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Motility and fertility of alginate encapsulated boar spermatozoa
Animal Reproduction Science 87 (2005) 111–120
Motility and fertility of alginate encapsulated boar spermatozoa San-Yuan Huanga , Ching-Fu Tua , Sieh-Hua Liub , You-Hai Kuoc,∗ a
Division of Biotechnology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan, Miaoli 35099, Taiwan, ROC b Department of Bioinformatics, Chung-Hua University, 30 Tung Shiang, Hsin Chu 30067, Taiwan, ROC c Division of Applied Biology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan, Miaoli 35099, Taiwan, ROC Received 30 March 2004; received in revised form 20 August 2004; accepted 17 September 2004
Abstract Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 ◦ C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16–18 ◦ C, and then were stored at 5 ◦ C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 ◦ C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24 h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 ◦ C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8 h incubation at
∗
Corresponding author. Tel.: +886 37 585936; fax: +886 37 585969. E-mail address: [email protected] (Y.-H. Kuo).
0378-4320/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2004.09.005
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37 ◦ C after storing for over three days (P < 0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability. © 2004 Elsevier B.V. All rights reserved. Keywords: Sperm releasing rate; Sperm motility; Fertility; Microencapsulation; Boar spermatozoa
1. Introduction Artificial insemination (AI) is conventionally adopted in pig reproduction. The percentage of sows using AI is steadily increasing in most major pig producing countries, and is predicted to increase from 48% in 1998 to 70% in 2005 (Weitze, 2000). This increase indicates excellent development potential for AI in this industry. Besides enhancing the AI and semen quality control techniques, the efficiency of utilizing semen from boars with superior genetic traits is another dimension of AI application. According to a Taiwanese survey, 25% of qualified semen was discarded (Chiang et al., 1997). To fully utilize semen from excellent boars, techniques for extending the storage time of left-over semen must be developed. Microencapsulation is the process of enclosing cells, tissues, or substances within a semipermeable membrane (Lim, 1984; Wheatley et al., 1985). Several types of cells and tissues have been successfully encapsulated, including hepatocytes, islet of Langerhans, tumor cells, adrenal cortical cells and sperm cells (Lim, 1984; Lim and Sun, 1980; Lim and Moss, 1981; Nebel et al., 1985; Esbenshade and Nebel, 1990). The encapsulated cells remain viable since the semipermeable membrane permits the exchange of nutrients and metabolites. Esbenshade and Nebel (1990) proposed that spermatozoa encapsulation may enhance the potential uses of AI by reducing the number of sperm required for each breeding. The potential role for microencapsulated semen in AI is to maintain spermatozoa viability while reducing susceptibility to retrograde action of the uterus and phagocytosis by leukocytes, and allowing their gradual release (Nebel et al., 1996). Few works have reported on microencapsulation of boar spermatozoa (Esbenshade and Nebel, 1990). This early result indicated that the motility of encapsulated sperm was similar to that of non-encapsulated (free) controls when examined immediately after encapsulation. However, the method adapted from the protocols used for bovine sperm encapsulation appears unsuitable for encapsulation of boar spermatozoa, and the encapsulated porcine sperm survived for no more than 16 h following encapsulation (Nebel and Saacke, 1996). A recent Italian study successfully encapsulated porcine spermatozoa (Torre et al., 1998), but in vivo fertility requires further evaluation. This study evaluates whether the alginate microencapsulization process can extend the longevity of boar spermatozoa stored at 5 ◦ C and further assesses the fertility of microencapsulated spermatozoa in vivo.
