Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws
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1 CSIRO PUBLISHING Reproduction, Fertility and Development, 2007, 19, Cryopreservation of epididymal alpaca (Vicugna pacos) sperm: a comparison of citrate-, Tris- and lactose-based diluents and pellets and straws Katherine M. Morton A,B, Roslyn Bathgate A, Gareth Evans A and W. M. Chis Maxwell A A Centre for Advanced Technologies in Animal Genetics and Reproduction (ReproGen), Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia. B Corresponding author. kmorton@vetsci.usyd.edu.au Abstract. Epididymal spermatozoa were harvested from male alpacas and frozen after extension and cooling to 4 Cin citrate-, Tris- and lactose-based diluents (Experiment 1) and as pellets in and 0.5-mL straws on either dry ice or over liquid nitrogen vapour (Experiment 2) to determine the effects diluents and packaging on their motility and acrosome integrity. In Experiment 1, sperm motility was higher after cooling to 4 C and after freeze thawing (0 but not 3 h postthaw) for spermatozoa extended in the lactose- than the citrate- or Tris-based diluent (P < 0.05). Post-thaw acrosome integrity after cooling to 4 C and post-thaw (0 h) was reduced for spermatozoa frozen in citrate- compared with lactoseor Tris-based diluents, but was similar for all groups 3 h after thawing. In Experiment 2, sperm motility immediately after thawing was higher for pellet freezing than for or 0.5-mL straws on dry ice or liquid nitrogen vapour (P < 0.05), although by 3 h post-thaw motility was similar for pellets and straws (P > 0.05). Acrosome integrity was similar for all groups immediately after thawing and 3 h post-thaw. Cryopreservation of epididymal alpaca spermatozoa is feasible, with retained motility and acrosome integrity post-thaw. Freezing as pellets in a lactose-based diluent is recommended. Additional keywords: artificial insemination, Camelidae, spermatozoa. Introduction Alpacas are members of the Camelidae family, which consists of Old World, Bactrian (Camelus bactrianus) and Dromedary (C. dromedarius) camels and New World camelids. New World camelids are native to South America and include the alpaca (Vicugna pacos), guanaco (Lama guanicoe), llama (L. glama) and vicuña (V. vicugna). Artificial reproductive technologies are not well established in alpacas, or any camelids, because their reproductive physiology is considerably different to that of other domestic animals (such as sheep and cattle). Alpacas copulate in sternal recumbency for up to 40 min and ejaculate throughout copulation (Bravo et al. 1997). The ejaculates are characterised by a highly viscous, low-volume semen containing a low concentration of spermatozoa that display an oscillatory motion (Bravo et al. 1997). The viscous seminal plasma traps spermatozoa, making handling, dilution and cryopreservation difficult (Bravo et al. 2000b). Previous attempts to cryopreserve alpaca spermatozoa have met with little success. Epididymal alpaca spermatozoa have not come in contact with the viscous seminal plasma and, therefore, provide a simpler model for establishing cryopreservation methods for ejaculated spermatozoa. Epididymal spermatozoa could be used to create gene banks for vicuña and guanaco, which are listed as threatened species in Appendix 2 of CITES (2005), and for spermatozoa from deceased male alpacas and racing camels with high genetic value. The aims of the present study were to investigate the feasibility of harvesting and cryopreserving epididymal alpaca spermatozoa after overnight transport to the laboratory. We further examined post-thaw sperm motility and acrosome integrity after dilution in citrate-, lactose- and Tris-based cryodiluents and freezing in pellets in or 0.5-mL straws on dry ice or over liquid nitrogen vapour. Materials and methods Animals and experimental design The pocedures described herein were approved by The University of Sydney s Animal Ethics Committee. For Experiment 1, epididymides and testes from