v), KCl 1.862% (v/v), NaH2PO4H2O 0.95% (v/v), HEPES 0.95% (v/v), H2O 75.24% (v/v), CaCl22H2O 0.95% (v/v), CaCl26H2O 1.045% (v/v), NaHCl3 9.5% (v/v), pyruvate 5% (v/v), Gentamycin 0.2% (v/v) and BSA 0.6% (w/v). The medium had pH of 7.4 and osmolarity was between 295 and 305 mOsm. Small incisions were made along the cauda epididymides to open the convoluted tubules, slight pressure was applied with forceps and the slices were washed with 5 mL sperm-TALP. Sperm motility was subjectively estimated by evaluating at least four microscopic fields by the same person (Table 1). Since there is no published information on the use of SYBR-14/PI viability stain in gazelles, a preliminary validation of the SYBR-14/PI stain for gazelle semen was performed, following protocols published previously [21,45]. An aliquot of fresh semen was placed in liquid nitrogen to kill the sperm cells. Fresh semen, with >60% motility, was then serially diluted with increasing percentages of the dead sperm cells and the viability was assessed by staining with SYBR-14/PI as was previously described [16,17]. Briefly, 100 mL aliquots were incubated in pre-warmed (37 8C) water bath, in the dark, with 3 mL of 2% SYBR-14 in DMSO and 1.5 mL of PI (LIVE/DEAD1 Sperm Viability Kit L-7011; Molecular Probes, Eugene, OR). Viability was determined by assessing at least 200 cells using a fluorescent microscope (Ninkon Eclipse 50i, equipped with Nikon digital camera DXM1200F and connected to IBM compatible computer; Nikon, Japan) at 400 magnification with excitation at 450–490 nm and emission at 520 nm. When excited at 488 nm, live cells fluorescence green and dead cells fluorescence orange. The percentage of undiluted live samples was adjusted to
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100% viability and percent data from the serially diluted samples were adjusted accordingly. Sperm morphology was assessed by evaluating smears which were fixed with 2% formaldehyde and stained with Giemsa stain under 1000 microscopic magnification. At least 200 spermatozoa of each of four gazelles were assessed according to the following categories: normal, abnormal head, abnormal tail, detached head, coiled tail and cytoplasmatic droplet. Sperm dimensions (head length and width, midpiece, principle piece and total length) were measured on at least 50 cells per gazelle by evaluating Giemsa stained smears. 2.2. Sperm freezing The large tissue debris were removed and the slurry was under-laid with 1 mL 60% Iodixanol solution (OptiPrepTM, Axis-Shield PoC AS, Oslo, Norway) in a 15 mL test tube and centrifuged at 1500 g for 10 min. After centrifugation the supernatant, the Iodixanol solution and the debris were aspirated. Total sperm concentrations were estimated using a Makler counting chamber [37] and phase-contrast microscope, after immobilizing the sperm in exact measure of water. The samples were split and re-suspended with eggyolk-free AndroMed1 freezing extender (MiniTu¨be, Tiefenbach, Germany) to a final concentration of between 28.9 and 62.8 106 mL1. The diluted semen was left to equilibrate for 1 h at room temperature. Semen was then chilled to 5 8C over 3 h at a rate of 0.1 8C min1 by submerging the test tubes in water bath inside a refrigerator. Once cooled, the semen was packaged into three to four pre-chilled 8.5 mL HollowTubesTM (IMT Ltd.) per animal. The tubes were then frozen using the multi-thermal gradient freezing device (MTG-516, IMT Ltd.) with start temperature (block A) set at 5 8C, end temperature (block B) set at 50 8C and collection chamber set at 100 8C as was previously described [3,4]. The frozen
Table 1 Participating gazelle’s details Parameter
G. gazella gazella 1
G. gazella gazella 2
G. gazella gazella 3
G. gazella gazella 4
G. dorcas
G. gazella acaiae
Death cause Time to sperm freezing (h) Subjective estimated motility (%) Time to sperm thawing (days)
Car accident 7 80 129
Car accident 24 60 235
Car accident 11 75 58 and 60
Car accident 23a 60 7 and 52
Euthanasia 6 75 159
Car accident N/A N/A 532
N/A: not available. a Testicles arrived at the laboratory 5 h after death and then kept at 4 8C for 15 h before processing. Freezing took place about 3 h after procession ensued.
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tubes were stored in two liquid nitrogen tanks, as a safety measure against possible accidents, till thawing. 2.3. Sperm post-thawing evaluation Some samples were kept as part of the cryobanking program and were not thawed. Sperm was thawed by holding the test tubes at room temperature (approximately 22–23 8C) for 90 s and then placing them in a 37 8C water bath for 60 s. Using computerassisted sperm analysis system (HTM-IVOS Software Version 12; Hamilton-Thorn, Beverly, MA, U.S.A.) the sperm was evaluated for percentage of motility and motility pattern, including path velocity (VAP), progressive velocity (VSL), track speed (VCL), lateral amplitude (ALH), beat frequency (BCF), straightness (STR) and linearity (LIN). Definitions of these variables were already described elsewhere [10,25]. The percentage of motile spermatozoa and the speed of progression (SOP) which was based on the progressive movement of the spermatozoa (static (0– 1), slow (2), medium (3) and rapid (4)), evaluated by the sperm analyzing system, were used to calculate the sperm motility index (SMI) as was previously described [27,32] as follows: SMI ¼
Present of motility þ ðSOP 25Þ 2
SOP was calculated by multiplying the mean percentage for each of the four sub-groups (static, slow, medium and rapid) by the number of the group and then summing up the numbers for the same animal. For the static group 0 was used. The mean value was used for percent of motility. To assess the viability of the cells, post-thaw motility was also evaluated, subjectively, by two experienced researchers 1, 2 and 3 h after thawing for samples kept at 37 8C. For morphology evaluation, smears were prepared, air-dried, fixed with 2% formaldehyde and then stained with Giemsa stain. At lease 200 cells per animal were evaluated under 1000 magnification. Morphology parameters included: normal morphology, abnormal head, abnormal tail, detached head, coiled tail and cytoplasmic droplet. Viability was assessed by staining with SYBR-14/PI viability staining kit as was described above. Acrosome integrity was assessed by staining sperm suspension with fluorescien isothiocyanate-conjugated Pisum sativum agglutinine (FITC-PSA) (Sigma– Aldrich, Israel) at a final concentration of 10 mg/mL
in combination with Hoechst 33342 (H33342, Sigma– Aldrich) at a final concentration of 200 mg/mL. Semen samples were incubated at 37 8C for 30 min. A 5 mL drop of stained sperm suspension was place on a microscope slide, covered with a cover slip and observed with fluorescence microscope. Spermatozoa heads with green fluorescence (FITC-PSA positive) over the acrosome cap were classified as cells with damaged acrosome. FITC-PSA negative were cells that were stained blue by the H33342 and were not stained green by the FITC-PSA. These cells were classified as cells with intact acrosome. 2.4. Cryopreservation apparatus The novel freezing aparatus used is based on ‘‘Multithermal gradient (MTG1, IMT, Nes Ziona, Israel)’’ directional freezing technology [2]. In brief, the device is built of four temperature domains within 270 mm copper blocks. The test tube is advanced at a constant velocity (V) through the predetermined temperature gradient (G = DT/d, where DT is temperature differences and d is the distance between temperatures) resulting in a cooling rate (B) according to the equation B = G V (Fig. 1). Seeding is performed at the tip of the test tube and ice interface propagate according to the freezing point of the solution. In the conventional freezing methods ice grows at an uncontrolled velocity and morphology and may, therefore, disrupt and kill cells in the sample. By moving the special 8.5 mL HollowTubeTM test tube with the semen at a constant velocity through a linear temperature gradient, using the MTG apparatus, one can control the ice crystal propagation, optimize their morphology, get continual seeding and homogenous cooling rate during the whole freezing process, thereby minimizing damages to the cells, even when freezing such large volume.