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2. Materials and methods 2.1. Experimental animals, semen collection and examination Ejaculated semen from sexually mature boars was collected by the gloved-hand technique (Hancock and Hovell, 1959), after which the sperm concentration was determined via the haemacytometer method. Sperm motility was visually assessed using a phase contrast microscope equipped with a 37 ◦ C heating plate (Huang et al., 2000) by the same welltrained technician to reduce variation. Briefly, fresh semen was dropped onto a pre-warmed (37 ◦ C) slide, on which a coverslip was overlaid. The motility was examined at 100–200× magnification, as described by Niwa (1961). Only ejaculate samples with sperm motility higher than 70% were further used for microencapsulation. Seven pure bred Landrace sows were used for inseminating encapsulated and nonencapsulated semen under natural estrus status. 2.2. Preparation of the concentrated semen The sperm-rich fraction of the semen was diluted with an equal volume of Modena extender (Swine Genetics International, Cambridge, IA, USA). The diluted semen was stored at 17 ◦ C for 4 h, then centrifuged at 15 ◦ C with 800 × g for 10 min. The supernatants were discarded and the sperm fraction was diluted with an equal volume of extender containing 20% egg yolk. The concentrated semen was used for microencapsulation. 2.3. Microencapsulation of porcine spermatozoa Sperm microcapsule was prepared as described in a previous report (Torre et al., 1998) with some modifications. Briefly, the concentrated semen was mixed with an equal volume of 15% alginic acid (Sigma Chemical Co., St. Louis, MO, USA). The sperm suspension was then forced through a 21-gauge hypodermic needle using a peristaltic pump (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) into a solution containing 100 mM CaCl2 and 50 nM HEPES. The microencapsulated sperm were kept in the same solution for 15 min, and then were rinsed three times with 0.9% NaCl. The microcapsules were transferred into 0.1% poly-l-lysine (Sigma), stirred for 15 min and then rinsed three times with 0.9% NaCl. The prepared microcapsules were stored at 5 ◦ C in Semengra extender (China Chemical & Pharmaceutical Co., Ltd., Taipei, Taiwan). 2.4. Evaluation of sperm releasing rate of microencapsulated semen The sperm release rate from the capsule was assessed by an in vitro release test (Torre et al., 2000). Following storage for 1, 3 and 7 days, the microcapsules were taken out and incubated at 37 ◦ C with or without 110 rpm stirring. The sperm release rate was determined by calculating the number of sperm in the medium at 2, 4, 6, 8 and 24 h after incubation. The sperm number was calculated using the haemacytometer method developed by Herrick and Self (1962).
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2.5. Evaluation of motility of microencapsulated sperm For evaluating the motility of microencapsulated sperm, the capsules were removed after 1–9 days of storage. The microcapsules were dissolved by repeat pipetting before sperm motility assessment. The motility was evaluated after 10 min, 8, 24, 32, 48, 56 and 72 h under 37 ◦ C incubation, as described above. The motility of the concentrated (free) semen was also evaluated. 2.6. Assessment of the fertility of microencapsulated semen Since the motility of the microencapsulated and non-encapsulated semen differed after 3 days of storage, the semen were further used to evaluate fertility after 4 days of storage. To evaluate the fertility of microencapsulated sperm, the microcapsules or free semen containing approximately 5 × 109 sperms were used for insemination. Both semen types were initially inseminated to sows at 24–28 h following the detection of standing heat. The second insemination was performed 8–12 h after the first one. Finally, the conception rate, farrowing rate and litter size were recorded. 2.7. Statistical analysis The percentage sperm release, and the effects of treatments on sperm motility and litter size, were analyzed using the ANOVA procedure of SAS (SAS, 1989). The significance of differences (P < 0.05) among treatments was determined by the Duncan multiple range test.