castrated male alpacas (n = 10; age 32.9 ± 5.8 months) were wrapped in gauze, which had been soaked previously in phosphate-buffered saline (PBS; Sigma, St Louis, MO, USA), and placed in a foam esky on top of a cold pack for transport to the laboratory overnight. Epididymal spermatozoa were then harvested, washed and divided into three aliquots for dilution in citrate-, Tris- and lactose-based cryodiluents, cooled to 4 C and pellet frozen. For Experiment 2, epididymides and testes from castrated male alpacas (n = 10; age 22.5 ± 0.9 months) were transported overnight to the laboratory using the method described for CSIRO /RD /07/070792
2 Cryopreservation of epididymal alpaca spermatozoa Reproduction, Fertility and Development 793 Experiment 1. Harvested epididymal spermatozoa were washed, diluted with lactose-based cryodiluent and cooled to 4 C. Cooled spermatozoa were then frozen as pellets or in or 0.5-mL straws on dry ice, or in or 0.5-mL straws over liquid nitrogen vapour. For both experiments, spermatozoa from individual males were kept separate throughout all procedures, and motility and acrosome integrity were assessed immediately after harvest, cooling to 4 C and at 0 and 3 h after thawing. Harvesting of spermatozoa Upon arrival at the laboratory, excess tissue was removed from the epididymides. The epididymides were cleaned thoroughly, surface blood vessels were punctured and the blood wiped away. Epididymides were then minced with a scalpel and the spermatozoa were allowed to swim out for 30 min into 4.0 ml warm Androhep (AH; Minitüb, Tiefenbach, Germany) in 60-mm Petri dishes (Bacto Laboratories, Liverpool, NSW, Australia) placed on heated (37 C) stages. Cryopreservation of epididymal spermatozoa For Experiment 1, spermatozoan suspensions were centrifuged at 300g for 10 min and the pellet resuspended in 1.5 ml AH. A two-step cryopreservation method was used for citrate- and lactose-based diluents, whereas a one-step method was used for the Tris-based diluent. Spermatozoan samples were divided into three aliquots and diluted 1 : 1 with citrate- or lactose-based cooling diluents, or 1 : 2 with the Tris-based freezing diluent. The citrate-based cooling diluent consisted of a mixutre of 80.6 mm sodium citrate and 33.3 mm glucose supplemented with 20% (v/v) hens egg yolk (Bravo et al. 1996). The citrate-based freezing diluent consisted of the cooling diluent supplemented with 5% (v/v) glycerol. The lactose-based cooling diluent consisted of 11% (w/v) lactose solution supplemented with 20% egg yolk. The lactose-based freezing diluent consisted of the cooling diluent supplemented with 9% (v/v) glycerol and 1.5% (v/v) Equex STM paste (IMV, L Aigle, France), as described previously by Bathgate et al. (2006). The Tris-based cryodiluent consisted of 360 mm Tris, mm citric acid and 33.3 mm glucose solution supplemented with 20% egg yolk and 6% glycerol (Evans and Maxwell 1987). The final glycerol concentration of all diluents was 3% (v/v). Diluted sperm were cooled to 4 Cover2h( 0.14 C/min). The citrate- and lactose-diluted spermatozoa were further diluted 2 : 1 with lactose-based freezing extender or 2 : 3 with citratebased freezing extender. Cooled spermatozoa were then frozen as pellets (250 µl) on dry ice as described by Evans and Maxwell (1987). For Experiment 2, spermatozoa were frozen using the twostep lactose method as described for Experiment 1. Briefly, spermatozoan suspensions were centrifuged (300g; 10 min) and the pellet resuspended to a final volume of 2 ml with cooling extender and cooled to 4 Cover2h( 0.14 C/min). Cooled spermatozoa were further diluted with 1.0 ml freezing extender and frozen as pellets (250 µl) on dry ice, or loaded into or 0.5-mL straws and frozen on dry ice (5 min) or in liquid nitrogen vapour. For the latter freezing method, straws were suspended 8.0 cm above the surface of the liquid nitrogen for 10 min and then 4 cm above the surface for 5 min. Straws were then plunged into liquid nitrogen and stored until use. Thawing of spermatozoa