Effect of sugar type on the survival of frozen-thawed rhesus monkey (Macaca mulatta) sperm.код для вставкиСкачать
American Journal of Primatology 68:103–108 (2006) BRIEF REPORT Effect of Sugar Type on the Survival of Frozen-Thawed Rhesus Monkey (Macaca mulatta) Sperm WEI SI1,2, HONG WANG1,2, CATHERINE REID3, THOMAS B. HILDEBRANDT3, and WEIZHI JI1,2n 1 Kunming Primate Research Center, the Chinese Academy of Sciences, Yunnan, China 2 Kunming Institute of Zoology, the Chinese Academy of Sciences, Yunnan, China 3 Institute for Zoo Biology and Wildlife Research, Berlin, Germany Sperm-freezing extenders supplemented with sugar or a combination of different sugars are widely used for the cryopreservation of nonhuman primate spermatozoa. Understanding which sugar or combination of sugars offers the highest level of cryoprotection would be beneficial for the development of sperm-freezing extenders. In the present study we aimed to investigate the effect of glucose, lactose, and raffinose separately or in combination on the cryosurvival of rhesus monkey spermatozoa. Toward that end, we prepared eight extenders by adding various types of sugars to a basic medium (BM): G-BM (0.3 M glucose), L-BM (0.3 M lactose), R-BM (0.3 M raffinose), LG-BM (0.15 M lactose+0.15 M glucose), RG-BM (0.15 M raffinose+0.15 M glucose), LR-BM (0.15 M lactose+0.15 M raffinose), and LRG-BM (0.1 M lactose+0.1 M raffinsoe+0.1 M glucose). A saline control (0.157 M sodium chloride) was also used. The results showed no significant difference in post-thaw motility when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRGBM, but the post-thaw motility was significantly lower when it was frozen with R-BM, LR-BM, and the saline control. The highest plasma membrane integrity was achieved when spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, and the highest acrosome integrity was achieved with G-BM, L-BM, LG-BM, RG-BM, LRG-BM, and the saline control. The results indicate that the various sugars offered different protective effects. For the cryopreservation of rhesus monkey spermatozoa, glucose (monosaccharide) and lactose (disaccharide) were shown to be more suitable than raffinose (trisaccharide) for preserving spermatozoal motility, plasma membrane, and acrosome. Specifically, Contract grant sponsor: Chinese Academy of Sciences; Contract grant number: KXCX1-05; Contract grant sponsor: Ministry of Science and Technology of China; Contract grant number: 2001DEA10009-09; Contract grant number: China National Science Foundation; Contract grant number: 30370166. n Correspondence to: Weizhi Ji, Kunming Institute of Zoology, Chinese Academy of Sciences, Jiaochang Donglu 32, Kunming, Yunnan 650223, China. E-mail: firstname.lastname@example.org Received 12 December 2004; revised 4 April 2005; revision accepted 7 April 2005 DOI 10.1002/ajp.20209 Published online in Wiley InterScience (www.interscience.wiley.com). r 2006 Wiley-Liss, Inc. 104 / Si et al. raffinose was detrimental to sperm acrosome integrity. Am. J. Primatol. r 2006 Wiley-Liss, Inc. 68:103–108, 2006. Key words: rhesus monkey; sperm; cryopreservation; sugar INTRODUCTION Supplementing semen-freezing extenders with sugar as a nonpenetrating cryoprotectant has demonstrated beneficial effects on the cryosurvival of spermatozoa. The protective effects of sugar have been reported to include cell dehydration, which reduces intracellular ice crystal formation [Chen et al., 1993], and the prevention of liposome fusion and leakage during dehydration and rehydration [Crowe et al., 1986]. Recently, it was found that the various types of sugars have different cryoprotective effects [Yildiz et al., 2000]. For the cryopreservation of nonhuman primate spermatozoa, the most extensively used sugars are glucose, lactose, and raffinose [Morrell & Hodges, 1998]. However, to our knowledge, no reports have been published on nonhuman primate spermatozoa cryopreservation comparing the effect of various types of sugars as cryoprotectants in one species, and the influence of these different sugars on nonhuman primate spermatozoa during freezing and thawing is not clear. Determining which sugar or combinations of sugars provide the highest postthaw parameters would be beneficial for the