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BOR - Papers in Press, published online ahead of print April 16, 2003.
Biol Reprod 2003, 10.1095/biolreprod.102.012914
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BIOLOGY OF REPRODUCTION 69, 602–611 (2003)
DOI: 10.1095/biolreprod.102.012914
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Sperm Cryopreservation in Three Species of Endangered Gazelles (Gazella cuvieri, G. dama mhorr, and G. dorcas neglecta)1

J.J. Garde3, A.J. Soler3, J. Cassinello4, C. Crespo4, A.F. Malo4, G. Espeso5, M. Gomendio4, and E.R.S. Roldan2,4

Departamento de Ciencia y Tecnología Agroforestal,3 ETSI Agrónomos, Universidad de Castilla-La Mancha, 02071-Albacete, Spain Museo Nacional de Ciencias Naturales (CSIC),4 28006-Madrid, Spain Estación Experimental de Zonas Aridas (CSIC), 5 04001-Almería, Spain


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Long-term storage of semen by cryopreservation, with high recovery rates on thawing, is essential for the establishment of genetic resource banks of endangered species. The purpose of the present study was to evaluate various diluents for the cryopreservation of spermatozoa from three species of gazelles (genus Gazella) in a captive breeding program. The diluents compared were Tes (N-tris(hydroxymethyl)methyl-2 aminoethane sulfonic acid)-Tris with 5% egg yolk and 6% glycerol (TEST) and Triladyl, yolk-citrate, Tris-trehalose, and Tris-lactose—all of them with 20% egg yolk and 6% (Triladyl) or 8% glycerol. Semen was obtained by electroejaculation from 12 G. cuvieri, 12 G. dama, and 13 G. dorcas males. Samples with less than 50% motile sperm, positive endosmosis, or acrosome integrity were not used. Diluted samples were loaded into 0.25-ml straws, cooled slowly to 5°C over 1.5 h (-0.16°C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapors for 10 min, and plunged into liquid nitrogen. Subsamples were assessed fresh, after refrigeration-equilibration, after freezing and thawing, and 2 h after thawing. Differences were seen between diluents, with best overall recovery rates after freezing and thawing found with Triladyl, TEST, and Tris-trehalose in G. cuvieri, TEST in G. dama, and Triladyl and TEST in G. dorcas. Differences were observed between species in the ability to withstand freezing and thawing, with best results seen in G. dorcas, intermediate results in G. dama, and worst results in G. cuvieri. These differences were inversely related to the average values of inbreeding of these populations. The underlying mechanism responsible for these differences may be a differential resistance to osmotic shock.

assisted reproductive technology, fertilization, gamete biology, sperm, sperm capacitation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
For an increasing number of species, captive breeding represents their best chance to avoid extinction. The World Conservation Union (IUCN) has recognized the potential role that captive breeding may play in conservation efforts and has recommended that vertebrate taxa numbering less than 1000 individuals in the wild should be considered for captive breeding [1]. Since 1971, the Estación Experimental de Zonas Aridas (EEZA) has developed captive-breeding programs for three endangered gazelle species: Gazella cuvieri, G. dama mhorr, and G. dorcas neglecta. These three species have reproduced successfully in captivity, allowing the translocation of animals to several zoos in Europe and the United States. The history and reproductive aspects of the populations of these three species have been described previously [2–5]. Gazella cuvieri is categorized in the IUCN Red List of Threatened Animals [6] as "Endangered." Gazella dama mhorr is also categorized as "Endangered," and no animals have been observed in the wild since 1968. Gazella dorcas (the species as a whole) is now categorized as "Vulnerable," but the Red List does not specify the status of G. dorcas neglecta, which is thought to be endangered.

The ultimate goal of captive-breeding programs dealing with endangered species should be to implement management policies that will maintain the potential of recreating a self-sustaining wild population [7]. Among the most important threats faced by captive populations are the loss of genetic variability and inbreeding. Because of the small founder populations for the gazelles kept at the EEZA, these factors imply serious risks. Detailed records kept at the EEZA since the founder populations were first established have allowed the calculation of inbreeding coefficients for all individuals. Based on this information, deleterious effects of inbreeding have been found on some components of female reproductive success in the three species of gazelles [4] and, also, on male reproductive function [8, 9]. In this context, artificial insemination and semen preservation have been identified as powerful tools in the breeding programs of gazelles [10], because this would allow the storage of semen from genetically valuable animals to extend generation times, to circumvent husbandry or medical factors that may prevent certain animals from breeding, and to transfer semen between subpopulations that may become geographically or biologically isolated [11, 12].

To our knowledge, no studies have been carried out on semen preservation in G. cuvieri. Previous work [10] has shown the feasibility of semen cryopreservation in G. dama mhorr but has also highlighted the difficulties encountered with available protocols for semen freezing. Attempts at artificial insemination in this species have resulted in no live offspring [10, 13]. Efforts to cryopreserve spermatozoa from G. dorcas have been reported [14], as have attempts to preserve sperm viability by incubation in culture media [15]. Semen preservation in related antelopes has received limited attention [16–19]. Better protocols for semen handling and cryopreservation are required in these antelope species to achieve the high percentages of sperm survival and fertility as obtained in other species of wild or semidomesticated ungulates [20–22]. In addition, it is important to understand what factors may influence sperm survival after cryopreservation to establish reliable cryopreservation protocols for the establishment of genetic resource banks for these species.