Spermatozoa frozen as pellets were thawed, two at a time, in clean glass test-tubes by vigorous agitation in a 37 C water bath until the pellets melted. Straws were thawed by agitating in a 37 C water bath for either 30 s or 1 min for the and 0.5-mL straws, respectively. Thawed spermatozoa were then washed by centrifugation (300g; 10 min), the pellet resuspended in 0.5 ml warm AH and maintained at 37 C. Assessment of spermatozoa Subjective assessments of motility were made by placing a 10-µL aliquot of resuspended spermatozoa on a prewarmed slide and covering with a warmed coverslip, as described by Evans and Maxwell (1987). Motility was examined using phase-contrast microscopy ( 100; Olympus, Tokyo, Japan) immediately after harvest (post-harvest), cooling to 4 C and immediately (0 h) and 3 h after thawing. Acrosome integrity was assessed by fluorescent isothiocyanate-conjugated peanut agglutin (FITC-PNA; Sigma), as described by Roth et al. (1998). Briefly, an aliquot of the spermatozoa was smeared on a slide and air-dried. Slides were then stored at 4 C for 2 4 weeks. Immediately before assessment, slides were stained with 100 µgml 1 FITC-PNA in PBS for 30 min in a humid 37 C atmosphere. Slides were then rinsed with PBS to remove excess stain and placed in the dark at 37 C to dry. Fade retardant, consisting of 90% (v/v) glycerol, 10% (v/v) PBS and 0.1% (w/v) p-phenylenediamine, was then placed on the stained area of the slide and covered with a coverslip. Spermatozoa were considered acrosome intact if the acrosome stained green, whereas those with no staining or a single band of green staining at the equatorial segment were considered as having non-intact acrosomes (Roth et al. 1998). Statistical analysis Statistical differences in the proportion of motile sperm and intact acrosomes were determined by ANOVA after arcsin transformation of data. Means were compared by least significant differences (l.s.d.) using Genstat (Release 7.2; Ceanet, Brisbane, Qld, Australia) with P < 0.05 considered significant. Results Experiment 1 Spermatozoa were successfully harvested from the epididymides of all males. Opaque white fluid was observed leaking into the medium after mincing of the epididymides. The fluid was not viscous and the spermatozoa therein displayed forward progressive motility (46.9 ± 4.5%). Considerable variation was observed between the epididymides of different males. On average, ± spermatozoa were recovered per male, but this ranged from 36.9 to spermatozoa. Sperm motility also varied between males, ranging from 30% to 60%. After dilution and cooling to 4 C, motility was higher for spermatozoa diluted in lactose- rather than citrate- or Tris-based
3 794 Reproduction, Fertility and Development K. M. Morton et al. Table 1. Motility of epididymal alpaca spermatozoa after harvesting (post-harvest), cooling to 4 C (Cooled), and immediately (0 h) and 3 h after freezing and thawing in citrate-, lactose- and Tris-based diluents Diluent Motility (%) Post-harvest Cooled Post-thaw Citrate 46.9 ± ± 3.8 a 6.9 ± 2.3 a 0.9 ± 0.6 a Lactose 31.9 ± 5.1 b 18.2 ± 5.7 b 3.2 ± 1.6 b Tris 18.8 ± 4.1 a 11.3 ± 3.0 a 1.4 ± 0.8 a,b Table 3. Motility of epididymal alpaca spermatozoa after harvesting (post-harvest), and immediately (0 h) and 3 h after freezing and thawing in pellets, or in and 0.5-mL straws frozen on dry ice or in liquid nitrogen vapour Freeze method Post-thaw Dry ice Pellets 27.0 ± 5.2 a 1.0 ± 0.7 a 0.25-mL straw 5.5 ± 1.1 b 0.6 ± 0.4 a 0.5-mL straw 5.0 ± 1.6 b 0.5 ± 0.5 a Liquid nitrogen vapour 0.25-mL straw 8.6 ± 1.8 b 0.6 ± 0.5 a 0.5-mL straw 10.0 ± 2.4 c 0.5 ± 0.5 a Table 2. Acrosome integrity of epididymal alpaca spermatozoa after harvesting (post-harvest),cooling to 4 C (Cooled),and immediately (0 h) and 3 h after freezing and thawing in citrate-, lactose- and Tris-based diluents Diluent Acrosome integrity (%) Post-harvest Cooled Post-thaw Citrate 90.6 ± ± 0.8 a 79.0 ± 1.0 a 75.0 ± 1.6 a Lactose 90.5 ± 0.5 b 80.0 ± 2.2 a,b 79.0 ± 0.9 a Tris 92.4 ± 1.1 b 83.4 ± 2.2 b 76.1 ± 1.4 a Table 4. Acrosome integrity of epididymal alpaca spermatozoa after harvesting (post-harvest), and immediately (0 h) and 3 h after freezing and thawing in pellets, or in and 0.5-mL straws frozen on dry ice or in liquid nitrogen vapour Freeze method Post-thaw Dry ice Pellets 87.5 ± 1.2 a 83.3 ± 2.5 a 0.25-mL straw 88.3 ± 1.1 a 82.3 ± 1.7 a 0.5-mL straw 81.0 ± 2.2 a 78.3 ± 2.1 a Liquid nitrogen vapour 0.25-mL straw 89.8 ± 1.6 a 83.0 ± 2.0 a 0.5-mL straw 89.0 ± 1.5 a 79.0 ± 1.9 a diluents. Sperm motility was also higher immediately after freezing and thawing for spermatozoa frozen in lactose- compared with Tris- or citrate-based diluents (P < 0.05; Table 1), but was higher 3 h post-thaw for sperm frozen in lactose- rather than citrate-based diluent, but not Tris. Acrosome integrity after harvesting was 90.6 ± 1.5% and was higher after cooling for spermatozoa diluted in lactose- and Trisbased diluent compared with those diluted in a citrate-based cryodiluent (Table 2). Immediately after thawing (0 h postthaw) acrosome integrity was higher for spermatozoa frozen in Tris-based diluent compared with citrate- but not lactosebased diluent. Three hours after thawing, acrosome integrity was similar for spermatozoa frozen in the three diluents. Experiment 2 As in Experiment 1, spermatozoa were successfully harvested from the epididymides of all males, with a mean motility of 47.7 ± 5.0%, which ranged between 10% and 70% for individual males. On average, ± spermatozoa (range ) were recovered per male. The motility of the spermatozoa after cooling to 4 Cwas similar immediately after harvest (data not shown). Immediately after thawing (0 h), motility was higher for spermatozoa frozen as pellets than all other groups (P < 0.05; Table 3). Motility immediately after thawing was higher for spermatozoa frozen in 0.5-mL straws in liquid nitrogen vapour compared with those frozen in 0.25-mL straws in liquid nitrogen and and 0.5- ml straws frozen on dry ice (P < 0.05). However, motility was similar between groups by 3 h post-thaw (P > 0.05). Acrosome integrity was similar immediately after harvest (91.2 ± 3.1%) and cooling to 4 C (89.3 ± 3.8%). After thawing, acrosome integrity was similar between groups at 0 h and 3 h post-thaw (P > 0.05; Table 4). Discussion In the present study, an efficient method was developed for the cryopreservation of epididymal alpaca spermatozoa after overnight transport to the laboratory. Post-thaw recovery of spermatozoa was superior in lactose- compared with citrateand Tris-based cryodiluents. Moreover, pellet-frozen sperm had higher post-thaw motility compared with and 0.5-mL straws frozen on dry ice or over liquid nitrogen vapour. Compared with other domestic livestock species, such as cattle, sheep and pigs, there have been few investigations of freezing methods for ejaculated or epididymal alpaca spermatozoa. Most investigations have focused on artificial insemination (AI) with frozen thawed semen and have neglected to determine the optimal freezing and thawing processes. Consequently, the
4 Cryopreservation of epididymal alpaca spermatozoa Reproduction, Fertility and Development 795 packaging, cryodiluent type and concentration of the cryoprotectant, as well as the freezing and thawing rates, remain to be elucidated for alpaca and other members of the Camelidae family. Dilution with sucrose egg yolk glycerol (Zhao et al. 2004) medium and freezing in glass ampoules (2 ml) has been the preferred method for semen from bactrian camels, whereas dromedary semen has been packaged in 0.25-mL straws after dilution in Green Buffer (IMV), or 4.0 ml straws after dilution with lactose-based cryodiluent (Bravo et al. 2000b). Freezing of alpaca and llama semen has been conducted in citrate (Bravo et al. 1996, 2000a; Burgel et al. 2000; Aller et al. 2003), biladyl (Vaughan et al. 2003), Green Buffer (Vaughan et al. 2003), triladyl (McEvoy et al. 1992; Vaughan et al. 2003) and Tris (Von Baer and Hellemann 1999; Burgel et al. 2000; Santiani et al. 2005) cryodiluents. Using