Fig. 1. Schematic drawing of multi-thermal gradient (MTG) freezing device.
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2.5. Statistical analysis Due to the small number of participating gazelles we elected to present the results for each of the four individuals. Where applicable, means and standard error of mean (S.E.M.) were calculated using DESCRIBE1 Version 1.49, a WINPEPI statistical program [1]. To evaluate the sperm dimensions, SPSS for windows, Version 10.0.7 (SPSS Inc., Chicago, IL) was used. The variables were evaluated for correlation using Pearson correlation coefficient. One-way ANOVA was then used in two ways. For items for which equal variance was assumed, Tukey HSD was used. For items for which equal variance was not assumed, Tamhane test was used. Differences were assumed to be significantly different if P < 0.05. Nonparametric tests were used to describe the results of the post-collection motility and acrosome evaluation. 3. Results Semen collected post-mortem from four G. gazella gazella, one G. gazella acaiae and one G. dorcas (Table 1). The time between death and freezing of spermatozoa ranged between 6 and 24 h. The mean S.E.M. sperm motility after extraction was 70.00 4.18% (median 75%, low/high quartiles 60% and 77.5%). Values do not include G. gazella acaiae for which pre-freezing data was not evaluated. Mean S.E.M. of spermatozoal head length and width, midpiece and principal piece length and total length of G. gazella gazella, G. gazella acaiae and G. dorcas are shown in Table 2. Using Pearson correlation test, strong correlation (P < 0.01) was found between sperm head length and width and, as can be expected, between the principal piece length and the total length. G. dorcas had shorter and narrower sperm head as compared to the other gazelles tested. G. gazella acaiae had the shortest principal piece and therefore also the shortest total length.
Fig. 2. Validation of the live/dead sperm viability kit for Gazelle semen by serial dilutions of frozen-thawed semen with killed sperm (r = 0.993, slope = 1.043). The percentage of undiluted live samples was adjusted to 100% viability and percent data from the serially diluted samples were adjusted accordingly.
Fresh semen serially diluted with increasing volumes of killed sperm showed a decreasing trend in the percentage of live spermatozoa in the sample (Fig. 2). The correlation coefficient (r) between proportion of fresh sperm:killed sperm (v/v), and the actual percentage of spermatozoa counted as live was r = 0.993 and the slope was 1.043. Results showed that up to 63% of the sperm survived the freezing and thawing process (Table 3). One sample (G. gazella acaiae) seems to have been contaminated with blood during sperm extraction and only 15% motility was observed after thawing. Post-thaw motility for the other samples, assessed by the sperm analyzing system, was (mean S.E.M.) 55.67 4.67%. Three samples were evaluated for motility 1, 2 and 3 h after thawing and all three showed no detectable decline in motility over the time of evaluation. Post-thaw viability, assessed by the SYBR-14/PI viability stain, was (mean S.E.M.) 57.49 3.24%. Interestingly, the viability of the G. gazella acaiae spermatozoa was 51.9% despite the very low motility observed. Acrosome integrity was assessed on samples of two G. gazella gazella. For each gazelle 8 microscopic
Table 2 Dimensions of spermatozoa in G. gazella gazella, G. gazella acaiae and G. dorcas Parameter measured (mean S.E.M.a) Head length Head width Midpiece length Principal piece length Total length
G. gazella gazella n = 100 (mm) b
8.13 0.37 5.44 0.33c 11.17 0.81b 42.50 1.97b 61.80 2.06b
G. gazella acaiae n = 50 (mm) c
8.55 0.53 4.91 0.38b 10.77 0.80a 40.46 4.40a 59.78 4.32a
G. dorcas n = 50 (mm) 6.41 0.42a 3.72 0.39a 10.53 0.70a 45.56 1.67c 62.50 1.78b
Measurements were conducted on Giemsa stained smears. Different letters in the same row indicate significantly different measurements (P 0.05). a S.E.M.: standard error of mean.
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Table 3 Post-thawing sperm motility, viability and morphology Evaluated parameter 6
G. gazella gazella 1 1
G. gazella gazella 2
G. dorcas
G. gazella acaiae
Concentration (10 mL ) Motile (%) Progressive (%)
62.77 11.95 63.00 3.21 15.33 2.67
42.55 1.97 46.50 3.28 13.75 2.66
28.85 5.62 57.25 4.77 21.00 4.60
60.4 0.764 15.00 1.73 2.33 0.33
Velocity distribution Static (0) (%) Slow (2) (%) Medium (3) (%) Rapid (4) (%)
22.67 6.36 14.67 3.38 28.33 3.28 34.67 1.33
33.00 2.38 20.50 1.66 31.5 0.65 15.00 2.68
13.00 1.78 30.00 5.60 18.00 4.74 39.00 9.39
39.33 14.4 46 14.05 10.67 0.88 4.33 0.88
Speed of progressiona SMI Viability (%) b
2.53 63.13 53.11
1.96 47.69 58.88
2.70 62.38 66.07
2.97 44.63 51.9
Motility characteristics Number of cells evaluated, n Path velocity (VAP) (mm/s) Prog. velocity (VSL) (mm/s) Track speed (VCL) (mm/s) Lateral amplitude (ALH) (mm) Beat frequency (BCF) (Hz) Straightness (STR) (%) Linearity (LIN) (%) Morphology (%) c Normal Abnormal head Abnormal tail Detached head Coiled tail Cytoplasmic droplet
378 90.67 4.90 64.30 4.80 171.33 2.39 8.00 0.35 24.33 4.14 71.00 1.53 41.33 2.03 65 2.5 8.5 2 5 17
693 63.55 2.97 56.96 2.42 96.23 4.48 4.70 0.29 25.03 2.22 88.00 0.71 60.75 1.60 41 6.5 8 29.5 11.5 4.5
470 94.75 13.48 73.90 8.04 175.28 26.77 6.43 0.34 36.03 4.32 72.25 3.09 41.75 2.25 43 2.5 7.5 7.5 25.5 14
490 62.53 4.51 44.27 4.26 111.3 6.97 5.2 0.85 23.77 2.28 68.67 2.03 40.33 1.86 41 8 3.5 16 28 3.5
Where applicable, values are means standard error of mean (S.E.M.). a Speed of progression: The sum of the percent of the velocity sub-groups multiplied by the number for the relevant group. b Viability: percent of live cells (stained green) after assessing at least 200 cells stained with viability dual stain SYBR-14/PI. c Morphology was assessed by staining with Giemsa stain under 1000 magnification, at least 200 cells per animal.
fields (at least 500 cells) on two slides were evaluated (see Fig. 3). For one gazelle 62.86% of the cells were FITC-PSA negative (intact acrosome) while for the other 66.88% of the cells were FITC-PSA negative.