We have undertaken the present study to explore the ability of various semen diluents to support cryopreservation of spermatozoa from the three species of gazelles, to compare the success of cryopreservation between the three species, and to examine if the level of inbreeding of the three populations affects semen cryopreservation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Animal manipulations were performed in accordance with the Spanish Animal Protection Regulation, RD223/1988, which conforms to European Union Regulation 86/609 and adheres to guidelines established in the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the Society for the Study of Reproduction.

Adult male gazelles of the three species used in the present study were maintained and managed by the EEZA at the Parque de Rescate de la Fauna Sahariana (PRFS) in the southern outskirts of Almeria. The PRFS, where animals are kept, is located at 36°50'10''N and 2°27'48'W and is 100 m above sea level. A total of 12 males of G. cuvieri (age, >3 yr; weight, >28 kg), 12 males of G. dama mhorr (age, >5 yr; weight, >55 kg), and 13 males of G. dorcas neglecta (age, >2 yr; weight, >14 kg) were used. All males were kept as isolated animals in individual enclosures and were healthy and reproductively mature at the time of the beginning of the present study.

Semen Collection

The time of the year chosen to collect semen depended on whether the species has a seasonal pattern of reproduction, as indicated by the breeding records of the EEZA. Thus, for G. cuvieri, which has peak mating activity during the autumn [2], semen collection was carried out in December 2000. For G. dama, which has peak mating activity during the spring and autumn, collection was carried out in April 2001, whereas for G. dorcas, which does not follow a seasonal pattern, collection was done in February 2001.

Semen collection was performed under surgical anesthesia using a combination of i.m. xylazine (0.2 mg/kg body wt; Bayer, Barcelona, Spain) and i.v. ketamine chlorohydrate (15 mg/kg body wt; Rhône Merieux, Lyon, France). When necessary, surgical anesthesia was maintained using halothane inhalation. After semen collection, the effects of anesthesia were reversed by i.v. administration of the antidote, yohimbine chlorohydrate (0.025 mg/kg body wt).

Semen was collected by electroejaculation using a sine-wave stimulator (P. T. Electronics, Boring, OR). The stimulator was capable of monitoring voltage and amperage and used an AC current of 220 V, 60 Hz, with a transformer producing a maximum of 55 V and 1.5 A. The stimulating voltage was delivered using rectal probes with three longitudinal, surface-mounted electrodes. Probe diameter, probe length, and electrode length for each species were as follows: for G. dorcas, 2.0, 27.7, and 5.0, respectively; for G. cuvieri, 2.7, 29.5, and 5.8 cm, respectively; and for G. dama, 3.2, 35.0, and 6.6 cm, respectively. The probe was lubricated, gently inserted into the rectum, and then orientated so that the electrodes were positioned ventrally. The penis was prolapsed beyond the prepuce, and semen was collected using a 30-ml, sterile plastic container, which was kept warm by covering it with the hand.

The electroejaculation regime used was based on that employed previously for ungulates [14, 23] with various modifications. It consisted of consecutive series of 5-sec pulses of similar voltage, each separated by a 5-sec break. Each series consisted of a total of four pulses. The initial voltage was 1 V for all species and was increased in each series. Increases of 1 V in each series were used until a level of 3 V (G. cuvieri and G. dorcas) or 4 V (G. dama) was reached. Thereafter, 0.5-V increases were used in each series for the three species. A standard stimulation period of 7 min was used for all males. If stimulation was interrupted, the series was restarted using 1 V less than that in the current series. Responses to each pulse were recorded.

Semen was placed at 30°C in a water bath pending analyses. Semen evaluation and freezing were carried out at laboratory facilities at the PRFS (hence, transport of the semen samples was not necessary).

Semen volume, sperm concentration, and subjective scores of motility (wave motion) were assessed shortly after collection. Also, within this interval, aliquots were diluted in PBS with bovine serum albumin (5 mg/ml) and used to assess individual sperm motility. Percentages of individual and progressively motile sperm were noted, and quality of motility was assessed using a scale of 0 (lowest) to 5 (highest). A sperm motility index (SMI) was calculated as follows

The sperm suspension was also used to assess viability, sperm morphology, and acrosome integrity. In addition, samples were taken to assess membrane integrity by means of the hypo-osmotic swelling (HOS) test.

The volume was measured by aspirating semen with a micropipette. Concentration was estimated using a hemocytometer. Motility (evaluated at 37°C), viability, and proportion of morphologically normal spermatozoa were assessed as described previously [24]. Acrosome integrity was evaluated after a 1:1 dilution in 2% glutaraldehyde in 0.165 M cacodylate/HCl buffer (pH 7.3). The percentage of spermatozoa with intact acrosomes (% NAR) was assessed by phase-contrast microscopy.

Sperm membrane integrity was assessed using the HOS test as described previously [25]. Briefly, 5 µl of semen or a diluted sperm suspension was mixed with 50 µl of a hypo-osmotic sodium citrate solution (100 mOsmol/kg) and incubating the mixture at room temperature for 30 min. The samples were then fixed in 2% glutaraldehyde/cacodylate, as described above, and evaluated under phase-contrast microscopy at 400x magnification. The sperm membrane was considered to be intact if the sperm tail was coiled at the end of the assay, and the result was expressed as the percentage positive endosmosis.

Semen Diluents

Various cryopreservation media (i.e., diluents) were used (Table 1): Triladyl, a commercially available extender (diluent A; Minitüb, Tiefenbach, Germany); Tes-Tris-yolk (TEST; diluent B), based on the zwitterionic buffers Tes (N-tris(hydroxymethyl)methyl-2 aminoethane sulfonic acid) and Tris (tris(hydroxymethyl) aminomethane); yolk-citrate (diluent C); Tris-trehalose (diluent D); and Tris-lactose (diluent E). The TEST was prepared with 5% egg yolk, based on previous findings of semen cryopreservation in G. dama [10], whereas the remaining diluents contained 20% egg yolk. Glycerol concentrations were used in the range of 6–8% (see Table 1).