a citrate-based cryodiluent, Bravo et al. (1996) reported one of the more successful methods of cryopreservation of alpaca spermatozoa. High rates of survival of the spermatozoa and an overall motility of 46.7% after thawing were reported, with 54.9% of initial motility retained after freezing and thawing. However, the details provided by the authors are not sufficient to permit replication of their methods. In a subsequent study, and in the only report on the cryopreservation of epididymal alpaca spermatozoa, Bravo et al. (2000a) froze epididymal spermatozoa from five llamas and four alpacas using the same method as for ejaculated semen (Bravo et al. 1996). Sperm from two of the five llamas did not survive freezing and thawing and the post-thaw motility of spermatozoa from one alpaca was 5%. The authors stated that the other samples were motile post-thaw, but did not present motility data. Subsequent studies have not met with similar success as reported by Bravo et al. (1996). Far lower post-thaw motility of alpaca and llama spermatozoa has been reported using citrate cryodiluent (12.5%, present study; 20 28%, Aller et al and Burgel et al. 2000). Other diluents used to cryopreserve alpaca and llama spermatozoa have also resulted in low post-thaw motilities: biladyl 21.3% (Vaughan et al. 2003), Green Buffer 17.4% (Vaughan et al. 2003), skim-milk 15 20% (Santiani et al. 2005), triladyl 10% (McEvoy et al. 1992;Vaughan et al. 2003),Tris 19% (present study) and 22 27% (Burgel et al. 2000). Lactose-based cryodiluents have not been used previously to freeze alpaca spermatozoa, but have been applied successfully in the freezing of camel spermatozoa (Anouassi et al. 1992; Musa et al. 1992; Bravo et al. 2000b) and are used routinely for cryopreservation of boar spermatozoa (Johnson et al. 2000). Alpaca and llama spermatozoa have been generally frozen in smaller volumes than camel semen, with most studies using ml straws frozen in liquid nitrogen vapour (Bravo et al. 1996, 2000a, 2000b). It would be expected that post-thaw motility would be improved for smaller straws, because they have a comparatively larger surface area to volume ratio than larger straws. This reduces variation in the freezing and thawing rates within the straw (Bwanga et al. 1990). However, Burgel et al. (2000) and Vaughan et al. (2003) concluded that the post-thaw motility of llama and alpaca spermatozoa, respectively, was superior with 0.5- compared with 0.25-mL straws. In contrast, the post-thaw quality of dromedary semen was improved by freezing in compared with 4.0-mL straws (Bravo et al. 2000b). Pellet freezing provides superior post-thaw motility of ram spermatozoa compared with straw freezing (Maxwell et al. 1995) and substantially increased the post-thaw motility of epididymal alpaca spermatozoa compared with both and 0.5-mL straws in the present study. Although pellet freezing is clearly more efficient, the requirements of the AI technique ultimately influence, if not dictate, the choice of freezing technique (Holt 2000), resulting in most spermatozoa being frozen in straws and, for camels, large-volume straws (4.0 ml) owing to the volume of seminal plasma required to induce ovulation (Bravo et al. 2000b). The addition of sodium dodecyl sulfate (SDS) in the form of Equex (IMV, L Aigle, France) or Orvus ES (MiniTüb, Tiefenbach b. Landshut, Germany) paste to the cryodiluent is routine for boar spermatozoa and has been demonstrated previously to improve post-thaw sperm quality in Eld s deer, elephants, greater kudu, scimitar-horned oryx, wildebeest and zebra (Holt 2000). The SDS enhances the protective effect of egg yolk by breaking down the lipid and making it more accessible to the membrane of the spermatozoan (Pontbriand et al. 1989). Equex and Orvus ES may be beneficial to camelid spermatozoa during cryopreservation, and have been used in cryodiluents for camel (Musa et al. 1992) and alpaca (lactose diluent in the present study) spermatozoa, but their possible benefits remain to be determined