Asian J Androl 2005; 7 (3): 303–309 DOI: 10.1111/j.1745-7262.2005.00021.x .Original Article . Effects on the quality of frozen-thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders Alexei Santiani1, Wilfredo Huanca1, Rómulo Sapana2, Teodosio Huanca2, Néstor Sepúlveda3, Raúl Sánchez3 1Veterinary Institute for Tropical and Highland Research, Faculty of Veterinary Medicine, Universidad Nacional Mayor de San Marcos, P.O. Box 03-5034, Lima 41, Perú 2 Quimsachata Experimental Station, National Institute of Agrarian Research, Puno, Perú 3 Reproductive Biotechnology Center, Universidad de La Frontera, P. O. Box 54-D, Temuco, Chile Abstract Aim: To evaluate two extenders and two cryoprotectant agents (CPA) for alpaca semen cryopreservation. Methods: Semen samples were obtained from four adult alpacas (Lama pacos) and frozen using extender I (TRIS, citrate, egg yolk and glucose) or extender II (skim milk, egg yolk and fructose), each containing either glycerol (G) or ethylene glycol (EG) as CPA. Consequently, four groups were formed: 1) extender I-G; 2) extender I-EG; 3) extender II-G; and 4) extender II-EG. Semen was diluted in a two-step process: for cooling to 5 °C (extenders without CPA), and for freezing (extenders with CPA). Viability and acrosome integrity were assessed using trypan blue and Giemsa stains. Results: When compared, the motility after thawing was higher (P < 0.05) in groups II-EG (20.0 % ± 6.7 %) and II-G (15.3 % ± 4.1 %) than that in groups I-G (4.0 % ± 1.1 %) and I-EG (1.0 % ± 1.4 %). Viable spermatozoa with intact acrosomes in groups II-EG (18.7 % ± 2.9 %) and II-G (12.7 % ± 5.9 %) were higher than that in groups I-G (5.7 % ± 1.5 %) and I-EG (4.0 % ± 1.0 %). Conclusion: The skim milk- and egg yolk-based extenders contain- ing ethylene glycol or glycerol to freeze alpaca semen seems to promote the survival of more sperm cells with intact acrosomes than the other extenders. (Asian J Androl 2005 Sep; 7: 303–309) Keywords: cryopreservation; glycerol; ethylene glycol; extenders; alpaca spermatozoa 1 Introduction portant animal resource in the altiplanic region of Sudamerica. In order to obtain a fast diffusion of indi- South American camelids, alpacas (Lama pacos) and viduals of higher genetic merit, artificial insemination (AI) llamas (Lama glama), are species with great economic has been used for many years in other species. Preg- importance in Peru and Bolivia, as they provide fiber and nancy rates higher than 60 % have been obtained by AI meat for High-Andean people. Nevertheless, low rates in alpacas using fresh semen, but this technique has not of fecundity affect the development of camelids, an im- been widely applied to camelids because of difficulties in developing suitable protocols for dilution and conserva- Correspondence to: Dr Néstor Sepúlveda, Reproductive Biotech- tion of alpaca semen [1, 2]. There are few reports con- nology Center, Universidad de La Frontera, P.O. Box 54-D, Temuco, Chile. cerning storage of South American camelid semen at low Tel: +56-45-325-462, Fax: +56-45-325-053 temperatures. More than 20 years ago, Graham et al. E-mail: nestor@ufro.cl [3] froze llama semen, reporting 45 % of motility after Received 2004-07-30 Accepted 2004-12-13 thawing. Nevertheless, in other recent studies [4, 5], © 2005, Asian Journal of Andrology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences. All rights reserved. .303. Alpaca semen cryopreservation motility after thawing was lower than 25 % in llama Institute for Agrarian Research (INIA-Puno) were used semen. in this study. Males were maintained on natural pastures In alpaca semen, motility after thawing was lower than at the Quimsachata Experimental Center, which is lo- 20 % [6] and Bravo et al. [7] reported between 30 % and cated in Puno, Peru, at an altitude of 4025 m above sea 40 % after freezing semen from both llamas and alpacas. level, 15°41´39" south latitude and 70°36´24" west This indicates that the spermatozoa of camelids suffer longitude. This locality belongs to the zone of altiplano. considerable damage during cryopreservation with the Semen samples were collected during the period of Feb- protocols used. ruary and March, which is the peak of the camelid breed- The use of cryoprotectant agents is