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  		TABLE 1. Composition of diluents employed in this study for cryopreservation of gazelle spermatozoa

Both G. cuvieri and G. dorcas had ejaculates with total numbers of spermatozoa that were much lower than those of G. dama (see Results). This allowed for a limited number of diluents to be tested on each ejaculate. Thus, we tested diluents A, B, and D in G. cuvieri and diluents A, B, C, and D in G. dorcas. For G. dama, with a larger ejaculate volume, all five diluents could be tested for each ejaculate.

With the exception of Triladyl, all diluents were prepared in the laboratory using reagent-grade chemicals purchased from Sigma or Merck (both of Madrid, Spain). All other chemicals were of reagent grade and were purchased from Sigma or Merck.

Semen Cryopreservation

Semen samples were diluted in one step with the chosen diluent(s) containing glycerol prewarmed to 30°C and then allowed to reach room temperature (~20°C). The extended semen was loaded into 0.25-ml plastic straws and then cooled slowly (-0.16°C/min) to 5°C and held for equilibration at that temperature for 2 h (total refrigeration time at 5°C, 3.5 h). At this point, subsamples were taken for evaluation of motility and of acrosome and membrane integrities using the methods described above. The straws were frozen in nitrogen vapors, 4 cm above the surface of the liquid nitrogen, for 10 min and then plunged into liquid nitrogen.

The straws were thawed for 30 sec at 37°C in a water bath, and the contents were poured into a glass tube and assessed for sperm motility, viability, and acrosome and membrane integrities. Thawed samples were incubated at 37°C for 2 h without dilution (i.e., in the diluent) or after dilution of 25 µl of thawed semen with 75 µl of a modified Tyrode medium (120 mM NaCl, 3.1 mM KCl, 2 mM CaCl2, 0.4 mM MgSO4, 20 mM Hepes, 5 mM glucose, 21.7 mM sodium lactate, 1 mM sodium pyruvate, 20 µg/ml of phenol red, and 5 mg/ml of bovine serum albumin). The medium had a pH 7.55 (adjusted with NaOH) at room temperature. At the end of this incubation period, sperm suspensions were again assessed for motility, viability, and acrosome and membrane integrities.

For sperm assessments, 100–200 spermatozoa were counted in each preparation. Results are presented as the mean ± SEM. Various rates were calculated to assess the cryoprotective abilities of the different diluents at different stages as follows:

In addition, a rate that would yield information regarding survival after dilution of thawed spermatozoa in Tyrode medium was calculated as follows:

Either ANOVA or paired Student t-tests were used for analyses, and the usual transformations were applied to those variables that were not normally distributed.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Semen Collection and Characteristics

Semen was collected successfully by electroejaculation from all males of the three species. No contamination with urine took place. Maximum voltage and amperage used in each species were as follows: G. cuvieri, 5 V, 70 mA; G. dama, 6.5 V, 130 mA; and G. dorcas, 4 V, 60 mA.

Considerable variations in semen quality were found between individuals of the three species. Because the main objective of the present study was to compare various diluents for cryopreservation, semen with initial poor quality was not included. Of the 12 males each of G. cuvieri and G. dama and the 13 males of G. dorcas, samples used in the present study came from 9, 10, and 10 males, respectively, after samples with less that 50% motility, 50% positive HOS test, or 50% acrosome integrity were rejected. The characteristics of the semen from males included in the present study are shown in Table 2.


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  		TABLE 2. Seminal parameters of fresh ejaculates obtained from three different gazelle species (genus Gazella) in the present study.a

Differences were noted in the seminal characteristics of the three species. Gazella dama yielded a larger total sperm number than the other two species. Overall, semen quality was worse in G. cuvieri, intermediate in G. dama, and best in G. dorcas (Table 2). In addition, considerable individual variations were found in semen quality, even after excluding low-quality semen samples.

Effect of Various Diluents on Cryopreservation of G. cuvieri Spermatozoa

Three diluents were examined with G. cuvieri spermatozoa: A, Triladyl; B, TEST; and D, Tris-trehalose (Table 3). In this species, prefrozen sperm motility (70%–77% motile sperm) was not different between the three diluents. At this stage, membrane integrity (as revealed by the HOS test) tended to be higher with Triladyl (63%), intermediate with TEST (51%), and lower with Tris-trehalose (45%), but the differences were not statistically significant (Table 3). Differences reached significance when the rate of spermatozoa that retained membrane integrity after refrigeration (i.e., refrigeration rate) were compared between diluents (A, 79.0 ± 5.0; B, 64.4 ± 8.7; D, 54.3 ± 4.4; P < 0.04).


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  		TABLE 3. Seminal parameters after cooling and equilibration, freezing and thawing, and incubation for 2 h after thawing of spermatozoa from Gazella cuvieri in different diluents.a

After freezing and thawing, motility (28%–35% motile sperm), viability (34%–46%), acrosome integrity (20%–28%), and membrane integrity (13%–17%) were low in the three diluents, but no statistically significant differences were detected between them. When spermatozoa were incubated for 2 h at 37°C, a marked loss in sperm motility was observed in all cases (final motility, 0%–5% motile sperm), although viability was similar to that seen after thawing. No differences were seen between diluents (Table 3).