3. Results 3.1. Releasing of the microencapsulated boar spermatozoa To evaluate the release of alginate microencapsulated spermatozoa, the sperm-rich fraction was microencapsulated and stored at 5 ◦ C. Following storage for 1, 3 and 7 days, the microcapsule was removed to assess sperm release. Table 1 lists the measurement results for sperm release kinetics. The results indicated whether, with or without stirring, the sperm release increased with the 37 ◦ C incubation time. Significant differences in sperm release existed after 2 h incubation on days 3 and 7. Although the difference was not significant, the sperm release of microcapsules in the stirring group following 24 h incubation was higher than for microcapsules in the non-stirring group (81 versus 60%). 3.2. Motility of the microencapsulated boar spermatozoa To examine whether alginate microencapsulization process can extend the longevity of boar semen, the concentrated and microencapsulated semen were removed every day to assess sperm motility. Table 2 lists the measurement results for sperm motility. Little difference in motility existed between the free and encapsulated semen after 10 min incubation at 37 ◦ C on every sampling day. The motility of encapsulated spermatozoa was significantly
Days of storage
Incubation without stirring (h) 2
4
Incubation with 110 rpm stirring 6
8
24
2
4
1 20.4 ± 6.9 40.9 ± 8.9 46.1 ± 10.1 48.3 ± 13.2 49.5 ± 13.5 15.2 ± 5.7 51.3 ± 4.3 3 40.8 ± 5.1a 57.7 ± 10.6 61.9 ± 10.2 67.2 ± 10.7 71.4 ± 10.0 11.9 ± 4.8b 39.1 ± 8.5 7 28.0 ± 3.3a 49.5 ± 8.4 53.0 ± 10.1 56.5 ± 9.4 60.9 ± 8.8 9.4 ± 1.7b 26.8 ± 4.6 a, b Sperm release differ significantly among treatments given the same storage and incubation time (P < 0.05).
6
8
24
59.2 ± 2.7 54.2 ± 8.5 47.6 ± 3.5
67.3 ± 4.8 62.4 ± 9.0 52.1 ± 5.5
83.5 ± 7.1 83.3 ± 12.9 76.5 ± 7.5
S.-Y. Huang et al. / Animal Reproduction Science 87 (2005) 111–120
Table 1 The spermatozoa release (%) of encapsulated semen cultured at 37 ◦ C after storage at 5 ◦ C for different periods (n = 3)
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Storage time (Day)
Treatment of semen
Duration of 37 ◦ C incubation 10 min
8h
24 h
Encapsulated 72.9 ± 5.2 60.0 ± 7.4 51.4 ± Free 82.1 ± 2.4 56.4 ± 5.2 37.1 ± 2 Encapsulated 67.1 ± 3.1b 57.9 ± 3.8 35.7 ± Free 76.4 ± 2.1a 47.1 ± 5.8 32.9 ± 3 Encapsulated 70.0 ± 6.9 55.7 ± 6.8 45.0 ± Free 78.6 ± 3.4 37.9 ± 6.4 25.0 ± 4 Encapsulated 70.0 ± 6.8 51.4 ± 6.5a 37.9 ± Free 72.1 ± 3.4 30.0 ± 4.4b 15.7 ± 5 Encapsulated 61.4 ± 6.1 45.7 ± 5.6a 34.3 ± Free 68.6 ± 2.6 23.6 ± 3.9b 14.7 ± 6 Encapsulated 60.0 ± 4.1 44.3 ± 4.4a 33.6 ± Concentrate 67.1 ± 1.8 21.4 ± 3.6b 10.9 ± 7 Encapsulated 55.7 ± 4.4 47.9 ± 4.6a 44.3 ± Free 62.1 ± 3.2 14.3 ± 3.2b 9.3 ± 8 Encapsulated 55.7 ± 6.8 48.6 ± 7.1a 42.9 ± Free 55.0 ± 4.8 14.4 ± 4.7b 9.4 ± 9 Encapsulated 51.4 ± 6.3 49.3 ± 7.0a 33.9 ± Free 48.6 ± 3.7 13.6 ± 3.7b 5.6 ± a,b Motility differ significantly among treatments given the same storage time (P < 0.05). 1
32 h 8.7 5.8 6.3 6.3 5.6a 6.2b 4.7a 4.7b 4.4a 4.6b 4.7a 3.9b 6.5a 3.6b 7.5a 3.5b 7.2a 2.1b
45.0 30.0 32.9 20.7 30.0 16.1 25.0 10.0 24.3 10.9 27.9 5.9 39.3 8.1 37.9 5.4 22.1 3.0
48 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
9.3 6.1 5.2 3.5 3.6a 3.4b 3.8a 3.6b 5.5a 2.7b 4.6a 2.2b 6.9a 4.0b 7.3a 2.8b 4.1a 1.3b
20.1 17.1 26.6 13.6 18.0 9.0 13.6 1.9 17.1 3.0 23.6 5.4 27.9 3.3 21.1 2.1 11.0 2.1
56 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.3 4.3 5.3 3.0 5.1 2.1 4.8a 0.9b 3.4a 1.3b 6.3a 2.8b 7.3a 2.1b 6.6a 1.5b 2.9a 0.8b
12.9 10.0 19.3 2.9 9.1 4.3 8.6 1.4 6.0 1.0 20.4 2.3 16.4 2.1 7.6 1.1 7.6 1.0
72 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.0 3.6 5.1a 1.5b 3.9 1.2 3.6 0.7 2.2 0.7 6.2a 1.4b 6.5 1.5 2.9 0.8 2.5a 0.7b
5.9 3.0 7.9 0.9 6.4 1.3 7.1 0.7 3.9 0.3 11.6 0.9 7.6 1.1 2.3 0.7 2.6 0.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.9 1.4 3.2 0.7 4.5 0.7 3.4 0.5 1.9 0.3 4.8a 0.7b 4.2 1.1 1.3 0.7 1.0a 0.1b