essential for the ing season in South America. viability of spermatozoa after thawing as these agents minimize intracellular ice formation and restrict the solu- 2.2 Semen collection tion effect [8]. The most frequently used cryoprotectant Semen was collected every second day, using a agent to freeze animal semen is glycerol [9], but ethylene modified ovine artificial vagina, wrapped in an electric glycol has also been applied to replace glycerol in many warming pad. The artificial vagina was mounted and species. Some studies have shown better effects pro- fixed a dummy inside, with the external access through duced by the use of ethylene glycol (e.g. in human [10] a hole. Samples from males who had copulated for more and bovine [11]). So far the only cryoprotectant agent than 15 min were processed, as this amount of time is used for cryopreservation of semen in camelids has been required to achieve camelids with semen good quality. glycerol [5, 6]. Semen samples were transported to the laboratory for Most extenders used for the freezing of camelid se- analysis. Due to the viscosity of the semen, samples were men have been prepared combining TRIS, citrate, glyc- put under mechanical action, passing them through a erol and egg yolk [3, 5, 6] . small-gauge needle. They were assessed for volume, In the majority of previous reports, assessment of sperm concentration, motility, viability and acrosomal sperm function after storage has been limited to sperm integrity. Only samples with normal seminal parameters motility [3, 6], with a single report describing the evalu- (at least 60 % of motility and 80 million cells/mL) [13] ation of viability using nigrosin-eosin stain [5]. However, were processed (n = 12). the acrosome integrity has been a useful parameter to evaluate fertility [12]. Using an immunogold-labeling tech- 2.3 Semen extenders nique to determine the stability of sperm acrosomes, Two extenders were used in the experiments. Ex- Valdivia et al. [6] indicated that only 5 % of spermatozoa tender I (10 mL) was prepared in autoclaved double dis- were immunolabeled after freezing-thawing. Von Baer tilled water (prepared in house) containing 0.244 g TRIS and Hellemann [4] utilized a spermatic stain to evaluate (Merck, Darmstadt, Germany), 0.136 g citric acid the acrosomal integrity, but viability was not measured. (Mallinckrodt, Xalostoc, Mexico), 0.082 g glucose There is no doubt that improvement of cryopreser- (Sigma-Aldrich, St. Louis, USA) and 20 % v/v egg yolk. vation techniques of camelid spermatozoa will contrib- Extender II (10 mL) consisted of skim cow milk (0.5 g/L ute to the development of programs of genetic improve- fat, Gloria S.A., La Victoria Perú), 5 % v/v egg yolk and ment in these species. To that end, a 2 × 2 factorial 0.485 g fructose (Sigma-Aldrich St. Louis, Mo, USA). experiment was designed to compare the efficiency of Each extender was divided into two equal fractions: frac- TRIS-citrate or skim milk-based extenders, containing tion A without the cryoprotectant used for cooling se- either glycerol or ethylene glycol as cryoprotectant agents men to 5 °C and fraction B containing 14 % v/v glycerol for the cryopreservation of alpaca semen. Trypan blue (G) (Ultra Pure Glycerol, Agtech, Manhattan, USA) or and Giemsa staining were used to assess the viability and 14 % v/v ethylene glycol (EG) (Biolife Freeze Medium acrosomal status. W/ 1.5 mol/L ethylene glycol, Agtech, Manhattan, USA) for freezing. Fraction B was added to an equal volume 2 Materials and methods to semen samples diluted in fraction A at 5 °C. Therefore, the final percentage of cryoprotectant agents was 7 %, 2.1 Animals which means 1 mol/L glycerol and 0.1 mol/L ethylene Four Huacaya-breed male alpacas from the National glycol. .304. Asian J Androl 2005; 7 (3): 303–309 2.4 Semen freezing and thawing zoa from other species was carried out in alpaca semen, After semen evaluation, ejaculates were divided into according to Didion et al. [14]. Due to the poor staining two groups in order to dilute them with equal volumes of of the acrosomal membrane, Giemsa 20 % was used extenders I or II (fraction A) at 35 °C. Tubes containing instead of 10 %. Briefly, 200 µL sperm suspension diluted semen were placed in a water bath