Cryopreservation of semen of tigers and lions: Computerized analysis of the motility parameters of the spermatozoa

S. B. Patil, D. Jayaprakash* and S. Shivaji

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India
*Dubai Gynaecology and Fertility Centre, PO Box No. 8729, Dubai

A computer-aided semen analyser was used to monitor motility and motility parameters of tiger and lion spermatozoa prior to and after cryopreservation to ascertain changes, if any, and to establish the quality of the semen sample. The data demonstrate a consistent decrease in various motility parameters of spermatozoa following freeze-thaw and unlike spermatozoa from unfrozen semen these are slower, less progressive and their trajectories are less planar. Such data could form the basis for discerning the quality of a semen sample using data of a semen sample from a proven fertile male as the baseline data.

SEMEN banking by cryopreservation is a great boon to wildlife conservationists since such preserved semen could be used when and where needed and more importantly to improve the genetic heterogeneity in a particular species or to introduce disease-resistant genes. Though protocols for cryopreservation of cattle and human semen are very well established, it is necessary to bear in mind that cryopreservation protocols are not universally applicable to all animals and need to be modified1,2. These protocols vary with respect to the semen diluent used, the cryoprotectant employed, the freezing regime and also dependent on whether the storage was to be done in straws, in ampules or as pellets3-5. Attempts have been made earlier to cryo- preserve semen from tigers6,7 and such spermatozoa have been evaluated with respect to per cent motility and fertilizing ability. Cryopreservation is also known to decrease percentage motility, increase acrosomal loss and decrease fertilizing ability8,9. However, many of the above parameters for evaluating cryopreserved sperm are subjective and are not easy to carry out.

In the present study, a computer-aided sperm analyser (CASA) was used to monitor motility and motility parameters such as curvilinear velocity (VCL), progressive velocity (VSL), path velocity (VAP), straightness (STR=VSL/VAP), linearity (LIN=VSL/VCL), the amplitude of lateral head displacement (ALH) and beat cross frequency (BCF) of tiger and lion semen samples prior to and after cryopreservation. Earlier studies have indicated that based on these motility parameters it has been possible to objectively discriminate an immature sperm from a mature one, a motile sperm from an immotile one, an hyperactivated sperm from a non- hyperactivated one and a slow moving sperm from a fast moving one10-17. Therefore, the main objectives of this study were to ascertain whether cryopreservation has any effect on the overall motility, motility pattern and motility parameters of spermatozoa with a view to using the data to evaluate the quality of the semen sample. The data revealed changes not only with respect to the percentage motile cells and the overall trajectory of the spermatozoon but a number of changes were also observed with respect to the various motility parameters.

Materials and methods

Collection of semen

Semen samples were collected from adult Indian tigers and Asiatic lions maintained at the Nehru Zoological Park (Hyderabad), Sakkarbaug Zoo (Junnagadh) and Nandankanan Zoological Park (Bhubaneshwar) following anaesthesia. Tigers and lions were anaesthetized using a combination of ketamine HCl (2.22 to 2.66 mg/kg body weight; Troy Labs, New South Wales, Australia) and xylazine (1.11 to 1.33 mg/kg body weight; Troy Labs, New South Wales, Australia) and a standard protocol was used to induce electroejaculation using a rectal probe (Shivaji et al., accompanying article).

Cryopreservation of semen

Attempts were made to cryopreserve only those semen samples which exhibited more than 40% initial sperm motility. The method of cryopreservation was essentially the same as that used for human semen with minor modifications. Briefly, the semen sample was diluted with an equal volume of Ham's F-10 at 37 C, spun at 800g for 10 min and the pellet suspended in Ham's F-10 to the original volume of the ejaculate. Aliquots (150micro l) of the suspended sperm sample were transferred to screw-capped cryotubes (1.8 ml volume) and mixed with an equal volume of the cryoprotectant medium containing TES (4.326 g), Tris (1.027 g), dextrose (0.200 g) in 100 ml distilled water to which 20 ml of heat inactivated egg yolk was added and pH adjusted to 7.4. The osmolality of the medium ranged between 290 and 320 mOsm. The cryotubes were then exposed to liquid nitrogen vapours at a height of 5 to 8 cm from the surface of liquid nitrogen for 45 min. This exposure reduced the temperature of the semen sample to -100 C. Subsequently, the tubes were stored immersed in liquid nitrogen until further use. The cryopreserved semen samples were thawed by plunging the cryotube into a 37 C water bath and assessed for percentage motile spermatozoa using the motility analyser.

Motility analysis

Assessment of spermatozoal motility using a CASA apart from being rapid and objective, would also indicate the number of motile cells and facilitate rapid analysis of motility parameters, such as VCL, VSL, VAP, STR, LIN, ALH and BCF13-15. Such parameters could be used to define spermatozoa which vary in their motility track or trajectory. In the present study a CASA HTM-IVOS (Version 10, Hamilton Thorne Research Inc., Danvers, MA, USA) was used for the motility analysis of tiger and lion spermatozoa prior to and after cryopreservation of semen.

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The parameter setup values (Table 1) were derived from preliminary experiments carried out to ascertain a combination of optimum values such that the analyser was identifying the immotile and motile spermatozoa correctly and was also tracking each motile spermatozoon following the guidelines of the manual and the recommendations of Working and Hurtt18 and Yeung et al.19. Using these parameter values and the playback facility in the analyser, it was observed that the analyser was identifying correctly the motile (a red dot on the sperm head) and immotile spermatozoon (a blue dot on the sperm head) and was also computing for a complete track of the spermatozoon.