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Table 2 Sperm motility (%) of microencapsulated and free semen cultured at 37 ◦ C after stored at 5 ◦ C for different duration (n = 5)
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Table 3 Reproductive performance of the sows inseminated with microencapsulated or free porcine semen Treatment
No. of sows inseminated (heads)
Conception rate (%)
Farrowing rate (%)
Average litter size (heads)
Encapsulated Free
4 3
75 100
75 100
12.3 ± 1.7 12.0 ± 0.6
higher than that of free semen after 8 h incubation at 37 ◦ C following storage for more than 3 days (P < 0.05). This effect continued until 48 h of incubation. When the 37 ◦ C incubation time exceeds 48 h, sperm motility decreased to a low level and the difference became non-significant. 3.3. Fertility of the microencapsulated boar spermatozoa For further evaluating the fertility of microencapsulated sperm, the concentrated and microencapsulated semen containing 5 × 109 sperm were inseminated to sows with natural estrus after 4 days of storage. The reproductive performance of the sows after insemination was shown in Table 3. No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and concentrated free semen.
4. Discussion Semen left-over is a common problem in the AI center practice. Microencapsulation of semen has been proposed as a method of choice for prolonged storage or sustained release of sperm within the female reproductive tract (Nebel and Saacke, 1996). However, this method of sperm storage appears more successful in bovines than that in pigs. This study evaluated the effect of alginate microencapsulization on extending the longevity of boar spermatozoa and on the fertility of microencapsulated spermatozoa in vivo. The results suggested that microencapsulation could prolong the storage time of boar spermatozoa and also sustain the fertility of microencapsulated sperm under common AI practice. The microencapsulation of bovine spermatozoa with calcium alginate has been successful and can achieve prolonged release (Nebel et al., 1985, 1993; Nebel and Saacke, 1994, 1996). However, the method used in bovines is not suitable for encapsulation of boar spermatozoa, and viability cannot be maintained for more than 16 h after encapsulation using this method (Nebel and Saacke, 1996). Consequently, a new encapsulation method specifically for swine has been developed (Torre et al., 1998, 2000). Torre et al. (2000) has assessed the release profile of microencapsulated spermatozoa under stirring. Torre et al. (2000) found that 0.12 M calcium concentration released approximately 40% cells in 6 h, and the lower concentration (0.07 M) released almost 100% cells at the same time. Using barium alginate and protamine–barium alginate for microencapsulation, Torre et al. (2002) found that encapsulation enhances semen preservation, providing protection against membrane damage on dilution. The in vitro release test of spermatozoa revealed massive cell delivery from barium alginate microcapsules within 6 h, and slow
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release from protamine–barium alginate capsules. This study evaluated the release of spermatozoa of capsules prepared with 0.1 M calcium under both static and stirring status. The result suggested that stirring can enhance sperm release (Table 1). However, the release rate was lower than reported in Torre et al. (2000). This inconsistency may result from the different equipment used for stirring the capsules and the method used for assessing the release. Previous studies have demonstrated that sodium alginate-poly-l-lysine cause minimal injury to the microencapsulated spermatozoa (Esbenshade and Nebel, 1990; Nebel et al., 1993, 1996). Nebel et al. (1996) showed that microcapsule wall thickness did not influence sperm motility, and no apparent cellular injury was noted following encapsulation. In boar spermatozoa, Torre et al. (2000) reported