at 35 °C. The aliquots were mixed 1:1 with 2 % trypan blue (T-0887 cooling rate was approximately 1 °C /3 min, from 35 °C Sigma, Sigma-Aldrich, St. Louis, Mo, USA) for 10 min to 5 °C. Once at 5 °C, each fraction A dilution (I and II) at 37 °C. Then, aliquots were washed two or three was divided into two samples. Equal volumes of frac- times with SP-Talp medium [11] (100 mmol/L sodium tion B (with glycerol or ethylene glycol) at 5 °C were chloride, 3.11 mmol/L potassium chloride, 25 mmol/L added in a second step, resulting in the following combi- sodium bicarbonate, 0.3 mmol/L potassium phosphate nations of diluent and cryoprotectant: extender I and glyc- monohydrate, 3 mL sodium lactate [60 % syrup], 2 mmol/L erol (I-G), extender I and ethylene glycol (I-EG), ex- calcium chloride dehydrate, 4 mmol/L magnesium chlo- tender II and glycerol (II-G) and extender II and ethyl- ride hexahydrate, 10 mmol/L HEPES, 1 mmol/L sodium ene glycol (II-EG). After mixing fractions A and B in pyruvate, 14 mmol/L penicillin G). Aliquots of 25 µL of each group, tubes were equilibrated for 30 min at 5 °C. the sperm suspension were placed on a slide, and air- Before freezing, samples of cooled diluted semen were dried at room temperature for 20 min. Thereafter, sper- rewarmed and incubated at 37 °C for 5 min for motility matozoa were stained for 40 min with Giemsa 20 % analysis. Meanwhile, the extended semen was loaded (Merck, Darmstadt, Germany) in distilled water prepared into 0.25 mL plastic French straws and exposed to liquid immediately before use. After staining, the slides were nitrogen vapor for 15 min. Finally, straws (three straws washed in distilled water, air-dried at room temperature per group) were plunged into liquid nitrogen. Straws for 30 min and mounted with permount and a coverslip. were thawed in a water bath at 37 °C for 5 min to assess At least 200 cells were assessed according to the follow- for motility, viability and acrosomal integrity. ing staining patterns: 1) dead spermatozoa with intact acrosomes, showing a blue or dark-blue post-acrosomal 2.5 Assessment of motility, viability and acrosomal in- region and a pink or purple acrosomal region; 2) dead tegrity spermatozoa with detached acrosomes, showing a blue Percentages of motile spermatozoa were assessed or dark-blue post-acrosomal region and a white acroso- under a coverslip (18 mm × 18 mm) on a warm glass mal region; 3) viable spermatozoa with intact acrosomes, slide (37 °C) by a trained technician, using phase con- showing a white post-acrosomal region and a purple trast microscopy (× 400 magnification) (CX31, Olympus, acrosomal region; and 4) viable spermatozoa with de- Tokyo, Japan). The double stain technique used to evalu- tached acrosomes showing white color in both post- ate sperm viability and acrosomal integrity in spermato- acrosomal and acrosomal region (Figure 1). A B C D Figure 1. Double stain tripan blue-Giemsa 20 % in alpaca spermatozoa. (A): Dead spermatozoa with intact acrosome; (B): Dead spermatozoa with detached acrosome; (C): Viable spermatozoa with detached acrosome; (D): Viable spermatozoa with intact acrosome. .305. Alpaca semen cryopreservation 2.6 Statistical analysis thawing. Extender II with either glycerol or ethylene Data in percentage (motility, viability and acrosomal glycol was found to bring less influence to sperm motil- integrity) were transformed (arcsine-square root trans- ity than extender I. formation) to approach a normal distribution. Compari- son between the four groups (I-G, I-EG, II-G and II- 3.2 Effect of cryoprotectant agents and extenders on EG) were analyzed using ANOVA (Prism version 3.0, sperm viability and acrosomal integrity San Diego, USA). Following a significant ANOVA, the The percentages of viable spermatozoa and viable Tukey multiple comparison test was used to determine spermatozoa with intact acrosomes after freezing and differences between means. Data for motility and viabil- thawing in extender II with glycerol or ethylene glycol ity were compared using the Pearson correlation test in were highest (Table 2). There was a significant differ- order to validate the double stain technique for alpaca ence (P < 0.05) between extenders but not between spermatozoa. P < 0.05 was condidered as significant. cryoprotectants. Viable spermatozoa also had intact acrosomes in both fresh and processed semen (Table 2). 