Taken together, these results suggested that one diluent (TEST) seemed to be marginally better for cryoprotection in G. cuvieri. The differences were not statistically significant, however, probably because of the high interindividual variation.

Effect of Various Diluents on Cryopreservation of G. dama Spermatozoa

Five diluents were examined with G. dama spermatozoa: A, Triladyl; B, TEST; C, yolk-citrate; D, Tris-trehalose; and E, Tris-lactose (Table 4). Prefrozen sperm motility was similar (63%–80% motile sperm) in the five diluents, although a trend toward a lower sperm motility was observed with diluents C, D, and E. Similar high proportions of spermatozoa with normal apical ridges (82%–90% NAR) were found with all five diluents. Membrane integrity at this stage was significantly lower with diluents D and E.


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  		TABLE 4. Seminal parameters after cooling and equilibration, freezing and thawing, and incubation for 2 h after thawing of spermatozoa from Gazella dama in different diluents.a

After freezing and thawing, a decrease in all parameters was observed with the five diluents. Motility was significantly higher with diluent B (43% motile sperm) than with the other diluents (A, 17%; C, 6%; D, 14%; and E, 15%). Membrane integrity was equally low with all diluents (15%–21%) with the exception of diluent C, which showed significantly higher values (28%). Acrosome integrity was similar with diluents A (43% NAR) and B (49% NAR), but with the other diluents, it showed significantly lower values (21%–28% NAR). When spermatozoa were incubated for 2 h at 37°C, a marked loss in sperm motility was observed with diluents A, C, D, and E (final motility, 0%–4% motile sperm), and these values were lower than those seen with diluent B (25% motile sperm). Viability also decreased with values at the end of the incubation, being even lower with diluent C. Acrosome integrity was better preserved during this incubation period by diluents A and B.

In summary, diluent B (TEST) afforded better motility preservation in this species, although no differences were found between this and diluent A (Triladyl) regarding membrane and acrosome integrities.

Effect of Various Diluents on Cryopreservation of G. dorcas Spermatozoa

Four diluents were examined with G. dorcas spermatozoa: A, Triladyl; B, TEST; C, yolk-citrate; and D, Tris-trehalose (Table 5). In this species, prefrozen sperm motility was equally high (82%–84% motile sperm) between the four diluents. Also, similar high proportions (92%–97% NAR) of spermatozoa with normal apical ridges were found. Membrane integrity at this stage was significantly lower with diluent D (Table 5).


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  		TABLE 5. Seminal parameters after cooling and equilibration, freezing and thawing, and incubation for 2 h after thawing of spermatozoa from Gazella dorcas in different diluents.a

After freezing and thawing, a decrease was observed in all parameters with all four diluents. No significant differences in motility were seen between diluents. Diluents A and C afforded better preservation of membrane integrity (as seen by % endosmosis). Acrosome integrity was not well preserved in diluent D. When spermatozoa were incubated for 2 h at 37°C, a decrease was noted in sperm motility and membrane and acrosome integrities. Differences were detected between diluents, with diluent D showing the lowest values (see Table 5).

In general terms, all diluents afforded similar good protection in this species with the exception of diluent D (Tris-trehalose), which had a tendency to perform worse during freezing and thawing and in the postthawing incubation.

Dilution and Incubation in Tyrode Medium after Thawing

On thawing, spermatozoa from G. cuvieri and G. dorcas were incubated in the same diluent (A, Triladyl; or B, TEST) for 2 h at 37°C, or they were diluted in a modified Tyrode medium and incubated under similar conditions.

For both species and for spermatozoa frozen-thawed in diluent A or diluent B, the results (Table 6) revealed a significant decrease in sperm motility on dilution in Tyrode medium. This reduction in sperm motility (as indicated by the calculated dilution survival rate) was similar for spermatozoa frozen-thawed in diluent A or B.


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  		TABLE 6. Sperm motility in Gazella cuvieri and G. dorcas after thawing or after a 2-h incubation in the freezing diluent or after dilution in Tyrode medium.a

When spermatozoa were incubated for 2 h in the same diluent used for freezing and thawing or after dilution in Tyrode medium, it was found that spermatozoa from G. cuvieri showed very poor motility at the end of the incubation, regardless of whether they were incubated in diluent A or B or in Tyrode medium. On the other hand, about half the G. dorcas spermatozoa survived incubation in diluent or in Tyrode medium (no statistical differences were found between the postthaw survival rates for % motility).

Comparison of dilution survival rates (i.e., the ability of sperm to resist dilution in Tyrode medium) between species revealed that spermatozoa from G. dorcas showed much higher values than those from G. cuvieri (see Table 6) and thus were better able to withstand dilution.

Comparison Between Species and Relation with Inbreeding

To assess if differences existed between species regarding their ability to survive the cryopreservation process, we compared the refrigeration, freezing, and overall freezing rates (see Materials and Methods). The use of rates rather than absolute values allows for direct comparison between species that differ in fresh semen parameters (Table 2). Comparisons were made between spermatozoa from the three species that were frozen-thawed in diluent A (Triladyl) and diluent B (TEST), because these diluents afforded the best cryopreservation results.

Refrigeration, freezing, and overall freezing rates calculated for diluents A and B and for spermatozoa from the three species are shown in Figure 1. The results showed that in general terms, all three species survived the refrigeration step equally well with both diluents; no major differences were noted in the refrigeration rates. Freezing and overall freezing rates showed similar trends when comparing species with either one or the other diluent. Gazella dorcas had the highest rates in all the parameters evaluated (% motility and sperm motility index, % endosmosis, and % intact acrosomes), with results that in most cases were significantly different from those seen in the other two species. Gazella cuvieri, on the other hand, had the lowest values for several of these parameters, with differences in the percentage acrosome integrity reaching significance between diluents.