that sperm motility reduced to 35–40% at 4 h after semen microencapsulation. This work found that encapsulated spermatozoa motility continued to exceed 40% at 8 h incubation at 37 ◦ C, even after 9 days of storage (Table 2). Recently, barium alginate and protamine alginate were used to encapsulate swine semen by Vigo et al. (2002). Morphological and functional characteristics (acrosome integrity and motility) were not found to differ between the free and encapsulated semen. Faustini et al. (2004) further reported that encapsulated boar spermatozoa had significantly higher acrosome integrity and generally higher in situ enzymatic activity than those of unencapsulated spermatozoa stored for 72 h at 18 ◦ C. The observations in this study demonstrated that the motility of encapsulated spermatozoa was significantly higher than that of free concentrated semen following 48 h of incubation at 37 ◦ C after more than 3 days of storage (Table 2). The results indicated that the microencapsulated spermatozoa could maintain their motility at 37 ◦ C for longer than the non-encapsulated spermatozoa, thus enabling boar semen storage time to be extended. The result also suggested that microencapsulation can extend the survival of boar spermatozoa following insemination. Nebel et al. (1996) found that pregnancy rates were the same for heifers inseminated with semen either unencapsulated or microencapsulated with microcapsules of various wall thicknesses. The results presented by Nebel et al. showed that extended bovine spermatozoa can survive in the female reproductive tract at least 40 h prior to fertilization. Vigo et al. (2002) evaluated the in vitro fertilization potency of barium alginate and protamine alginate encapsulated swine semen. The results presented by Vigo et al. suggested that the technological encapsulation process did not compromise in vitro fertilization potency of the spermatozoa. Though this study contains a limited sample size, sows inseminated with non-encapsulated and microencapsulated semen after 4 days of storage displayed similar reproductive performances (Table 3). This preliminary finding indicated that encapsulated boar spermatozoa may maintain their ability to fertilize the ova in the sow reproductive tract. Vishwanath et al. (1997) indicated that the fertility of heifers with encapsulated semen increased with reducing interval between insemination and estrus. Consequently, the time window in which insemination could be conducted without reducing fertility was smaller with the encapsulated semen than with the control semen. Whether the same reproductive performance could be reached by inseminating the contact microcapsule into sows and also the optimal insemination time remained to be evaluated. This investigation found that some of the microcapsules ruptured during the insemination, and thus designing a suitable device for inseminating microcapsules into sows will be another issue that must be resolved for microencapsulation application.
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In conclusion, the findings of this study further confirmed that microencapsulation could prolong the storage time of boar spermatozoa and might also sustain the fertility of microencamsulated spermatozoa after 4 days of storage. However, due to limited number of sows inseminated, whether the microencapsulization could prolong spermatozoal retention time in the reproductive tract of sows and also fertilization ability requires further elucidation.
Acknowledgements The authors would like to thank the Council of Agriculture Executive Yuan, Republic of China, for financially supporting this research under Contract No. 88AST-1.5-AID-08. The ATIT transgenic team is also appreciated for its technical assistance related to artificial insemination using both microencapsulated and concentrated semen. Technical support from Dr. C.W. Liao and Miss E.C. Huang is also appreciated.