because neither study included control groups (same cryodiluent without SDS). The initial motility of the epididymal sperm in the present study was high considering the interval between castration and harvesting (16 24 h). Epididymal spermatozoa are relatively easy to handle and dilute, being free from the viscous seminal plasma that impedes progress in developing efficient methods for preserving ejaculated alpaca spermatozoa. Forward progressive motion was displayed by epididymal spermatozoa immediately after harvest, in contrast with the oscillatory motility of ejaculated spermatozoa. In contrast, Bravo et al. (2000a) reported the absence of forward progressive motility for alpaca or llama epididymal spermatozoa. The effects of centrifugation on camelid spermatozoa have not been investigated extensively, but Ferre et al. (2000) reported that centrifugation (300g, 10 min) was highly detrimental to the longevity of fresh diluted llama spermatozoa. Therefore, the post-thaw survival of epididymal alpaca spermatozoa may be improved by the removal of the prefreeze centrifugation step. Parrilla et al. (2006) used sedimentation instead of centrifugation to concentrate sex-sorted boar spermatozoa. Given the beneficial effect of sedimentation on sex-sorted boar spermatozoa reported by the authors, attempts to modify this technique for inclusion during cryopreservation of alpaca spermatozoa may be warranted. There was minimal loss of motility of epididymal alpaca spermatozoa after cooling to 4 C in the present study. In another experiment, epididymal alpaca spermatozoa were liquid stored at 4 C for h without loss of motility (K. M. Morton, unpubl. data). These observations suggest that alpaca spermatozoa may be resistant to cold shock, although further larger-scale studies using both ejaculated and epididymal alpaca spermatozoa are required to confirm this hypothesis. Furthermore, the minimal loss in motility after cryopreservation of epididymal alpaca spermatozoa when pellet frozen in lactose cryodiluent
5 796 Reproduction, Fertility and Development K. M. Morton et al. was unexpected, given the lack of knowledge about freezing camelid spermatozoa and the general perception that camelid spermatozoa do not survive well after freezing and thawing (Vaughan et al. 2003). These results highlight the need for systematic investigation of the effects of the various freezing and thawing processes using ejaculated alpaca spermatozoa, similar to those described by Salamon and Maxwell (1995), which will undoubtedly improve the survival of ejaculated alpaca spermatozoa after freezing and thawing and lead to the development of efficient preservation procedures for alpacas and eventually all camelids. In conclusion, the present research demonstrates the feasibility of harvesting and cryopreserving epididymal alpaca spermatozoa with a long interval between castration and harvesting. Pellet freezing epididymal alpaca spermatozoa in lactose-based cryodiluent produces superior post-thaw survival compared with citrate- and Tris-based cryodiluents, and freezing in and 0.5-mL straws. Furthermore, the present research challenges the perception that alpaca spermatozoa are highly susceptible to cold shock and do not tolerate cryopreservation. Although these results are encouraging, further systematic studies are required. Acknowledgements This research was supported by the Rural Industries Research and Development Corporation (RIRDC) and the Australian Alpaca Association. The authors thank Ms K. J. Heasman for her excellent technical assistance and Mrs Iona McKinnon for organizing and assisting with the collection of testes. References Aller, J. F., Rebuffi, G. E., Cancino, A. K., and Alberio, R. H. (2003). Influence of cryopreservation on the motility, viability and fertility of llama (Lama glama) spermatozoa. Arch. Zootec. 52, [in Spanish]. 