Fig. 3. G. gazella gazella post-thaw acrosome staining with FITCPSA. Only damaged acrosomes were stained.
G. gazella gazella 1 spermatozoa were highly active and progressively motile as was revealed by high values of VAP, VCL and VSL. Similar values were also observed with the G. dorcas spermatozoa. On the other hand the values of the same parameters for G. gazella gazella 2 and G. gazella acaiae were low, indicating low activity and poorly progressive spermatozoa. The SMI values for G. gazella gazella 1 and G. dorcas were 63.13 and 62.38, respectively, while those for G. gazella gazella 2 and G. gazella acaiae were only 47.69 and 44.63, respectively. The morphology of spermatozoa was assessed by evaluating at least 200 cells per gazelle by assessing slides that were fixed with formaldehyde and stained with Giemsa stain. The proportions of spermatozoa with either normal morphology, or with various types of abnormalities are shown in Table 3. G. gazella gazella 1 had the highest proportion of normal cells (65%) with similar proportion of between 41% and 43% for the other three gazelles. G. gazella gazella 2 had a 29.5% of detached heads.
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4. Discussion The number of studies conducted on the characterization and cryopreservation of gazelle species spermatozoa is very limited and, to the best of our knowledge, none was published on the Mountain gazelle. In the work described here we have characterized the spermatozoa of Mountain, Desert and Acacia gazelles and have demonstrated that large volume cryopreservation of gazelle sperm collected post-mortem is possible, with satisfactory post-thaw characteristics. Epididymal sperm used in this study was extracted by making cuts in the cauda epididymis. When using the cutting method, however, one must be cautious to avoid blood contamination. In the current study one sample got contaminated with blood (G. gazella acaiae) and the post-thawing motility of this sample was only 15%. Blood contamination was showed to inflict damage to cryopreserved spermatozoa because the cryopreservation process cause lyses to the red blood cells, exposing the spermatozoa to the hemoglobin which have a negative effect on them [50]. Sperm dimensions of G. dorcas were reported before [7,26] and the results reported here are comparable despite the facts that the measurements in this study were conducted after thawing and that they are based on such a small number of samples. This is the first time the dimensions of spermatozoa in the Mountain gazelle are reported. Keeping in mind the small sample number, the values reported here suggest that the G. gazella gazella spermatozoa is slightly smaller than those of the Gazella dama mhorr and Gazella cuvieri [7,25,26]. The sperm dimensions of the G. gazella acaiae seem to be comparable to those reported for the goat [9]. It is said that sperm size is inversely correlated to body size [9]. This rule seems not to hold for the gazelle species if the results reported here and by others are representative. The gazelle with the smallest body size, G. dorcas, also has the smallest sperm head size and G. dama mhorr has the largest body size and sperm head size. The extender used in this study was egg-yolk-free. When comparing the results obtained in the present study for post-thaw motility and SMI with those of the G. dorcas, G. cuvieri and G. dama mhorr tested in several egg-yolk-based extenders, our results are similar to those reported by Garde et al. [15] for G. dorcas and better for the other two species. In another study [25] it was found that acrosomal damage of G. dama mhorr spermatozoa was significantly and linearly increased by increasing concentration of egg-yolk. It is assumed that the egg-yolk confers protection to the sperm plasma
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membrane by the incorporation of lipids into it and reducing the sperm sensitivity to chilling [53]. Since egg-yolk originates from animals, its composition is instable due to its dependence on many biological variables beyond our control. One must also keep in mind that there is always the risk of transmitting pathogens along with it. If egg-yolk can be avoided, as was shown in the present study—good viability, motility and little acrosomal damage, the whole process can be better controlled and the risk of pathogen transmission can be eliminated, making cryopreservation and artificial insemination significantly better and safer. There was strong linear correlation between the proportion of fresh sperm:killed sperm and the number of viable spermatozoa with intact membrane identified by the SYBR-14/PI viability staining. This suggests that this viability stain can effectively identify and differentiate between live and dead spermatozoa of gazelles, similar to its performance in other mammalian species [8,11,16,21,45]. It was shown in previous studies that freezing with the MTG apparatus minimize damage to cells [3,4,13]. The motility following sperm extraction was 71.67% while the mean post-thaw motility was 55.67%, viability was 59.35% and intact acrosome averaged at 64.87% of the cells. These results, excluding those for G. gazella acaiae, are similar to those reported for G. dorcas by Garde et al. [15]. This may indicate that only about 22% of the cells lost their motility or membrane integrity during the pre-freezing processing, freezing and thawing. Post-thawing sperm were also nicely progressively motile as indicated by their high SMI values between 47.69 and 63.13 and viable as demonstrated by the fact that no deterioration in motility was observed over 3 h of incubation at 37 8C. These similarities bring forward the advantage of the MTG apparatus, namely the possibility to cryopreserve large volume samples. Utilizing the MTG apparatus, we have showed that regardless of the species—domestic farm animals [3] or wildlife [22], the same freezing protocol resulted in good post-thaw parameters. For certain species, it was suggested that whole ejaculate can be cryopreserved in a single HollowTubeTM [3] making it yet another advantage of this large volume freezing technique. Previous studies on post-mortem collection of epididymal spermatozoa showed that even several days after the death of the animal, viable spermatozoa can still be found in the cauda epididymis [23,30,32,42]. In a preliminary study done in our laboratory it was shown that motility of ram spermatozoa collected from the cauda epididymis of testes stored at room temperature
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for 24 h was similar to the motility of spermatozoa obtained from fresh testes [18]. In the present study we could divide the animals into two pairs based on postthaw motility and motility pattern parameters (VCL, VSL, VAP, BCF, ALH and LIN). The spermatozoa of G. gazella gazella 1 and G. dorcas were highly motile and highly active while those of G. gazella gazella 2 and G. gazella acaiae had low motility and motility pattern. Interestingly, these results closely correlate with the time that has elapsed between the time of death and the time of freezing the cells. While the spermatozoa of the G. gazella gazella 1 and G. dorcas were frozen within 6–7 h of death, those of the other two were frozen 11 or 24 h after the animal was killed in the car accident. This may indicate that although acceptable motility can still be obtained 24 h post-mortem (G. gazella gazella 2 was with 60% motility), shortening the time between death and cryopreservation of the cells can be beneficial. Since this difference was observed between two individuals of the same species (G. gazella gazella) it negates the possibility that this motility difference is due to species variations. It may very well be that while motility is preserved for many hours after the death of the animal, other processes make them more sensitive to chilling, resulting in poor survival following cryopreservation. If collection and cryopreservation can not be performed within a short while from the death of the animal, so as to minimize tissue deterioration, it would be beneficial to keep the testes (or the whole animal) in refrigeration, at 4–5 8C, till processing, preferably within 24 h from the animal’s death, as was seen in G. gazella gazella 4 and reported by others [23,30,32]. It is worthwhile noting that all samples collected for this study were obtained during the winter months (between the end of November and early March) and out of the breeding season. It is therefore possible that samples collected during the reproductive season will show even better results, and yet, we have showed here that even out of the season samples can still be collected and preserved with satisfactory results. The population of the gazelle species in Israel are dwindling because of high fawn mortality rate, habitat fragmentation and destruction, diseases, predation and, possibly, also illegal hunting [5,31,40,47,54,55]. The aim or our present work was to show that creating a genome resource bank for the gazelle species in Israel is possible and that even dead animals are not lost for the genetic pool of the population. This is specifically important for the G. gazella acaiae with only 12 individuals left in the wild. Gametes collected from both sexes can be preserved for later use in artificial insemination or in vitro fertilization of salvaged oocytes to generate