References Chiang, J.C., Ho, R.J., Kuo, Y.H., Lee, K.H., 1997. 1996 Annual Report of Fresh Boar Semen Extension and Catalog of Superior Boars, Pig Research Institute, Taiwan. Esbenshade, K.L., Nebel, R.L., 1990. Encapsulation of porcine spermatozoa in poly-l-lysine microsphere. Theriogenology 33, 499–508. Faustini, M., Torre, M.L., Stacchezzini, S., Norberti, R., Consiglio, A.L., Porcelli, F., Conte, U., Munari, E., Russo, V., Vigo, D., 2004. Boar spermatozoa encapsulated in barium alginate membranes: a microdensitometric evaluation of some enzymatic activities during storage at 18 ◦ C. Theriogenology 61, 173–184. Hancock, J.L., Hovell, G.L.R., 1959. The collection of boar semen. Vet. Res. 71, 664–669. Herrick, J.L., Self, H.L., 1962. Evaluation of Fertility in the Bull and Boar. University Press, Iowa, Iowa State. Huang, S.Y., Kuo, Y.H., Lee, Y.P., Tsou, H.L., Lin, E.C., Ju, C.C., Lee, W.C., 2000. Association of heat shock protein 70 with semen quality in boars. Anim. Reprod. Sci. 63, 231–240. Lim, F., Sun, A.M., 1980. Microencapsulated islets as bioartificial endocrine pancreas. Science 210, 908–910. Lim, F., Moss, R.D., 1981. Microencapsulation of living cells and tissue. J. Pharm. Sci. 70, 351–354. Lim, F., 1984. Microencapsulation of living cells and tissues—theory and practice. In: Lim, F. (Ed.), Biomedical Applications of Microencapsulation. CRC Press, Boca Ration, FL, pp. 137–154. Nebel, R.L., Saacke, R.G., 1994. Technology and applications for encapsulated spermatozoa. Biotechnol. Adv. 12, 41–48. Nebel, R.L., Saacke, R.G., 1996. Spermatozoa microencapsulation and capsule behavior in female tract. Reprod. Dom. Anim. 31, 75–85. Nebel, R.L., Bame, J.H., Saacke, R.G., Lim, F., 1985. Microencapsulation of bovine spermatozoa. J. Anim. Sci. 60, 1631–1639. Nebel, R.L., Vishwanath, R., McMillan, W.H., Saacke, R.G., 1993. Microencapsulation of bovine spermatozoa for use in artificial insemination: a review. Reprod. Fertil. Dev. 5, 701–712. Nebel, R.L., Vishwanath, R., McMillan, W.H., Pitt, C.J., 1996. Microencapsulation of bovine spermatozoa: effect of capsule membrane thickness on spermatozoal viability and fertility. Anim. Reprod. Sci. 44, 79–90. Niwa, T., 1961. Researches and practices in artificial insemination of pig. Proc. 4th Int. Congr. Anim. Reprod. 1, 83–88. SAS Institute, 1989. SAS User’s Guide: Statistics, Release 6.03 SAS Institute Inc., Cary, NC. Torre, M.L., Maggi, L., Giunchedi, P., Conte, U., Maffeo, G., 1998. Calcium alginate beads containing a hydrophilic polymer for the encapsulation of swine spermatozoa. S.T.P. Pharm. Sci. 8, 233–236. Torre, M.L., Maggi, L., Vigo, D., Galli, A., Bornaghi, V., Maffeo, G., Conte, U., 2000. Controlled release of swine semen encapsulated in calcium alginate beads. Biomaterials 21, 1493–1498.