The motility analyser was used to determine the sperm count, the number of motile spermatozoa in a semen sample, and all the seven motility parameters for any single spermatozoon. The motility track or trajectory of the tiger and lion spermatozoon was also observed using the `zoom' function which presents an enlarged view and also provides   x and y axis coordinates of each track.

Statistical analysis

Significant differences between the data was determined by Student's t-test.

Results and discussion

Cryopreservation of semen

Artificial insemination using cryopreserved semen is extensively used in cattle to avoid inbreeding and facilitate genetic heterogeneity. Further, it is now an accepted procedure in human infertility clinics to use cryopreserved semen for intrauterine insemination in certain types of human infertility2.

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In fact cryopreserved semen pregnancies have been observed in different types of deers20-25. But, such studies in Indian tigers and lions are lacking and there is a need to develop methods for cryopreservation of felid spermatozoa prior to attempting artificial insemination. Table 2 summarizes results of our attempts to cryopreserve semen from tigers and lions. The data indicated a decrease in percentage motility of tiger and lion spermatozoa following thawing by 15 to 50%. Donoghue et al.6 had shown that freeze-thawing reduced tiger sperm motility by about 5-45% and their study also indicated that the decrease in percentage motility after cryopreservation was independent of the percentage initial motility prior to cryopreservation, since the 4 animals used in their study had an initial motility >80%. However, it has been generally observed by earlier investigators and even in the current study that the percentage motility after cryopreservation was consistently > 50% when the initial motility of the ejaculate was >70%. Byers et al.7 also showed that in Siberian tigers the motility decreased dramatically from 61.12+\-15.4% to 40.4+\-11.3% immediately after thawing.

Cryopreservation of spermatozoa could result in damage to spermatozoa and thus affect its fertilizing ability8,9. Thus, it is important to assess sperm function using in vitro bioassays such as the zona-free oocyte penetration test. Using such a bioassay it was demonstrated that the penetration of the zona-free hamster oocyte was decreased when frozen-thawed spermatozoa were used. But Donoghue et al.6 demonstrated that fresh and thawed tiger spermatozoa were equally capable of penetrating domestic cat eggs. Our results indicate that frozen-thawed spermatozoa of Indian tigers also consistently penetrate 83 to 90% of the oocytes (see Shivaji et al., accompanying article).

Computer-aided sperm analysis

Computer-aided analysis of spermatozoa facilitates rapid analysis of various motility parameters, viz. VSL, VAP, VCL, STR, LIN, BCF and ALH. In the present study CASA system was used to monitor changes in the motility parameters of tiger and lion spermatozoa prior to and after cryopreservation (Table 3). The results indicate a significant decrease in VAP, VSL, STR and LIN and increase in ALH and BCF in cryopreserved tiger spermatozoa compared to spermatozoa from neat semen. Lion spermatozoa following cryopreservation also showed a decrease in all the velocity parameters (VAP, VSL and VCL) and LIN.

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Unlike the cryopreserved tiger spermatozoa, the cryopreserved lion did not show any increase in ALH and BCF. Further, the decrease in STR and LIN was not as prominent as in the tiger. Based on these results it may be concluded that cryopreserved tiger and lion spermatozoa are slower (due to a consistent decrease in the velocity parameters) and their trajectories are probably more curved and less planar (due to consistent decrease in STR and LIN) and are thus less progressive as indicated by a decrease in VSL (Figure 1).

The present CASA data are not sufficient to understand or establish the basis for the changes observed in the motility parameters of tiger and lion spermatozoa but it may be speculated that such changes may decrease sperm quality. Freezing and thawing has been demonstrated to affect human sperm quality by severe impairment of sperm motility and the range of post thaw motility varied by 15-80% from individual to individual26-30. It was also observed that velocity, linearity and ALH were only slightly affected30.

Table 3 shows the pooled data of a number of spermatozoa from eight semen samples from tigers and five semen samples from lions and thus do not reflect the individual variations if any in the semen from individuals of the same species. In fact we did not observe significant variations from individual to individual following post thaw (Table 4). Therefore, it may be concluded that the overall trend in the alteration in the motility parameters following freeze thaw is similar from animal to animal, thus indicating that pooling of data is acceptable and does not in any way bring about discrepancy in the results.

This is the first study on the motility parameters of tiger and lion spermatozoa and such studies could form the basis for evaluating semen samples with respect to their quality. For instance, alterations in the motility parameters of tiger and lion spermatozoa may not only affect its motility pattern but also its fertilizing ability. Majority of the spermatozoa prior to and after freeze-thaw exhibited `planar' motility pattern that is the trajectory of motility was linear. But, following freeze thaw the spermatozoa travelled a shorter distance in unit time (as indicated by decrease in VSL) probably due to decrease in VAP and due to slightly curved trajectories (as indicated by decrease in STR and LIN). But such changes did not affect the fertilizing ability of cryopreserved semen of tiger (see our previous report) lion and leopard (unpublished results).



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ACKNOWLEDGEMENTS.We thank Directors and staff of Hyderabad, Sakkarbaug and Bhubaneshwar zoos for their support in immobilizing the animals and for collecting blood and semen samples. S.S. gratefully acknowledges the Central Zoo Authority, Ministry of Environment and Forests, Government of India for financial support for the work. We also thank Mr S. C. Sharma, Member Secretary, Central Zoo Authority for his constant support and encouragement for the present work and Mr K. Santosh Kumar and S. Sudha for their involvement in the earlier stages of this project. S.S. specially thanks Dr K. C. Majumdar, Scientist, CCMB for his involvement and support in the project. We thank Dr Lalji Singh, Director, CCMB for his valuable suggestions.

Received 19 August 1998; accepted 29 September 1998