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  		FIG. 1. Comparison of refrigeration rate (A and B), freezing rate (C and D), and overall freezing rate (E and F) of spermatozoa from three different gazelle species (genus Gazella) frozen and thawed in Triladyl (A, C, and E) or TEST (B, D, and F). Refrigeration rate is equal to the value after refrigeration and equilibration divided by the value in fresh semen multiplied by 100. Freezing rate is equal to the value after thawing divided by the value after refrigeration and equilibration multiplied by 100. Overall freezing rate is equal to the value after thawing divided by the value in fresh semen multiplied by 100. Values are the mean ± SEM. Data were examined using analysis of variance and Fisher post-hoc tests after value transformation. Bars with different letters indicate statistically significant differences. % motility, percentage motile spermatozoa; SMI, sperm motility index; HOST +ve, percentage spermatozoa with coiled tail (positive response) after the HOS test; % NAR, percentage spermatozoa with intact acrosomes

The three species have different average levels of inbreeding because of differences in the number of founding individuals [9]. For the males included in the present study, the average coefficients of inbreeding were as follows: G. cuvieri, 0.1493 ± 0.0096; G. dama, 0.1012 ± 0.0133; and G. dorcas, 0.0454 ± 0.0125 (ANOVA: F2,26 = 18.71, P < 0.0001; Fisher post-hoc tests revealed differences between the three species, P <= 0.01). A comparison between average values of inbreeding and the freezing or overall freezing rates of the three species (Fig. 1) revealed that spermatozoa from the species with the highest level of inbreeding (G. cuvieri) had a poor ability to survive freezing and thawing, whereas spermatozoa from the species with the lowest level of inbreeding (G. dorcas) had the highest capacity to survive the cryopreservation process.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The results of the present study indicate that differences occur both when using various semen diluents and between species during cryopreservation of gazelle spermatozoa. Species differences seem to be related to the level of inbreeding in the populations.

Semen was collected by electroejaculation using a protocol previously employed in other ungulates [14, 23], although it was different from the method previously used in these species by us [8, 9, 24] and in G. dama by others [10, 13]. Furthermore, semen samples used in the present study were selected by rejecting those that were below a quality threshold. Thus, it is not possible to compare the results of ejaculate quality of the present study with those reported previously. Clear differences were found between the ejaculate quality of the three species, with G. cuvieri showing lower quality ejaculates, a fact that has been noted earlier [9, 24].

No live offspring has been produced after cryopreservation of semen from gazelle species. There have been attempts at cryopreservation and artificial insemination in the Speke gazelle (G. spekei), but no births have been obtained using frozen-thawed semen [16]. Earlier attempts concerning semen preservation in G. dorcas met with moderate success [14, 15] and were not followed by artificial insemination trials. Efforts to cryopreserve G. dama spermatozoa resulted in limited success, and artificial insemination trials have so far been unsuccessful [10]. To our knowledge, sperm cryopreservation in G. cuvieri has not been reported. Semen from other antelopes (addax, blackbuck, and scimitar-horned oryx) has been collected and frozen successfully, with live offspring being obtained after artificial insemination [19]. This has also been the case with other species of nondomestic ungulates [19]. We have therefore attempted to improve the success of semen cryopreservation in G. dorcas and G. dama and have explored, to our knowledge for the first time, the possibility of freezing G. cuvieri spermatozoa. To evaluate the ability of various cryoprotective media, we quantified sperm survival after cooling and after freezing and thawing. In some species, the motility of recently thawed samples is not a good indicator for the success of in vitro fertilization, whereas longevity after sperm incubation is a much more reliable parameter [26]. Therefore, we also assessed sperm parameters 2 h after thawing and incubation at 37°C.

We found that spermatozoa from the three gazelle species could be successfully cryopreserved, although survival varied depending on cryoprotective media and species. Differences were mainly noted during the freezing-thawing stage. Our results also revealed that better cryoprotection was obtained in comparison to previous studies. In G. dorcas, we obtained an average of 55% motile sperm and 71% intact acrosomes after thawing, as opposed to 40% motile sperm and 36% intact acrosomes in previous work [14]. In G. dama, our results (43% motile sperm, 49% intact acrosomes) were an improvement over earlier findings (36% motile sperm, 41% acrosome integrity) [10].

One critical factor during cryopreservation of spermatozoa from different species is the composition of the various diluents. Various complex cryoprotective buffer systems are capable of preserving sperm function during freezing and thawing [27],and several have been used in wild ungulates, with varying degrees of success [19]. All the diluents examined provided similar cryoprotection in G. cuvieri and G. dorcas, with a trend for better performance of TEST in G. cuvieri and of Triladyl in G. dorcas. On the other hand, in G. dama, media composition strongly influenced the results. The TEST was better than Triladyl, with the other three diluents tested showing poor results. Therefore, no diluent was consistently better across species. This suggests that comparative semen diluent testing, similar to that used in the present study, may be a prerequisite to cryobanking of spermatozoa from any endangered species. It should be noted that although it was possible to cryopreserve G. cuvieri spermatozoa, the results were poor. The low recovery after thawing and the low survival during the postthaw incubation strongly suggest that spermatozoa cryopreserved using these diluents may not perform well after artificial insemination. Our results therefore indicate that the diluents used with G. cuvieri spermatozoa are far from ideal and that further work is needed to improve cryopreservation in this species. Similarly, in G. dama, additional studies are needed to improve results, because only approximately half the total number of spermatozoa survived cryopreservation. On the other hand, in G. dorcas, good cryopreservation was achieved and, furthermore, all diluents afforded good protection in this species. This is important, because it provides alternatives that are better suited for field conditions, such as the possibility of using a commercially available diluent.