development of freezing extenders and the cryosurvival of spermatozoa. Thus, the present study was designed to examine the effect of adding glucose (monosaccharide), lactose (disaccharide), and raffinose (trisaccharide) separately or in combination on sperm motility and plasma membrane/acrosome integrity of rhesus monkey spermatozoa during freezing and thawing. Eight sperm-freezing extenders containing various types of sugars in different concentrations and combinations were tested. MATERIALS AND METHODS Sperm-Freezing Extender Preparation We prepared the freezing extenders by dissolving various types of sugars in different concentrations and combinations in a basic medium (BM), which was prepared by the addition of 10% (v/v) fresh egg yolk to Milli-Q water. The compositions of the extenders are listed in Table I. Table I. The Composition of the Sperm-Freezing Extendersn Concentration (M) Components Glucose Lactose Raffinose NaCl Egg yolk Glycerol G-BM L-BM R-BM LG-BM RG-BM LR-BM LRG-BM Saline control 0.3 – – – 10% 10% – 0.3 – – 10% 10% – – 0.3 – 10% 10% 0.15 0.15 – – 10% 10% 0.15 – 0.15 – 10% 10% – 0.15 0.15 – 10% 10% 0.1 0.1 0.1 – 10% 10% – – – 0.157 10% 10% n Note. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless indicated otherwise. The extenders were centrifuged at 7000g for 1 hr to separate egg yolk droplets. Before use, glycerol was added to the concentration of 10%. Am. J. Primatol. DOI 10.1002/ajp Effect of Sugar Type on Cryopreservation / 105 Semen Collection and Freezing Ten semen samples were collected by penile electro-ejaculation from four male rhesus macaques (8–10 years old) provided by the Kunming Primate Research Center, Chinese Academy of Sciences. To avoid disturbing sugar from the seminal plasma, the semen samples were washed with TALP-Hepes containing 3 mg/mL BSA by centrifugation at 150 g for 10 min. Each sample was divided into eight aliquots and diluted with nine volumes of the eight experimental freezing extenders without glycerol at room temperature. The extended samples were equilibrated at 41C for 2 hr, and further diluted with an equal volume of the same freezing extender variant containing 10% glycerol to reach a final concentration of 5% glycerol. The samples were loaded in 0.25-ml cryostraws (IMV, L’Aigle, France) and sealed. To freeze the specimens, the straws were held horizontally 5 cm above liquid nitrogen for 10 min and then submerged directly into the liquid nitrogen for storage [Sankai et al., 1994]. For thawing, the straws were plunged directly into a 371C water bath for 2 min. The spermatozoa sample was then diluted with 5 volumes of TALP-Hepes containing 3 mg/mL BSA, for 5 times at 30-sec intervals. Sperm Motility With the use of a prewarmed hemocytometer counting chamber, we assessed the fresh unwashed sperm samples and thawed sperm samples for percentage of progressive motility by counting 200 in duplicate. The sperm motility recovery rate was calculated by the following formula: (post-thaw motility % 100)/fresh sperm motility %. Assessment of Sperm Membrane and Acrosome Integrity Nuclei stain Hoechst 33258 and fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) was used to determine the integrity of the sperm plasma membrane and acrosome, as described by Cross et al.  and Esteves et al. . At least 200 spermatozoa were scored for each sample. Statistical Analysis All data were expressed as the means7SD. The percentage data of sperm motility and membrane/acrosome integrity were subjected to arcsine square root transformation before a statistical analysis was performed. An analysis of variance (ANOVA), followed by Tukey’s test, was used to analyze the differences among different groups. Values with Po0.05 were considered statistically different. RESULTS Sperm Motility After Freezing and Thawing As shown in Table II, similar post-thaw spermatozoa motility and motility recovery rates were found in G-BM, L-BM, LG-BM, RG-BM, and LRG-BM groups (P40.05). These rates were significantly higher than those of the R-BM, LR-BM, and saline control groups (Po0.05). The extender containing only raffinose (R-BM) and the saline control group provided similar cryoprotection for sperm motility (P40.05). This was significant lower than that obtained from