embryos that can either be preserved or laparoscopically transferred to surrogate mothers. It is our aim to explore the possibility of using the closely related G. gazella gazella as a surrogate mother for G. gazella acaia embryos produced via IVF in case oocytes from this later subspecies will become available. Acknowledgements We thank Dr. Nili Avni-Magen from the Tisch Family Zoological Gardens, Jerusalem, Israel and Dr. Limor Miara from the Hai-Kef Zoological Gardens, Rishon Lezion, Israel, for informing us when dead gazelles were available. Special thanks are due to Ms. Olga Shneorson and Mr. Itzik Dekel for invaluable laboratory assistance. References [1] Abramson JH. WINPEPI (PEPI-for-Windows): computer programs for epidemiologists. Epidemiol Perspect Innovations 2004;1:10 pp. [2] Arav A. Device and methods for multigradient directional cooling and warming of biological samples. US Patent: 5,873,254, Amir Arav; 1999. [3] Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H. New trends in gamete’s cryopreservation. Mol Cell Endocrinol 2002;187:77–81. [4] Arav A, Zeron Y, Shturman H, Gacitua H. Successful pregnancies in cows following double freezing of a large volume of semen. Reprod Nutr Dev 2002;42:583–6. [5] Baharav D. Notes on the population structure and biomass of the mountain gazelle, Gazella gazella gazella. Isr J Zool 1974; 23:39–44. [6] Blash S, Melican D, Gavin W. Cryopreservation of epididymal sperm obtained at necropsy from goats. Theriogenology 2000; 54:899–905. [7] Cassinello J, Abaigar T, Gomendio M, Roldan ERS. Characteristics of the semen of three endangered species of gazelles (Gazella dama mhorr, G. dorcas neglecta and G. cuvieri). J Reprod Fertil 1998;113:35–45. [8] Chalah T, Brillard JP. Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI). Theriogenology 1998;50:487–93. [9] Cummins JM, Woodall PF. On mammalian sperm dimensions. J Reprod Fertil 1985;75:153–75. [10] Davis RO, Siemers RJ. Derivation and reliability of kinematic measures of sperm motion. Reprod Fertil Dev 1995;7:857–69. [11] Ericsson SA, Garner DL, Thomas CA, Downing TW, Marshall CE. Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa. Theriogenology 1993;39:1009–24. [12] Foote RH. Fertilizing ability of epididymal sperm from dead animals. J Androl 2000;21:355. [13] Gacitua H, Arav A. Successful pregnancies with directional freezing of large volume buck semen. Theriogenology 2005; 63:931–8.
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Post-mortem semen cryopreservation and characterization in two different endangered gazelle species (Gazella gazella and Gazella dorcas) and one subspecies (Gazella gazelle acaiae) Joseph Saragusty a,b, Haim Gacitua b, Roni King c, Amir Arav b,* a
Department of Animal Sciences, Faculty of Agricultural, Food and Environmental Quality Sciences, Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76000, Israel b Institute of Animal Science, Agriculture Research Organization, The Volcani Center, P.O. Box 6, Bet-Dagan 50250, Israel c Israel Nature and Parks Authority, 3 Am Ve’Olamo Street, Jerusalem 95463, Israel Received 18 August 2005; received in revised form 16 January 2006; accepted 20 January 2006
Abstract Both Gazella gazella and Gazella dorcas are endangered species with continually dwindling population size, yet basic knowledge on their spermatozoa is missing. Semen collected post-mortem (PM) from the cauda epididymis of five adult gazelles (three Gazella gazella gazella, one Gazella gazella acaiae and one G. dorcas) was cryopreserved using directional freezing of large volumes (8 mL) with egg-yolk-free extender. Sperm size measurements and SYBR-14/propodium iodide (PI) viability stain validation for use in gazelles were conducted. Post-thaw characterization included motility, viability, acrosome damage evaluation, computerized motility characterization and morphology and sperm motility index (SMI) was calculated. Extracted sperm motility was 71.67 11.67% (mean S.E.M.). Post-thaw motility ranged between 15% and 63%, viability was 57.49 3.24%, intact acrosome was detected in 63.74 2.6% (median 64.8%, upper/lower quartiles 71.79%, 61.82%), and normal morphology ranged between 41% and 63%. Motility characterization showed two sub-groups—highly active and progressively motile spermatozoa with SMI of 62.75 0.38 and low activity and poorly progressive with SMI of 46.16 1.53. Our results indicate that PM preservation of gazelle spermatozoa with satisfactory post-thaw viability is possible and cryobanking is achievable. # 2006 Elsevier Inc. All rights reserved. Keywords: Cryopreservation; Spermatozoa; Genome resource bank (GRB); Epididymal sperm; Gazelle
1. Introduction Gazella gazella or Mountain gazelle, and Gazella dorcas or Desert gazelle, are two of several closely related species found in the Middle East. Both are listed as vulnerable in the World Conservation Union’s * Corresponding author. Tel.: +972 8 9484423; fax: +972 8 9475075. E-mail address: [email protected] (A. Arav). 0093-691X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2006.01.055
(IUCN) red list of threatened species and the Red Book of Vertebrates in Israel [54], with a decline in their world wide population of more than 20% (Desert gazelle) or 30% (Mountain gazelle) and up to 50% decline in the population of the Mountain gazelle in northern Israel in the last decade [31,38,39]. The main causes for the population decline include habitat loss or degradation, over-hunting and predation [38–40,44]. A severe outbreak of foot and mouth disease among the Mountain gazelles in natural reserves in the north of
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Israel resulted in the death of approximately 1500–2000 animals, constituting over 50% of the population there [55,56]. Such an outbreak demonstrates how vulnerable small populations can be. A tiny population of a highly endangered subspecies of the Mountain gazelle has been described recently and was named Acacia gazelle (Gazella gazella acaiae) [43]. This subspecies was found to be closer genetically to the Gazella gazella cora found in Saudi Arabia than to the Israeli Gazella gazella gazella (Mokady and Geffen, manuscript in preparation). During the last decades, even though the females are reproducing, the population is declining due to low survival rate of the fawns. According to the last survey, conducted in May– June 2005, only 12 individuals were counted. All efforts taken thus far to change this dire demographic trend proved fruitless. Recognizing these risks and the potential of captive and ex situ breeding, the World Conservation Union (IUCN) has approved in 2002 new guidelines for the conservation and maintenance of existing genetic diversity and viable populations of all taxa [28]. Captive breeding programs for various gazelle species exist for more than 3 decades now. To maintain biodiversity and to limit the negative consequences of small and fragmented populations, such as inbreeding depression [51] and susceptibility to disease outbreaks [55], there is an imperative need for the development of techniques to preserve the gametes of these endangered species. With the advances in reproduction technologies—artificial insemination (AI) and cryopreservation, and with the recognition for the need to protect many species from the danger of extinction, the establishment of genome resource banks (GRB) has been gaining increasing acceptance in recent years [19,20,34,35,46,57,58]. The number of studies carried out on semen preservation in the gazelle species is very limited and, to the best of our knowledge, none have been published on the G. gazella semen. To-date, no live offspring has been produced following insemination with cryopreserved semen from gazelle species [15] despite several attempts [25]. The limited research done on the preservation of other gazelle species and related antelopes can be helpful but, in cryobiology, species variations is a well recognized issue [24,36] and the need to develop specific techniques to preserve the gametes of each of the gazelle species is therefore due. Epididymal sperm collection and preservation is a well documented collection method [6,14,27,29,33,36, 41,48,49]. Probably the main advantage of this method is that it enables us to collect sperm post-mortem and,