3 Results 3.3 Correlation between motility and viability 3.1 Effect of cryoprotectant agents and extenders on Motility and viability were strongly correlated in fresh sperm motility and frozen-thawed semen (P < 0.01), with a correlation The results of the present study suggested that sperm coefficient of r = 0.96 (Figure 2). motility tended to decrease following incubation at 5 ºC for 30 min. The decrease was significant compared with 4 Discussion the activity of fresh semen for extender II and glycerol (Table 1). In our study, the double stain technique (trypan blue Sperm motility decreased further after freezing and and Giemsa 20 %) was reliable to evaluate both the vi- Table 1. Mean percentages ± SD of motility of alpaca spermatozoa in fresh semen, after 30 min at 5 °C and after thawing in various extenders. b,e Data with different superscript letters within columns differ significantly (P < 0.05). Extender I: TRIS, citric acid + glucose + egg yolk; extender II: skim milk + fructose + egg yolk. Extenders and cryoprotectant agents Before thawing After thawing Motility (%) Motility (%) Fresh semen (before any dilution) 72.0 ± 13.0b After 30 min of incubation at 5 °C: Extender I + glycerol 56.6 ± 7.6b 4.0 ± 1.1b Extender I + ethylene glycol 66.5 ± 6.4b 1.0 ± 1.4b Extender II + glycerol 49.2 ± 7.5e 15.3 ± 4.1e Extender II + ethylene glycol 64.0 ± 13.9b 20.0 ± 6.7e Table 2. Mean percentages ± SD of viable spermatozoa and viable spermatozoa with intact acrosome in fresh semen and after freezing- thawing in various extenders. b,e,h Data with different superscript letters within columns differ significantly (P < 0.05). Extender I: TRIS, citric acid + glucose + egg yolk; extender II: skim milk + fructose + egg yolk. Extenders and cryoprotectant agents Viable spermatozoa (%) Viable with intact acrosome (%) Fresh semen (before any dilution) 80.7 ± 10.1b 78.3 ± 10.0b After freezing-thawing: Extender I + glycerol 6.2 ± 1.5e 5.7 ± 1.5e Extender I + ethylene glycol 4.3 ± 4.3e 4.0 ± 1.0e Extender II + glycerol 13.0 ± 6.1e,h 12.7 ± 5.9e,h Extender II + ethylene glycol 21.0 ± 1.0h 18.7 ± 2.9h .306. Asian J Androl 2005; 7 (3): 303–309 100 (66.5 %) and II-EG (64.0 %), sperm motility rates post- 8 0 thaw in groups II-G (15.3%) and II-EG (20.0 %) were 6 0 better (P < 0.05) than that in groups I-G (4.0 %) and I- 4 0 EG (1.0 %). It is possible that certain compounds of 2 0 skim milk such as casein or lactose protects alpaca sperm 0 better than TRIS-citric acid. It is concluded that the 0 20 40 60 80 100 extender based on skim milk, egg yolk and fructose pro- sperm motility (%) vides a higher protection of alpaca spermatozoa than the Figure 2. Correlation between percentages of motility and viable extender based on TRIS, citric acid, egg yolk and glu- spermatozoa of alpacas (r = 0.96) (P < 0.0001). cose as measured by results obtained post-thaw. In relation to cryoprotectants, varying degrees of susceptibility and resistance in spermatozoa have been reported using different agents and concentrations of ability and acrosomal integrity in alpaca spermatozoa. cryoprotectants. This can be attributed to differences Preliminary trials using Didion’s stain [14] produced only between species with regard to the structure, permeabil- poor staining of the acrosomal membrane of alpaca ity and function of the plasmatic membrane of sperma- spermatozoa. We therefore tried Giemsa 20 % which tozoa [9,16]. achieved good staining of the acrosomal membrane The use of a final glycerol concentration of 7 % (Figure 1). (1 mol/L) to freeze camelid semen has probably been In an immunogold labeling technique for acrosomes adapted from protocols to freeze semen from rams or with human anti-proacrosin/acrosin monoclonal antibody bulls. Holt [16] indicated that bovine spermatozoa are rou- (C5F10) described by Valdivia et al. [6], only 5 % of thawed tinely cryopreserved with 4 %–8 % glycerol concentra- alpaca spermatozoa were labeled. It is probable that the tions , and these concentrations have been successfully results corresponded only to viable spermatozoa, because used to cryopreserve spermatozoa of some wild rumi- we