the medium containing lactose and raffinose together (LR-BM) (Po0.05). Am. J. Primatol. DOI 10.1002/ajp 106 / Si et al. Table II. Motility of Rhesus Monkey Spermatoza After Freezing and Thawing With Various Types of Sugarsn Sperm-freezing extender Saline control G-BM L-BM R-BM LG-BM RG-BM LR-BM LRG-BM Fresh sperm motility (%) Post-thaw sperm motility (%) Motility recovery rate (%) 72.976.5 18.573.9a 41.274.6b 38.176.0b 16.073.1a 39.775.6b 38.173.6b 28.175.9c 42.378.1b 25.776.6a 55.878.5b 52.678.8b 22.174.6a 54.576.1b 52.574.9b 39.179.8c 58.4712.1b n Different superscripts in a column indicate significant difference (Po0.05). Table III. Plasma Membrane Integrity and Acrosoem Integrity of Rhesus Monkey Spermatozoa after Freezing and Thawing with various Types of Sugarsn Integrity (%) Sperm-freezing extender Fresh sperm Saline control G-BM L-BM R-BM LG-BM RG-BM LR-BM LRG-BM Plasma membrane Acrosome 77.475.3a 18.975.5b 48.772.4c 45.474.0c,d 23.776.7b 48.874.4c 45.374.8c,d 38.976.2d 47.275.4c 89.771.9a 76.474.1b 77.874.4b 78.173.2b 62.9710.6c 80.376.2b 77.378.9b 68.975.7c 77.574.5b n Different superscripts in a column indicate significant different (Po0.05). Sperm Plasma Membrane and Acrosome Integrity of Fresh and Cryopreserved Sperm The status of the plasma membrane and acrosome of the rhesus spermatozoa cryopreserved with various types of sugars is shown in Table III. Compared to fresh sperm, cryopreservation damaged the plasma membrane significantly (Po0.05). The plasma membrane integrity obtained with G-BM, L-BM, LG-BM, RG-BM, and LRG-BM was significantly higher than that obtained with R-BM and the saline control after cryopreservation (Po0.05). No statistical difference was found among LR-BM, L-BM, and RG-BM (P40.05). R-BM and the saline control provided the worst cryoprotection of the plasma membrane among the eight groups (Po0.05), but no difference was found between these two groups (P40.05). Compared to fresh sperm, the percentage of cryopreserved spermatozoa with intact acrosome decreased significantly (Po0.05). G-BM, L-BM, LG-BM, RG-BM, LRG-BM, and the saline control showed similar protection values for sperm acrosome (P40.05), but they were significantly higher than those obtained with R-BM and LR-BM (Po0.05). Am. J. Primatol. DOI 10.1002/ajp Effect of Sugar Type on Cryopreservation / 107 DISCUSSION Sugars have been widely used in animal spermatozoa cryopreservation. Previous studies indicated that monosaccharide is more effective than disaccharide for freezing ram sperm [Molinia et al., 1994], and trisaccharide is a less effective cryoprotective agent than monosaccharide for bull spermatozoa [Garcia & Graham, 1989]. In our study, the individual sugars glucose (G-BM), and lactose (L-BM) were more effective in protecting sperm motility and plasma membrane/ acrosome integrity compared to raffinose (R-BM) for rhesus monkey spermatozoa cryopreservation. No difference was found between monosaccharide and disaccharide for the rhesus monkey. However, both monosaccharides and disaccharides were more effective than trisaccharide. On the other hand, it has been reported that some types of sugars damage the spermatozoa acrosome during freezing and thawing [Yildiz et al., 2000]. In dogs, glucose, lactose, and raffinose increased the percentage of damaged acrosomes after freezing and thawing, but fructose, galactose, xylose, trehalose, malthose, and sucrose decreased the percentage of damaged acrosomes. However, in the rhesus monkey, of the three sugars examined (G-BM, L-BM, and R-BM), only raffinose (R-BM) increased the percentage of spermatozoa with damaged acrosome compared to the saline control (without sugar). The results indicate that the cryoprotective effects of various types of sugars may be species dependent. Sugars usually do not penetrate the sperm membrane, and they provide cryoprotection outside of the membrane by stabilizing cell membranes via the loose, electrostatic binding of saccharide hydroxyl groups to phosphate groups on the membrane lipid head [Chen et al., 1993]. In our study, compared to the saline control group (without sugar), the post-thaw sperm motility and plasma membrane integrity of spermatozoa cryopreserved by G-BM, L-BM, LG-BM, RGBM, and LRG-BM were