if stored, it can be used to extend the reproductive ‘‘life span’’ of that individual. When dealing with endangered species, this may enable us to preserve the spermatozoa of wild and genetically valuable captive males who die in an accident or otherwise. The spermatozoa accumulated in the cauda epididymis is already mature and fertile [12] making it a useful source. Several methods were described as to how to extract the sperm out of the cauda epididymis. These include squeezing the cauda epididymis [33], making cuts in the cauda epididymis [23,33,41], cutting and squeezing [49], extrusion by air pressure [27,32] and flushing the vas deferens [41]. Flushing the vas deference, when compared with the cutting method [41], was showed to be superior, yet it seems to be less suitable for field work. In directional freezing technique, utilizing the multithermal gradient device (MTG1, IMT Ltd., Nes Ziona, Israel), damage to the cells, even when freezing in large volumes, can be minimized [2–4]. Using this technology, several studies have demonstrated the viability and fertility of frozen-thawed semen in a variety of species [4,13,52]. Large volume freezing has numerous additional advantages, making it an attractive alternative to freezing in straws. Risks of contamination, mix ups or loss of straws can all be avoided and storage, handling costs and space can all be reduced. A single insemination dose in a single large tube makes it easy for use in artificial insemination and easy to mark and control. Identifying samples stored in straws exposes them to high temperatures as soon as they are taken out of the liquid nitrogen, incurring further damage to the cells. For a large volume sample taken out of liquid nitrogen to reach a temperature of 100 8C takes over 2 min at room temperature (unpublished observation), giving the handler ample time to identify it and decided if to take it out or return it back to the liquid nitrogen. The aim of this study was to characterize and evaluate the feasibility of cryopreserving post-mortem epididymal sperm. Achieving this goal may provide us with the means to preserve genetic material from the last few individuals left from G. gazella acaiae and hopefully assist in preventing its extinction. 2. Materials and methods 2.1. Semen collection, processing and evaluation Testicles were obtained post-mortem from each of six sexually mature gazelles that were killed or severely injured (and were later euthanized) in car accidents in
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Israel. These testicles were submerged in saline and transported in ambient temperature to the laboratory within 24 h of death. Using a sterile scalpel, the skin and parietal tunic were removed, leaving the cauda epididymides exposed. These were isolated and placed in pre-warmed (30 8C) sperm-TALP medium containing NaCl 4.18% (v/v), KCl 1.862% (v/v), NaH2PO4H2O 0.95% (v/v), HEPES 0.95% (v/v), H2O 75.24% (v/v), CaCl22H2O 0.95% (v/v), CaCl26H2O 1.045% (v/v), NaHCl3 9.5% (v/v), pyruvate 5% (v/v), Gentamycin 0.2% (v/v) and BSA 0.6% (w/v). The medium had pH of 7.4 and osmolarity was between 295 and 305 mOsm. Small incisions were made along the cauda epididymides to open the convoluted tubules, slight pressure was applied with forceps and the slices were washed with 5 mL sperm-TALP. Sperm motility was subjectively estimated by evaluating at least four microscopic fields by the same person (Table 1). Since there is no published information on the use of SYBR-14/PI viability stain in gazelles, a preliminary validation of the SYBR-14/PI stain for gazelle semen was performed, following protocols published previously [21,45]. An aliquot of fresh semen was placed in liquid nitrogen to kill the sperm cells. Fresh semen, with >60% motility, was then serially diluted with increasing percentages of the dead sperm cells and the viability was assessed by staining with SYBR-14/PI as was previously described [16,17]. Briefly, 100 mL aliquots were incubated in pre-warmed (37 8C) water bath, in the dark, with 3 mL of 2% SYBR-14 in DMSO and 1.5 mL of PI (LIVE/DEAD1 Sperm Viability Kit L-7011; Molecular Probes, Eugene, OR). Viability was determined by assessing at least 200 cells using a fluorescent microscope (Ninkon Eclipse 50i, equipped with Nikon digital camera DXM1200F and connected to IBM compatible computer; Nikon, Japan) at 400 magnification with excitation at 450–490 nm and emission at 520 nm. When excited at 488 nm, live cells fluorescence green and dead cells fluorescence orange. The percentage of undiluted live samples was adjusted to
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100% viability and percent data from the serially diluted samples were adjusted accordingly. Sperm morphology was assessed by evaluating smears which were fixed with 2% formaldehyde and stained with Giemsa stain under 1000 microscopic magnification. At least 200 spermatozoa of each of four gazelles were assessed according to the following categories: normal, abnormal head, abnormal tail, detached head, coiled tail and cytoplasmatic droplet. Sperm dimensions (head length and width, midpiece, principle piece and total length) were measured on at least 50 cells per gazelle by evaluating Giemsa stained smears. 2.2. Sperm freezing The large tissue debris were removed and the slurry was under-laid with 1 mL 60% Iodixanol solution (OptiPrepTM, Axis-Shield PoC AS, Oslo, Norway) in a 15 mL test tube and centrifuged at 1500 g for 10 min. After centrifugation the supernatant, the Iodixanol solution and the debris were aspirated. Total sperm concentrations were estimated using a Makler counting chamber [37] and phase-contrast microscope, after immobilizing the sperm in exact measure of water. The samples were split and re-suspended with eggyolk-free AndroMed1 freezing extender (MiniTu¨be, Tiefenbach, Germany) to a final concentration of between 28.9 and 62.8 106 mL1. The diluted semen was left to equilibrate for 1 h at room temperature. Semen was then chilled to 5 8C over 3 h at a rate of 0.1 8C min1 by submerging the test tubes in water bath inside a refrigerator. Once cooled, the semen was packaged into three to four pre-chilled 8.5 mL HollowTubesTM (IMT Ltd.) per animal. The tubes were then frozen using the multi-thermal gradient freezing device (MTG-516, IMT Ltd.) with start temperature (block A) set at 5 8C, end temperature (block B) set at 50 8C and collection chamber set at 100 8C as was previously described [3,4]. The frozen
Table 1 Participating gazelle’s details Parameter
G. gazella gazella 1
G. gazella gazella 2
G. gazella gazella 3
G. gazella gazella 4
G. dorcas
G. gazella acaiae
Death cause Time to sperm freezing (h) Subjective estimated motility (%) Time to sperm thawing (days)
Car accident 7 80 129
Car accident 24 60 235
Car accident 11 75 58 and 60
Car accident 23a 60 7 and 52
Euthanasia 6 75 159
Car accident N/A N/A 532
N/A: not available. a Testicles arrived at the laboratory 5 h after death and then kept at 4 8C for 15 h before processing. Freezing took place about 3 h after procession ensued.