The success of cryopreservation could be affected by diluent components, such as the buffer system, osmotic pressure, and concentration of glycerol or egg yolk. Interactions between the various components also may occur. The diluents that tended to perform better (Triladyl and TEST) included Tris-citrate or Tes-Tris as a buffering system. Zwitterionic buffers have been shown to differ in protective capacity for bovine spermatozoa [28, 29]. Tris-citrate did not always result in good preservation, because two diluents with this buffering system (Tris-trehalose and Tris-lactose) gave poorer results, indicating that other factors are important. Triladyl and TEST also had lower osmotic pressure than the other diluents. This alone may not explain all the differences, however, because the two diluents performed differently in one species.

Glycerol concentration can affect cryosurvival. Bull spermatozoa are routinely cryopreserved using 4%–8% glycerol, and acceptable results generally have been obtained in wild ruminants using this range [19, 30]. In the present study, diluents did not vary greatly in glycerol concentration (6%–8%), so it is possible that this component was not a source of variation. It remains to be established whether other cryoprotective agents are more suitable for gazelle spermatozoa [18].

Regarding egg-yolk concentration, Triladyl had 20% yolk, whereas TEST was made with 5% yolk, the latter based on a previous report indicating that a lower concentration of egg yolk resulted in better cryoprotection in G. dama [10]. Whereas this may be true for G. dama when using TEST, this may not necessarily be the case in general, because in our hands, diluents with 20% yolk were good in the other two species. In addition, it is not clear whether high concentrations of egg yolk would have detrimental effects, because no differences were found between TEST (5% yolk) and Triladyl (20% yolk) after cooling and equilibration (i.e., after 3.5-h exposure of spermatozoa to egg yolk). Future studies should address this issue in more detail, because a high egg-yolk concentration may nevertheless have toxic effects on spermatozoa (brindled gnu [31] and Murrah buffalo [32]) or interactions may occur between egg yolk and the buffer system [31].

One important factor affecting cryopreservation is species differences, as seen in domestic animals and in related nondomestic ungulates [33]. We have compared the survival of spermatozoa of the three species during cooling-equilibration and during freezing-thawing. Survival after cooling was somewhat lower in G. cuvieri and tended to be better in G. dorcas. Similarly, during freezing-thawing, G. cuvieri tended to perform worse and G. dorcas to perform better (differences in several parameters were statistically significant). The same was true when survival of the overall freezing process (cooling plus freezing and thawing) was analyzed. Taken together, our results indicate that the species more resistant to cryopreservation is G. dorcas and that the species most sensitive is G. cuvieri. This agrees with earlier findings showing interspecies differences of cryosusceptibility in deer species [22] and African antelope [18].

One possible mechanism that may explain differences between gazelle species is a differential resistance of spermatozoa to osmotic shock [34]. This was suggested by differences noted in fresh ejaculates from the three species when evaluating the proportions of spermatozoa that were positive in the HOS test (lowest values in G. cuvieri and highest values in G. dorcas). We compared the osmotic tolerance of spermatozoa from G. cuvieri (the least resistant to the freezing process) and G. dorcas (the most resistant) to rapid removal of glycerol on thawing (Table 6). The one-step decrease of glycerol by dilution with Tyrode medium resulted in a decline in motility parameters in both species, but the decrease was more pronounced in G. cuvieri than in G. dorcas, regardless of the diluent employed for freezing-thawing. Osmotic stress after the rapid removal of glycerol [35] is related to differences in the relative permeability of glycerol and water across the sperm membrane. Mammalian spermatozoa appear to behave as linear osmometers, and cell death occurs if the spermatozoa swell or shrink beyond species-specific osmotic tolerances [36–38]. In addition, membrane damage may occur secondary to rapid water movement across the membrane [39]. The difference in response subsequent to a return to isosmotic conditions may therefore reflect differences in the relative osmotic tolerance of gazelle spermatozoa. Our results thus indicate that the spermatozoa of G. dorcas, in addition to being the ones that better resist the freezing process, are also those with a higher resistance to the osmotic shock caused by rapid removal of the glycerol, with the opposite being true for G. cuvieri spermatozoa. Therefore, differences in survival after cryopreservation between spermatozoa from these gazelle species agree well with their sensitivity to osmotic shock. The observation that tolerance to osmotic stress is highly predictive of sperm survival after cryopreservation is important for two reasons. First, it shows that differences in membrane structure, permeability, and elasticity influence the extent to which sperm will survive the cryopreservation process. Second, it suggests that sperm survival can be optimized by minimizing osmotic shock, perhaps by a gradual addition and removal of glycerol [40].

The source of variation in sperm cryosusceptibility at the species or individual level is not clear, but a role for a genetic component is suggested by work in cattle [41], horses [42], pigs [37, 43] and mice [34, 44–46]. In mice, cryopreservation of spermatozoa from inbred strains is less successful than with outbred strains [34, 46], which suggests a genetic basis for the sensitivity of mouse spermatozoa to osmotic shock and freezing injury. It is possible that a genetic basis also exists for the differences in cryopreservation of the gazelle species. The three species examined in the present study differ in the average level of inbreeding of the populations (because they descend from a different number of founders). Gazella cuvieri is the species with the highest average level of inbreeding, whereas G. dorcas is the one with the lowest. Because G. cuvieri was the species with the least ability to survive cryopreservation and G. dorcas was the best, an inverse relation between the level of inbreeding and the ability to survive cryopreservation in these species was evident. It is therefore likely that inbreeding has led to the enhanced expression of deleterious recessive alleles, which substantially reduce ejaculate quality and increase cryosusceptibility.