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Theriogenology 47, doi: /s x(97) Bravo, P. W., Alarcon, V., and Bondurant, R. H. (2000a). Epididymal spermatozoa characteristics and its use on artificial insemination of llamas and alpacas. In Proceedings of the 14th International Congress on Animal Reproduction, Stockholm, Sweden. p. 92 [Abstract]. (International Congress on Animal Reproduction: Stockholm.) Bravo, P. W., Skidmore, J. A., and Zhao, X. X. (2000b). Reproductive aspects and storage of semen in Camelidae. Anim. Reprod. Sci. 62, doi: /s (00) Burgel, H., Erhardt, G., and Gauly, M. (2000). Cryopreservation of llama (Lama glama) semen. Reprod. Domest. Anim. 35, 26. [Abstract] Bwanga, C. O., de Braganca, M. M., Einarsson, S., and Rodriguez-Martinez, H. (1990). Cryopreservation of boar semen in mini- and maxi-straws. Zentralbl Veterinarmed A. 37, CITES (2005). Appendices I, II and III. Convention on International Trade in Endangered Species of Wild Fauna and Flora. (CITES Secretariat: Geneva.) Available online at shtml [Accessed on 19 February 2007.] Evans, G., and Maxwell, W. M. C. (1987). Salamon sartificial Insemination of Sheep and Goats. (Butterworths: Sydney.) Ferre, L. B., Malik de Tchara, G., Aller, J. F., and Alberio, J. R. (2000). Effects of diluent, seminal plasma and centrifugation on quality traits of llama semen. In Proceedings of the 14th International Congress on Animal Reproduction, Stockholm, Sweden. p. 101 [Abstract]. (International Congress on Animal Reproduction: Stockholm.) Holt, W. V. (2000). Basic aspects of frozen storage of semen. Anim. Reprod. Sci. 62, doi: /s (00) Johnson, L.A., Weitze, K. F., Fiser, P., and Maxwell, W. M. C. (2000). Storage of boar sperm. Anim. Reprod. Sci. 62, doi: /s (00) Maxwell, W. M. C., Landers, A. J., and Evans, G. (1995). Survival and fertility of ram spermatozoa frozen in pellets, straws and minitubes. Theriogenology 43, doi: / x(95)00092-m McEvoy,T. G., Kyle, C. E., Slater, D.,Adam, C. L., and Bourke, D.A. (1992). Collection, evaluation and cryopreservation of llama semen. J. Reprod. Fertil. Abstr. Ser. 9, 48. Musa, B., Sieme, H., Merkt, H., and Hago, B. E. D. (1992). Artificial insemination in dromedary camels. In Proceedings of the First International Camel Conference, Dubai, 2 6 February (Eds W. R. Allen, A. J. Higgins, I. G. Mayhew, D. H. Snow and J. F. Wade.) pp (R&W Publications: Newmarket, UK.) Parrilla, I., Vazquez, J. M., Gil, M. A., Garcia, E. M., Caballero, I., Roca, J., and Martinez, E. A. (2006). Improving the functionality of stored flow cytometrically sex-sorted boar spermatozoa: sedimentation vs. centrifugation. Reprod. Fertil. Dev. 18, 282. [Abstract] doi: /rdv18n2ab349 Pontbriand, D., Howard, J. G., Schieve, M. C., Stuart, L. D., and Wildt, D. E. (1989). Effect of cryoprotective diluent and method of freeze thawing on survival and acrosomal integrity of ram spermatozoa. Cryobiology 26, doi: / (89) Roth, T. L., Weiss, R. B., Buff, J. L., Bush, L. M., Wildt, D. E., and Bush, M. (1998). Heterologous in vitro fertilization and sperm characteristics in an endangered African antelope, the scimitar horned oryx (Oryx dammah). Biol. Reprod. 58, doi: /biolreprod Salamon, S., and Maxwell, W. M. C. (1995). Frozen storage of ram semen I. Processing, freezing, thawing and fertility after cervical insemination. Anim. Reprod. Sci. 37, doi: / (94)01327-i Santiani, A., Huanca, W., Sapana, R., Huanca, T., Sepulveda, N., and Sanchez, R. (2005). Effects on the quality of frozen thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders. Asian J. Androl. 7, doi: /j x Vaughan, J. L., Galloway, D., and Hopkins, D. (2003). Artificial Insemination in Alpacas (Lama pacos). (Rural Industries Research and Development Corporation: Kingston, ACT.) Von Baer, L., and Hellemann, C. (1999). Cryopreservation of llama (Lama glama) semen. Reprod. Domest. Anim. 34, doi: /j x Zhao, X. X., Huang,Y. M., Nie, Q. C., Zhang,Y. K., and Chen, B. X. (2004). Effect of different extenders on motility survival and acrosomal integrity of camel spermatozoa frozen in ampoules. J. Camel Pract. Res. 1, Manuscript received 13 March 2007, accepted 16 May
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