significant higher. However, no statistical difference in sperm acrosome integrity was found between these groups and the saline control. The results indicate that during freezing and thawing, the motility and plasma membrane of rhesus monkey spermatozoa was mostly damaged when extender without sugar was used, and compared to sodium chloride, the sugars provided effective protection outside of the membrane to the sperm plasma membrane. It has been suggested that disaccharide is more effective than monosaccharide because the former has a lower carbon : hydrogen (C:H) ratio and a greater number of hydroxyl groups per molecule [Bakas & Disalvo, 1991]. However, our results revealed no significant difference between glucose and lactose (G-BM vs. L-BM). The discrepancy may due to the fact that in addition to the effect of stabilizing the cell membrane, sugars may also affect the pattern of crystallization, the shape and width of the channels in the unfrozen solution, and the mechanical properties of the solidified medium, which could relieve or prevent damage to spermatozoa [Nicolajsen & Hvidt, 1994]. The sugar-type-dependent properties and the different mechanisms of cryoprotection could contribute to the different cryoprotective properties of various types of sugars. Because of the different cryoprotective mechanisms found in various types of sugars, it may be hypothesized that the combined use of monosaccharide and disaccharide in proper concentrations could provide better protection than that achieved with the use of monosaccharide or disaccharide alone [Yildiz et al., 2000]. In the present study, the results indicate that the combined use of monosaccharide, disaccharide, and trisaccharide could provide better protection compared to the use of trisaccharide alone (LG-BM, RG-BM, LRG-BM, and Am. J. Primatol. DOI 10.1002/ajp 108 / Si et al. LR-BM vs. R-BM), and in a low concentration (0.15 M), glucose combined with raffinose could provide better cryoprotection than lactose combined with raffinose (RG-BM vs. LR-BM). In conclusion, this study shows that glucose and lactose have better protective effects than raffinose for preserving sperm motility, plasma membrane function, and acrosome integrity. Raffinose was detrimental to the acrosome integrity of rhesus monkey spermatozoa. These results can help researchers design more effective sperm-freezing extenders and improve the cryopreservation of nonhuman primate spermatozoa. REFERENCES Bakas LS, Disalvo EA. 1991. Effect of Ca2+ on the cryoprotective action of trehalose. Cryobiology 28:347–353. Chen Y, Foote RH, Brockett CC. 1993. Effect of sucrose, trehalose, hypotaurine, taurine, and blood serum on survival of frozen bull sperm. Cryobiology 30:423–431. Cross NL, Morales P, Overstreet JW, Hanson FW. 1986. Two simple methods for detecting acrosome reacted human sperm. Gamete Res 15:213–226. Crowe LM, Crowe JH, Womersley C, Reid D, Appel L, Rudolph A. 1986. Prevention of fusion and leakage in freeze-dried liposomes by carbohydrates. Biochim Biophys Acta 861:131–140. Esteves SC, Sharma RK, Thomas Jr AJ, Agarwal A. 2000. Improvement in motion characteristics and acrosome status in cryopreserved human spermatozoa by swim-up processing before freezing. Hum Reprod 15:2173–2179. Garcia MA, Graham EF. 1989. Development of a buffer system for dialysis of bovine Am. J. Primatol. DOI 10.1002/ajp spermatozoa before freezing. II. Effects of sugars and sugar alcohols on post thaw motility. Theriogenology 31:1029–1037. Molinia FC, Evans G, Maxwell WM. 1994. In vitro evaluation of zwitterion buffers in diluents for freezing ram spermatozoa. Reprod Nutr Dev 34:491–500. Morrell JM, Hodges JK. 1998. Cryopreservation of non-human primate sperm: priorities for future research. Anim Reprod Sci 53:43–63. Nicolajsen H, Hvidt A. 1994. Phase behavior of the system trehalose-NaCl-water. Cryobiology 31:199–205. Sankai T, Terao K, Yanagimachi R, Cho F, Yoshikawa Y. 1994. Cryopreservation of spermatozoa from cynomolgus monkeys (Macaca fascicularis). J Reprod Fertil 101:273–278. Yildiz C, Kaya A, Aksoy M, Tekeli T. 2000. Influence of sugar supplementation of the extender on motility, viability and acrosomal integrity of dog spermatozoa during freezing. Theriogenology 54:579–585.