   ACKNOWLEDGMENTS
 
We thank Eulalia Moreno for permission to study the gazelles and access to facilities at the EEZA. We also thank the staff of the PRFS (Ramon Escamilla, Francisco Hernandez, Oscar Salinas, Juan Belzunces, Alfonso Lopez, Gema Morales, and Marcos Gonzalez) for expert handling of the animals and assistance.


   FOOTNOTES
 
1 Supported by the Spanish Ministry of Science and Technology (Project REN2000-1470/GLO) Back

2 Correspondence: E.R.S. Roldan, Museo Nacional de Ciencias Naturales (CSIC), c/Jose Gutierrez Abascal 2, 28006-Madrid, Spain. FAX: 34 91 564 5078; roldane{at}mncn.csic.es Back

Received: 31 October 2002.

First decision: 25 November 2002.

Accepted: 8 April 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. IUCN. The IUCN Policy Statement on Captive Breeding. Cambridge: IUCN Publications Service; 1987
  2. Olmedo G, Escos J, Gomendio M. Reproduction de gazella cuvieri en captivité. Mammalia 1985 49:501-507
  3. Alados CL, Escos J, Vericad JR. Captive populations of Northwest African Antilopinae and Caprinae at the Estacion Experimental de Zonas Aridas. In: Dixon A, Jones D (eds.), Conservation and Biology of Desert Antelopes. London: Christopher Helm; 1988:199–211
  4. Alados CL, Escos J. Phenotypic and genetic characteristics affecting lifetime reproductive success in female Cuvier's, dama and dorcas gazelles (Gazella cuvieri, G. dama and G. dorcas). J Zool 1991 223:307-321
  5. Cano M. El Antílope Mohor Gazella (=Nanger) dama mhorr Bennett 1832 en Cautividad. Taxonomía, Biología, Ecología, Etología, Técnicas de Salvamento y Reintroducción, y Posibilidades de Aprovechamiento Económico. Granada, Spain: Universidad de Granada; 1991. Thesis
  6. Hilton-Taylor C. IUCN Red List of Threatened Species. Gland, Switzerland: IUCN; 2000
  7. Ebenhard T. Conservation breeding as a tool for saving animal species from extinction. Trends Ecol Evol 1995 10:438-443
  8. Roldan ERS, Cassinello J, Abaigar T, Gomendio M. Inbreeding, fluctuating asymmetry and ejaculate quality in an endangered ungulate. Proc R Soc Lond B 1998 265:243-248[Medline]
  9. Gomendio M, Cassinello J, Roldan ERS. A comparative study of ejaculate traits in three endangered ungulates with different levels of inbreeding: fluctuating asymmetric as an indicator of reproductive and genetic stress. Proc R Soc Lond B 2000 267:875-862[Medline]
  10. Holt WV, Abaigar T, Jabbour HN. Oestrus synchronization, semen preservation and artificial insemination in the Mohor Gazelle (Gazella dama mhorr) for the establishment of a genome resource bank programme. Reprod Fertil Dev 1996 8:1215-1222[CrossRef][Medline]
  11. Wildt DE. Genetic resource banks for conserving wildlife species: justification, examples and becoming organized on a global basis. Anim Reprod Sci 1992 28:247-257[CrossRef]
  12. Watson PF, Holt WV, (eds.) Cryobanking the Genetic Resource. London: Taylor and Francis; 2001
  13. Abaigar T, Holt WV. Towards the development of a genetic resource bank for the Mohor gazelle: Putting theory into practice. In: Watson PF, Holt WV (eds.), Cryobanking the Genetic Resource. London: Taylor and Francis; 2001:123–139
  14. Howard J, Pursel VG, Wildt D, Chakraborty P, Bush M. Comparative of various extenders for freeze-preservation of semen from selective captive wild ungulates. J Am Vet Med Assoc 1981 179:1157-1161[Medline]
  15. Howard JG, Wildt DE, Chakraborty P, Bush M. Reproductive traits including seasonal observations on semen quality and serum hormone concentrations in the dorcas gazelle. Theriogenology 1983 20:221-234[CrossRef]
  16. Boever J, Knox D, Merilan C, Read B. Estrus induction and artificial insemination with successful pregnancy in Speke's gazelle. In: Proceedings of the 9th International Congress of Animal Reproduction and Artificial Insemination, Madrid, Spain. 1980; 2:565–569
  17. Holt WV, Moore HDM, North RD, Hartman TD, Hodges JK. Hormonal and behavioural detection of oestrus in blackbuck (Antilopre cervicapra) and successful artificial insemination with fresh and frozen semen. J Reprod Fertil 1988 82:717-725[Abstract]
  18. Loskutoff NM, Simmons HA, Goulding M, Thompson G, Jongh TD, Simmons LG, Species and individual variations in cryoprotectant toxicities and freezing resistance of epididymal sperm from African antelope. Anim Reprod Sci 1996 42:527-535[CrossRef]
  19. Holt WV. Germplasm cryopreservation in elephants and wild ungulates. In: Watson PF, Holt WV (eds.), Cryobanking the Genetic Resource. London: Taylor and Francis; 2001:317–348
  20. Jabbour HN, Argo CMcG, Brinklow BR, Loudon ASI, Hooton J. Conception rates following intrauterine insemination of European (Dama dama dama) fallow deer does with fresh or frozen-thawed Mesopotamian (Dama dama mesopotamica) fallow deer spermatozoa. J Zool 1993 230:379-384