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tubes were stored in two liquid nitrogen tanks, as a safety measure against possible accidents, till thawing. 2.3. Sperm post-thawing evaluation Some samples were kept as part of the cryobanking program and were not thawed. Sperm was thawed by holding the test tubes at room temperature (approximately 22–23 8C) for 90 s and then placing them in a 37 8C water bath for 60 s. Using computerassisted sperm analysis system (HTM-IVOS Software Version 12; Hamilton-Thorn, Beverly, MA, U.S.A.) the sperm was evaluated for percentage of motility and motility pattern, including path velocity (VAP), progressive velocity (VSL), track speed (VCL), lateral amplitude (ALH), beat frequency (BCF), straightness (STR) and linearity (LIN). Definitions of these variables were already described elsewhere [10,25]. The percentage of motile spermatozoa and the speed of progression (SOP) which was based on the progressive movement of the spermatozoa (static (0– 1), slow (2), medium (3) and rapid (4)), evaluated by the sperm analyzing system, were used to calculate the sperm motility index (SMI) as was previously described [27,32] as follows: SMI ¼
Present of motility þ ðSOP 25Þ 2
SOP was calculated by multiplying the mean percentage for each of the four sub-groups (static, slow, medium and rapid) by the number of the group and then summing up the numbers for the same animal. For the static group 0 was used. The mean value was used for percent of motility. To assess the viability of the cells, post-thaw motility was also evaluated, subjectively, by two experienced researchers 1, 2 and 3 h after thawing for samples kept at 37 8C. For morphology evaluation, smears were prepared, air-dried, fixed with 2% formaldehyde and then stained with Giemsa stain. At lease 200 cells per animal were evaluated under 1000 magnification. Morphology parameters included: normal morphology, abnormal head, abnormal tail, detached head, coiled tail and cytoplasmic droplet. Viability was assessed by staining with SYBR-14/PI viability staining kit as was described above. Acrosome integrity was assessed by staining sperm suspension with fluorescien isothiocyanate-conjugated Pisum sativum agglutinine (FITC-PSA) (Sigma– Aldrich, Israel) at a final concentration of 10 mg/mL
in combination with Hoechst 33342 (H33342, Sigma– Aldrich) at a final concentration of 200 mg/mL. Semen samples were incubated at 37 8C for 30 min. A 5 mL drop of stained sperm suspension was place on a microscope slide, covered with a cover slip and observed with fluorescence microscope. Spermatozoa heads with green fluorescence (FITC-PSA positive) over the acrosome cap were classified as cells with damaged acrosome. FITC-PSA negative were cells that were stained blue by the H33342 and were not stained green by the FITC-PSA. These cells were classified as cells with intact acrosome. 2.4. Cryopreservation apparatus The novel freezing aparatus used is based on ‘‘Multithermal gradient (MTG1, IMT, Nes Ziona, Israel)’’ directional freezing technology [2]. In brief, the device is built of four temperature domains within 270 mm copper blocks. The test tube is advanced at a constant velocity (V) through the predetermined temperature gradient (G = DT/d, where DT is temperature differences and d is the distance between temperatures) resulting in a cooling rate (B) according to the equation B = G V (Fig. 1). Seeding is performed at the tip of the test tube and ice interface propagate according to the freezing point of the solution. In the conventional freezing methods ice grows at an uncontrolled velocity and morphology and may, therefore, disrupt and kill cells in the sample. By moving the special 8.5 mL HollowTubeTM test tube with the semen at a constant velocity through a linear temperature gradient, using the MTG apparatus, one can control the ice crystal propagation, optimize their morphology, get continual seeding and homogenous cooling rate during the whole freezing process, thereby minimizing damages to the cells, even when freezing such large volume.
Fig. 1. Schematic drawing of multi-thermal gradient (MTG) freezing device.
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2.5. Statistical analysis Due to the small number of participating gazelles we elected to present the results for each of the four individuals. Where applicable, means and standard error of mean (S.E.M.) were calculated using DESCRIBE1 Version 1.49, a WINPEPI statistical program [1]. To evaluate the sperm dimensions, SPSS for windows, Version 10.0.7 (SPSS Inc., Chicago, IL) was used. The variables were evaluated for correlation using Pearson correlation coefficient. One-way ANOVA was then used in two ways. For items for which equal variance was assumed, Tukey HSD was used. For items for which equal variance was not assumed, Tamhane test was used. Differences were assumed to be significantly different if P < 0.05. Nonparametric tests were used to describe the results of the post-collection motility and acrosome evaluation. 3. Results Semen collected post-mortem from four G. gazella gazella, one G. gazella acaiae and one G. dorcas (Table 1). The time between death and freezing of spermatozoa ranged between 6 and 24 h. The mean S.E.M. sperm motility after extraction was 70.00 4.18% (median 75%, low/high quartiles 60% and 77.5%). Values do not include G. gazella acaiae for which pre-freezing data was not evaluated. Mean S.E.M. of spermatozoal head length and width, midpiece and principal piece length and total length of G. gazella gazella, G. gazella acaiae and G. dorcas are shown in Table 2. Using Pearson correlation test, strong correlation (P < 0.01) was found between sperm head length and width and, as can be expected, between the principal piece length and the total length. G. dorcas had shorter and narrower sperm head as compared to the other gazelles tested. G. gazella acaiae had the shortest principal piece and therefore also the shortest total length.
Fig. 2. Validation of the live/dead sperm viability kit for Gazelle semen by serial dilutions of frozen-thawed semen with killed sperm (r = 0.993, slope = 1.043). The percentage of undiluted live samples was adjusted to 100% viability and percent data from the serially diluted samples were adjusted accordingly.