found that most of the viable spermatozoa maintained nants and primates. However, other species (pigs and intact acrosomes when examined by the trypan blue and mice) do not tolerate concentrations higher than 3 % and Giemsa 20 % stain technique. Other authors [4] found 1.75 %, respectively, and the effect seems to be spe- that 50 % of llama spermatozoa had normal acrosomes cies-specific instead of taxon-specific. For these reasons, after using the Spermac stain method, with percentages alpaca and llama spermatozoa probably each require a of motility between 20 % and 30 %. specific concentration of cryoprotectant agent to obtain In relation to the extenders used, the best viability good post-thaw motility and viability rates, which are results after thawing were observed in the groups di- not necessarily the same optimal concentrations used for luted with extender II (skim milk, egg yolk and fructose), rams or bulls. despite the fact that most extenders utilized to freeze We utilized a final concentration of cryoprotectants of camelid semen have been prepared based on TRIS, citrate, 1.0 mol/L glycerol in groups I-G and II-G and 0.1 mol/L eth- glucose or egg yolk [4–6]. We found that motility and ylene glycol in groups I-EG and II-EG. Valdivia et al. [6] viability in groups frozen in an extender containing TRIS, using 1 mol/L glycerol, obtained a similar motility recov- citric acid, glucose and egg yolk (I-G and I-EG) were ery after thawing to that obtained in the present study. lower than 10 % post-thawing. Using 6 % glycerol (approximately 0.86 mol/L), Aller et Accordingly, Ratto et al. [15] reported better motil- al. [5] found 32 % viability in thawed llama spermatozoa. ity after conserving llama spermatozoa at 4 °C during 72 h On the other hand, using diluted semen with TRIS-ex- using Kenney extender (skim milk and glucose) and Colo- tender and EDTA freezing extender, von Baer and rado extender (skim milk and salts) in comparison with Hellemann [4] obtained motility rates ranging from 20 % TRIS- and egg yolk-based extender. Our results for to 30 % after thawing. It seems that South American sperm motility were similar after the cooling process, camelid spermatozoa cannot tolerate final cryoprotectant prior to freezing. Despite the fact that the percentages concentrations of 1 mol/L or more and that lower con- of motility were lower in group II-G (49.2 %) prior to centrations would have better cryoprotectant effects and/ freezing in comparison with groups I-G (56.6 %), I-EG or less toxicity; similar results have been indicated in this .307. sperm viability (%) Alpaca semen cryopreservation study but further research is required in this area. way for their critical and linguistic review. Alpaca spermatozoa diluted in extender II were simi- larly cryoprotected by ethylene glycol and glycerol. Eth- References ylene glycol has been successfully utilized as a cryopro- tectant agent, replacing glycerol in many species. This 1 Bravo PW, Flores U, Garnica J, Ordoñez C. Collection of is the first study to use ethylene glycol as a cryopro- semen and artificial insemination of alpacas. Theriogenology1997; 47: 619–26. tectant in camelid semen. Ethylene glycol has shown 2 Huanca W, Gauly M. Conservación de semen refrigerado de better cryoprotectant effects than glycerol in human [10] llamas. Rev Inv Vet Perú 2001; Suppl 1: 460–1. and bovine [11] spermatozoa, as it permeates sperm 3 Graham EF, Schmell MK, Eversen BK, Nelson DS. Semen membranes faster than glycerol [10]. In dogs, ethylene preservation in nondomestic mammals. Symp Zool Soc Lon- glycol can be used as a cryoprotectant agent at concen- don 1978; 43: 153–82. trations of 0.25, 0.5 and 1.0 mol/L as opposed to glyc- 4 Von Baer L, Hellemann C. Cryopreservation of llama (Lamaglama) semen. Reprod Domest Anim 1999; 34: 95–6. erol at 0.8 mol/L [17]. In addition, ethylene glycol could 5 Aller JF, Rebulfi GE, Cancino AK, Alberio RH. Influence of substitute glycerol as cryoprotectant agent if used at the cryopreservation on the motility, viability and fertility of llama same or lower concentrations to freeze horse semen [18]. (Lama glama). Arch Zootec 2003; 52: 15–23. Apparently, ethylene glycol fewer detrimental effects on 6 Valdivia M, Suyo M, Manosalva I, Ruiz M, Romero S, Olazabal J, the viability and motility of spermatozoa [19], providing et al. Cryopreservation and immunoreactivity of proacrosin/acrosinsystem in alpaca spermatozoa. Biol Reprod 2000; 62: 103. a better protective effect to the acrosome than glycerol. 