  21. Monfort SL, Asher GW, Wildt DE, Wood TC, Schiewe MC, Williamson LR, Bush M, Rall WF. Successful intrauterine insemination of Eld's deer (Cervus eldi thamin) with frozen-thawed spermatozoa. J Reprod Fertil 1993 99:459-465[Abstract]
  22. Asher GW, Berg DK, Evans G. Storage of semen and artificial insemination in deer. Anim Reprod Sci 2000 62:195-211[CrossRef][Medline]
  23. Roth TL, Weiss RB, Buff JL, Bush LM, Wildt DE, Bush M. Heterologous in vitro fertilization and sperm capacitation in an endangered African antelope, the scimitar-horned oryx (Oryx dammah). Biol Reprod 1998 58:475-482[Abstract/Free Full Text]
  24. 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[Abstract]
  25. Garde JJ, Ortiz N, García A, Gallego L, Landete-Castillejos T, López A. Postmortem assessment of sperm characteristics of the red deer during the breeding season. Arch Androl 1998 41:195-202[Medline]
  26. Roth TL, Bush LM, Wildt DE, Weiss RB. Scimitar-Horned Oryx (Oryx dammah) spermatozoa are functionally competent in a heterologous bovine in vitro fertilization system after cryopreservation on dry ice, in a dry shipper, or over liquid nitrogen vapor. Biol Reprod 1999 60:493-498[Abstract/Free Full Text]
  27. Watson PF. The causes of reduced fertility with cryopreserved semen. Anim Reprod Sci 2000 60: –61 481-492
  28. Graham EF, Grabo BG, Brown KI. Effect of some zwitter ion buffers on the freezing and storage of spermatozoa. J Dairy Sci 1972 55:372-378
  29. Rasul Z, Anzar M, Jalali S, Ahmad N. Effect of buffering systems on post-thaw motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa. Anim Reprod Sci 2000 59:31-41[CrossRef][Medline]
  30. Leibo SP, Songsasen H. Cryopreservation of gametes and embryos of non-domestic species. Theriogenology 2002 57:303-326[CrossRef][Medline]
  31. Watson PF. Electroejaculation, semen characteristics and semen preservation of the brindled gnu. J Reprod Fertil 1976 47:123-126[CrossRef][Medline]
  32. Kumar S, Sahni KL, Mohan G. Effect of different extender formulations on acrosomal maintenance of buffalo spermatozoa frozen in milk, tris and sodium-citrate dilutors. Ind J Anim Sci 1993 63:1233-1239
  33. Holt WV. Fundamental aspects of sperm cryobiology: the importance of species and individual differences. Theriogenology 2000 53:47-58[CrossRef][Medline]
  34. Songsasen H, Leibo SP. Cryopreservation of mouse spermatozoa. II. Relationship between survival after cryopreservation and osmotic tolerance of spermatozoa from three strains of mice. Cryobiology 1997 35:255-269[CrossRef][Medline]
  35. Gao DY, Mazur P, Critser JK. Fundamental cryobiology of mammalian spermatozoa. In: Karow AM, Critser JK, (eds.), Reproductive Tissue Banking: Scientific Principles. San Diego: Academic Press; 1997:263–328
  36. Gilmore JA, McGann LE, Liu J, Gao DY, Peter AT, Kleinhans FW, Critser JK. Effect of cryoprotectant solutes on water permeability of human spermatozoa. Biol Reprod 1995 53:985-995[Abstract]
  37. Gilmore JA, Du J, Tao J, Peter AT, Critser JK. Osmotic properties of boar spermatozoa and their relevance to cryopreservation. J Reprod Fertil 1996 107:87-95[Abstract]
  38. Gilmore JA, Liu J, Peter AT, Critser JK. Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol Reprod 1998 58:28-36[Abstract/Free Full Text]
  39. Curry MR, Watson PF. Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology 1994 31:39-46[CrossRef][Medline]
  40. Gao DY, Liu J, Liu C, McGann LE, Watson PF, Kleinhans FW, Mazur P, Critser ES, Critser JK. Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum Reprod 1995 10:1109-1122[Abstract/Free Full Text]
  41. Thomas CA, Garner DL. Post-thaw bovine spermatozoal quality estimated from fresh samples. J Androl 1994 15:489-500[Abstract/Free Full Text]
  42. Amann RP, Pickett BW. Principles of cryopreservation and a review of cryopreservation of stallion spermatozoa. Equine Vet Sci 1987 7:145-173
  43. Thurston LM, Siggins K, Mileham AJ, Watson PF, Holt WV. Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation. Biol Reprod 2002 66:545-554[Abstract/Free Full Text]
  44. Tada N, Sato M, Yamanoi J, Mizorogi T, Kasai K, Ogawa S. Cryopreservation of mouse spermatozoa in the presence of raffinose and glycerol. J Reprod Fertil 1990 89:511-516[Abstract]
  45. Nakagata N, Takeshima T. Cryopreservation of mouse spermatozoa from inbred and F1 hybrid strains. Exp Anim 1993 42:317-320
  46. Sztein JM, Farley JS, Mobraaten LE. In vitro fertilization with cryopreserved inbred mouse sperm. Biol Reprod 2000 63:1774-1780[Abstract/Free Full Text]



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