Fresh semen serially diluted with increasing volumes of killed sperm showed a decreasing trend in the percentage of live spermatozoa in the sample (Fig. 2). The correlation coefficient (r) between proportion of fresh sperm:killed sperm (v/v), and the actual percentage of spermatozoa counted as live was r = 0.993 and the slope was 1.043. Results showed that up to 63% of the sperm survived the freezing and thawing process (Table 3). One sample (G. gazella acaiae) seems to have been contaminated with blood during sperm extraction and only 15% motility was observed after thawing. Post-thaw motility for the other samples, assessed by the sperm analyzing system, was (mean S.E.M.) 55.67 4.67%. Three samples were evaluated for motility 1, 2 and 3 h after thawing and all three showed no detectable decline in motility over the time of evaluation. Post-thaw viability, assessed by the SYBR-14/PI viability stain, was (mean S.E.M.) 57.49 3.24%. Interestingly, the viability of the G. gazella acaiae spermatozoa was 51.9% despite the very low motility observed. Acrosome integrity was assessed on samples of two G. gazella gazella. For each gazelle 8 microscopic
Table 2 Dimensions of spermatozoa in G. gazella gazella, G. gazella acaiae and G. dorcas Parameter measured (mean S.E.M.a) Head length Head width Midpiece length Principal piece length Total length
G. gazella gazella n = 100 (mm) b
8.13 0.37 5.44 0.33c 11.17 0.81b 42.50 1.97b 61.80 2.06b
G. gazella acaiae n = 50 (mm) c
8.55 0.53 4.91 0.38b 10.77 0.80a 40.46 4.40a 59.78 4.32a
G. dorcas n = 50 (mm) 6.41 0.42a 3.72 0.39a 10.53 0.70a 45.56 1.67c 62.50 1.78b
Measurements were conducted on Giemsa stained smears. Different letters in the same row indicate significantly different measurements (P 0.05). a S.E.M.: standard error of mean.
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Table 3 Post-thawing sperm motility, viability and morphology Evaluated parameter 6
G. gazella gazella 1 1
G. gazella gazella 2
G. dorcas
G. gazella acaiae
Concentration (10 mL ) Motile (%) Progressive (%)
62.77 11.95 63.00 3.21 15.33 2.67
42.55 1.97 46.50 3.28 13.75 2.66
28.85 5.62 57.25 4.77 21.00 4.60
60.4 0.764 15.00 1.73 2.33 0.33
Velocity distribution Static (0) (%) Slow (2) (%) Medium (3) (%) Rapid (4) (%)
22.67 6.36 14.67 3.38 28.33 3.28 34.67 1.33
33.00 2.38 20.50 1.66 31.5 0.65 15.00 2.68
13.00 1.78 30.00 5.60 18.00 4.74 39.00 9.39
39.33 14.4 46 14.05 10.67 0.88 4.33 0.88
Speed of progressiona SMI Viability (%) b
2.53 63.13 53.11
1.96 47.69 58.88
2.70 62.38 66.07
2.97 44.63 51.9
Motility characteristics Number of cells evaluated, n Path velocity (VAP) (mm/s) Prog. velocity (VSL) (mm/s) Track speed (VCL) (mm/s) Lateral amplitude (ALH) (mm) Beat frequency (BCF) (Hz) Straightness (STR) (%) Linearity (LIN) (%) Morphology (%) c Normal Abnormal head Abnormal tail Detached head Coiled tail Cytoplasmic droplet
378 90.67 4.90 64.30 4.80 171.33 2.39 8.00 0.35 24.33 4.14 71.00 1.53 41.33 2.03 65 2.5 8.5 2 5 17
693 63.55 2.97 56.96 2.42 96.23 4.48 4.70 0.29 25.03 2.22 88.00 0.71 60.75 1.60 41 6.5 8 29.5 11.5 4.5
470 94.75 13.48 73.90 8.04 175.28 26.77 6.43 0.34 36.03 4.32 72.25 3.09 41.75 2.25 43 2.5 7.5 7.5 25.5 14
490 62.53 4.51 44.27 4.26 111.3 6.97 5.2 0.85 23.77 2.28 68.67 2.03 40.33 1.86 41 8 3.5 16 28 3.5
Where applicable, values are means standard error of mean (S.E.M.). a Speed of progression: The sum of the percent of the velocity sub-groups multiplied by the number for the relevant group. b Viability: percent of live cells (stained green) after assessing at least 200 cells stained with viability dual stain SYBR-14/PI. c Morphology was assessed by staining with Giemsa stain under 1000 magnification, at least 200 cells per animal.
fields (at least 500 cells) on two slides were evaluated (see Fig. 3). For one gazelle 62.86% of the cells were FITC-PSA negative (intact acrosome) while for the other 66.88% of the cells were FITC-PSA negative.
Fig. 3. G. gazella gazella post-thaw acrosome staining with FITCPSA. Only damaged acrosomes were stained.
G. gazella gazella 1 spermatozoa were highly active and progressively motile as was revealed by high values of VAP, VCL and VSL. Similar values were also observed with the G. dorcas spermatozoa. On the other hand the values of the same parameters for G. gazella gazella 2 and G. gazella acaiae were low, indicating low activity and poorly progressive spermatozoa. The SMI values for G. gazella gazella 1 and G. dorcas were 63.13 and 62.38, respectively, while those for G. gazella gazella 2 and G. gazella acaiae were only 47.69 and 44.63, respectively. The morphology of spermatozoa was assessed by evaluating at least 200 cells per gazelle by assessing slides that were fixed with formaldehyde and stained with Giemsa stain. The proportions of spermatozoa with either normal morphology, or with various types of abnormalities are shown in Table 3. G. gazella gazella 1 had the highest proportion of normal cells (65%) with similar proportion of between 41% and 43% for the other three gazelles. G. gazella gazella 2 had a 29.5% of detached heads.
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4. Discussion The number of studies conducted on the characterization and cryopreservation of gazelle species spermatozoa is very limited and, to the best of our knowledge, none was published on the Mountain gazelle. In the work described here we have characterized the spermatozoa of Mountain, Desert and Acacia gazelles and have demonstrated that large volume cryopreservation of gazelle sperm collected post-mortem is possible, with satisfactory post-thaw characteristics. Epididymal sperm used in this study was extracted by making cuts in the cauda epididymis. When using the cutting method, however, one must be cautious to avoid blood contamination. In the current study one sample got contaminated with blood (G. gazella acaiae) and the post-thawing motility of this sample was only 15%. Blood contamination was showed to inflict damage to cryopreserved spermatozoa because the cryopreservation process cause lyses to the red blood cells, exposing the spermatozoa to the hemoglobin which have a negative effect on them [50]. Sperm dimensions of G. dorcas were reported before [7,26] and the results reported here are comparable despite the facts that the measurements in this study were conducted after thawing and that they are based on such a small number of samples. This is the first time the dimensions of spermatozoa in the Mountain gazelle are reported. Keeping in mind the small sample number, the values reported here suggest that the G. gazella gazella spermatozoa is slightly smaller than those of the Gazella dama mhorr and Gazella cuvieri [7,25,26]. The sperm dimensions of the G. gazella acaiae seem to be comparable to those reported for the goat [9]. It is said that sperm size is inversely correlated to body size [9]. This rule seems not to hold for the gazelle species if the results reported here and by others are representative. The gazelle with the smallest body size, G. dorcas, also has the smallest sperm head size and G. dama mhorr has the largest body size and sperm head size. The extender used in this study was egg-yolk-free. When comparing the results obtained in the present study for post-thaw motility and SMI with those of the G. dorcas, G. cuvieri and G. dama mhorr tested in several egg-yolk-based extenders, our results are similar to those reported by Garde et al. [15] for G. dorcas and better for the other two species. In another study [25] it was found that acrosomal damage of G. dama mhorr spermatozoa was significantly and linearly increased by increasing concentration of egg-yolk. It is assumed that the egg-yolk confers protection to the sperm plasma