7 Bravo PW, Ordoñez C, Alarcón V. Processing and freezing of However, in other species such as ovine, ethylene glycol semen and artificial insemination of alpacas and llamas. Pro- is not as good as glycerol [20] for semen cryopreservation, ceeding of the 13th International Congress on Animal but the best cryoprotectant effects of ethylene glycol Reproduction; 30 June–4 July 1996 Sydney, Australia. have been obtained using lower concentrations (3 %) than 8 Medeiros CM, Forell F, Oliveira AT, Rodrigues JL. Current glycerol (7 %) [20]. status of sperm cryopreservation: why isn’t it better? Therio-genology 2002; 57: 327–44. In conclusion, alpaca spermatozoa are highly suscep- 9 McGonagle LS, Goldstein M, Feldschuh J, Foote RH. The tible to cryopreservation. Under our working conditions, influence of cryoprotective media and processing procedures this study suggests that ethylene glycol at lower concen- on motility and migration of frozen-thawed human sperm. trations with skim milk, fructose-based extender may Asian J Androl 2002; 4: 137–41. have a better cryoprotectant effect than glycerol with 10 Gilmore JA, Liu J, Woods EJ, Peter AT, Critser JK. Cryopro-tective agent and temperature effects on human sperm mem- TRIS-citric acid-based extender on alpaca spermatozoa. brane permeabilities: convergences of theoretical and empiri- Because of the species-specific cryoprotectant effects, cal approaches for optimal cryopreservation methods. Hum an optimization of specific concentrations of cryopro- Reprod 2000; 15: 335–43. tectant agents for alpaca spermatozoa is necessary. 11 Guthrie HD, Liu J, Critser JK. Osmotic tolerance limits and Double stain with trypan blue and Giemsa 20 % turned effects of cryoprotectants on motility of bovine spermatozoa. out to be a valid technique for the concomitant evalua- Biol Reprod 2002; 67: 1811–6.12 Schill WB, Topfer-Petersen E, Heissler E. The sperm acrosome: tion of viability and acrosomal integrity in alpaca sper- functional and clinical aspects. Hum Reprod 1988; 3: 139–45. matozoa . Finally, our results in conjunction with those 13 Bravo PW, Skidmore JA, Zhao XX. Reproductive aspects of others indicate the need for continued research into and storage of semen in Camelidae. Anim Reprod Sci 2000; 62: semen cryopreservation in alpacas. 173–93. 14 Didion BA, Dabrinsky JR, Giles JR, Graves CN. Staining procedure to detect viability and true acrosome reaction in Acknowledgment spermatozoa of various species. Gamete Res 1989; 22: 51–7. 15 Ratto MH, Wolter M, Gómez C, Berland M. Refrigeration of This research was supported by the National Sci- epididymal sperm from lama with three different extenders. ence and Technology Council (CONCYTEC) of Perú, Proceeding of the II World Congress of South Americans Veterinary Institute for Tropical and Highland Research Camelids, 2-6 November 1999, Cusco, Perú. p. 79-80. (IVITA) and Dirección de Investigación de la Universidad 16 Holt WV. Fundamental aspects of sperm cryobiology: theimportance of species and individual differences. Theriogenology de La Frontera (Research Project DIUFRO 130204). We 2000; 53: 47–58. wish to thank Dr Marcelo Bertolini and Dr David Gallo- 17 Pereira SM, Rigon RM, Mezzalira A, Cecim M. Ethylene .308. Asian J Androl 2005; 7 (3): 303–309 glycol on canine semen cryopreservation. Ciencia Rural 2002; 19 Ball BA, Vo A. Osmotic tolerance of equine spermatozoa and 32: 649–55. the effects of soluble cryoprotectants on equine sperm motility, 18 Mantovani R, Rora A, Falomo ME, Bailoni L, Vicenti L. Com- viability and mitochondrial membrane potential. J Androl parison between glycerol and ethylene glycol for the cryo- 2001; 22: 1061–9. preservation of equine spermatozoa: semen quality assess- 20 Molinia FC, Evans G, Maxwell WM. Incorporation of pen- ment with standard analyses and with the hypoosmotic swell- etrating cryoprotectants in diluents for pellet-freezing ram ing test. Reprod Nutr Dev 2002; 42: 217–26. spermatozoa. Theriogenology 1994; 42: 849–58. .309.