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membrane by the incorporation of lipids into it and reducing the sperm sensitivity to chilling [53]. Since egg-yolk originates from animals, its composition is instable due to its dependence on many biological variables beyond our control. One must also keep in mind that there is always the risk of transmitting pathogens along with it. If egg-yolk can be avoided, as was shown in the present study—good viability, motility and little acrosomal damage, the whole process can be better controlled and the risk of pathogen transmission can be eliminated, making cryopreservation and artificial insemination significantly better and safer. There was strong linear correlation between the proportion of fresh sperm:killed sperm and the number of viable spermatozoa with intact membrane identified by the SYBR-14/PI viability staining. This suggests that this viability stain can effectively identify and differentiate between live and dead spermatozoa of gazelles, similar to its performance in other mammalian species [8,11,16,21,45]. It was shown in previous studies that freezing with the MTG apparatus minimize damage to cells [3,4,13]. The motility following sperm extraction was 71.67% while the mean post-thaw motility was 55.67%, viability was 59.35% and intact acrosome averaged at 64.87% of the cells. These results, excluding those for G. gazella acaiae, are similar to those reported for G. dorcas by Garde et al. [15]. This may indicate that only about 22% of the cells lost their motility or membrane integrity during the pre-freezing processing, freezing and thawing. Post-thawing sperm were also nicely progressively motile as indicated by their high SMI values between 47.69 and 63.13 and viable as demonstrated by the fact that no deterioration in motility was observed over 3 h of incubation at 37 8C. These similarities bring forward the advantage of the MTG apparatus, namely the possibility to cryopreserve large volume samples. Utilizing the MTG apparatus, we have showed that regardless of the species—domestic farm animals [3] or wildlife [22], the same freezing protocol resulted in good post-thaw parameters. For certain species, it was suggested that whole ejaculate can be cryopreserved in a single HollowTubeTM [3] making it yet another advantage of this large volume freezing technique. Previous studies on post-mortem collection of epididymal spermatozoa showed that even several days after the death of the animal, viable spermatozoa can still be found in the cauda epididymis [23,30,32,42]. In a preliminary study done in our laboratory it was shown that motility of ram spermatozoa collected from the cauda epididymis of testes stored at room temperature
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for 24 h was similar to the motility of spermatozoa obtained from fresh testes [18]. In the present study we could divide the animals into two pairs based on postthaw motility and motility pattern parameters (VCL, VSL, VAP, BCF, ALH and LIN). The spermatozoa of G. gazella gazella 1 and G. dorcas were highly motile and highly active while those of G. gazella gazella 2 and G. gazella acaiae had low motility and motility pattern. Interestingly, these results closely correlate with the time that has elapsed between the time of death and the time of freezing the cells. While the spermatozoa of the G. gazella gazella 1 and G. dorcas were frozen within 6–7 h of death, those of the other two were frozen 11 or 24 h after the animal was killed in the car accident. This may indicate that although acceptable motility can still be obtained 24 h post-mortem (G. gazella gazella 2 was with 60% motility), shortening the time between death and cryopreservation of the cells can be beneficial. Since this difference was observed between two individuals of the same species (G. gazella gazella) it negates the possibility that this motility difference is due to species variations. It may very well be that while motility is preserved for many hours after the death of the animal, other processes make them more sensitive to chilling, resulting in poor survival following cryopreservation. If collection and cryopreservation can not be performed within a short while from the death of the animal, so as to minimize tissue deterioration, it would be beneficial to keep the testes (or the whole animal) in refrigeration, at 4–5 8C, till processing, preferably within 24 h from the animal’s death, as was seen in G. gazella gazella 4 and reported by others [23,30,32]. It is worthwhile noting that all samples collected for this study were obtained during the winter months (between the end of November and early March) and out of the breeding season. It is therefore possible that samples collected during the reproductive season will show even better results, and yet, we have showed here that even out of the season samples can still be collected and preserved with satisfactory results. The population of the gazelle species in Israel are dwindling because of high fawn mortality rate, habitat fragmentation and destruction, diseases, predation and, possibly, also illegal hunting [5,31,40,47,54,55]. The aim or our present work was to show that creating a genome resource bank for the gazelle species in Israel is possible and that even dead animals are not lost for the genetic pool of the population. This is specifically important for the G. gazella acaiae with only 12 individuals left in the wild. Gametes collected from both sexes can be preserved for later use in artificial insemination or in vitro fertilization of salvaged oocytes to generate
embryos that can either be preserved or laparoscopically transferred to surrogate mothers. It is our aim to explore the possibility of using the closely related G. gazella gazella as a surrogate mother for G. gazella acaia embryos produced via IVF in case oocytes from this later subspecies will become available. Acknowledgements We thank Dr. Nili Avni-Magen from the Tisch Family Zoological Gardens, Jerusalem, Israel and Dr. Limor Miara from the Hai-Kef Zoological Gardens, Rishon Lezion, Israel, for informing us when dead gazelles were available. Special thanks are due to Ms. Olga Shneorson and Mr. Itzik Dekel for invaluable laboratory assistance. References [1] Abramson JH. WINPEPI (PEPI-for-Windows): computer programs for epidemiologists. Epidemiol Perspect Innovations 2004;1:10 pp. [2] Arav A. Device and methods for multigradient directional cooling and warming of biological samples. US Patent: 5,873,254, Amir Arav; 1999. [3] Arav A, Yavin S, Zeron Y, Natan D, Dekel I, Gacitua H. New trends in gamete’s cryopreservation. Mol Cell Endocrinol 2002;187:77–81. [4] Arav A, Zeron Y, Shturman H, Gacitua H. Successful pregnancies in cows following double freezing of a large volume of semen. Reprod Nutr Dev 2002;42:583–6. [5] Baharav D. Notes on the population structure and biomass of the mountain gazelle, Gazella gazella gazella. Isr J Zool 1974; 23:39–44. [6] Blash S, Melican D, Gavin W. Cryopreservation of epididymal sperm obtained at necropsy from goats. Theriogenology 2000; 54:899–905. [7] Cassinello J, Abaigar T, Gomendio M, Roldan ERS. Characteristics of the semen of three endangered species of gazelles (Gazella dama mhorr, G. dorcas neglecta and G. cuvieri). J Reprod Fertil 1998;113:35–45. [8] Chalah T, Brillard JP. Comparison of assessment of fowl sperm viability by eosin-nigrosin and dual fluorescence (SYBR-14/PI). Theriogenology 1998;50:487–93. [9] Cummins JM, Woodall PF. On mammalian sperm dimensions. J Reprod Fertil 1985;75:153–75. [10] Davis RO, Siemers RJ. Derivation and reliability of kinematic measures of sperm motion. Reprod Fertil Dev 1995;7:857–69. [11] Ericsson SA, Garner DL, Thomas CA, Downing TW, Marshall CE. Interrelationships among fluorometric analyses of spermatozoal function, classical semen quality parameters and the fertility of frozen-thawed bovine spermatozoa. Theriogenology 1993;39:1009–24. [12] Foote RH. Fertilizing ability of epididymal sperm from dead animals. J Androl 2000;21:355. [13] Gacitua H, Arav A. Successful pregnancies with directional freezing of large volume buck semen. Theriogenology 2005; 63:931–8.
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