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Torre, M.L., Faustini, M., Norberti, R., Stacchezzini, S., Maggi, L., Maffeo, G., Conte, U., Vigo, D., 2002. Boar semen controlled delivery system: storage and in vitro spermatozoa release. J. Control Rel. 85, 83–89. Vigo, D., Faustini, M., Torre, M.L., Pecile, A., Villani, S., Asti, A., Norbert, R., Maggi, L., Conte, U., Cremonesi, F., Stacchezzini, S., Maffeo, G., 2002. Boar semen controlled-delivery system: morphological investigation and in vitro fertilization test. Reprod. Fertil. Dev. 14, 307–314. Vishwanath, R., Nebel, R.L., McMillan, W.H., Pitt, C.J., Macmillian, K.L., 1997. Selected time of insemination with microencapsulated bovine spermatozoa affect pregnancy rates of synchronized heifers. Theriogenology 48, 369–376. Wheatley, M.A., Eisen, H., Langer, R., 1985. Alginate/ poly-l-lysine microcapsules as a vehicle for controlled release of macromolecules. Proc. Int. Symp. Contr. Rel. Bioreact. Mater. 12, 245–246.
Motility and fertility of alginate encapsulated boar spermatozoa San-Yuan Huanga , Ching-Fu Tua , Sieh-Hua Liub , You-Hai Kuoc,∗ a
Division of Biotechnology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan, Miaoli 35099, Taiwan, ROC b Department of Bioinformatics, Chung-Hua University, 30 Tung Shiang, Hsin Chu 30067, Taiwan, ROC c Division of Applied Biology, Animal Technology Institute Taiwan, P.O. Box 23, Chunan, Miaoli 35099, Taiwan, ROC Received 30 March 2004; received in revised form 20 August 2004; accepted 17 September 2004
Abstract Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 ◦ C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16–18 ◦ C, and then were stored at 5 ◦ C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 ◦ C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24 h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 ◦ C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8 h incubation at
∗
Corresponding author. Tel.: +886 37 585936; fax: +886 37 585969. E-mail address: [email protected] (Y.-H. Kuo).
0378-4320/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2004.09.005
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37 ◦ C after storing for over three days (P < 0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability. © 2004 Elsevier B.V. All rights reserved. Keywords: Sperm releasing rate; Sperm motility; Fertility; Microencapsulation; Boar spermatozoa
1. Introduction Artificial insemination (AI) is conventionally adopted in pig reproduction. The percentage of sows using AI is steadily increasing in most major pig producing countries, and is predicted to increase from 48% in 1998 to 70% in 2005 (Weitze, 2000). This increase indicates excellent development potential for AI in this industry. Besides enhancing the AI and semen quality control techniques, the efficiency of utilizing semen from boars with superior genetic traits is another dimension of AI application. According to a Taiwanese survey, 25% of qualified semen was discarded (Chiang et al., 1997). To fully utilize semen from excellent boars, techniques for extending the storage time of left-over semen must be developed. Microencapsulation is the process of enclosing cells, tissues, or substances within a semipermeable membrane (Lim, 1984; Wheatley et al., 1985). Several types of cells and tissues have been successfully encapsulated, including hepatocytes, islet of Langerhans, tumor cells, adrenal cortical cells and sperm cells (Lim, 1984; Lim and Sun, 1980; Lim and Moss, 1981; Nebel et al., 1985; Esbenshade and Nebel, 1990). The encapsulated cells remain viable since the semipermeable membrane permits the exchange of nutrients and metabolites. Esbenshade and Nebel (1990) proposed that spermatozoa encapsulation may enhance the potential uses of AI by reducing the number of sperm required for each breeding. The potential role for microencapsulated semen in AI is to maintain spermatozoa viability while reducing susceptibility to retrograde action of the uterus and phagocytosis by leukocytes, and allowing their gradual release (Nebel et al., 1996). Few works have reported on microencapsulation of boar spermatozoa (Esbenshade and Nebel, 1990). This early result indicated that the motility of encapsulated sperm was similar to that of non-encapsulated (free) controls when examined immediately after encapsulation. However, the method adapted from the protocols used for bovine sperm encapsulation appears unsuitable for encapsulation of boar spermatozoa, and the encapsulated porcine sperm survived for no more than 16 h following encapsulation (Nebel and Saacke, 1996). A recent Italian study successfully encapsulated porcine spermatozoa (Torre et al., 1998), but in vivo fertility requires further evaluation. This study evaluates whether the alginate microencapsulization process can extend the longevity of boar spermatozoa stored at 5 ◦ C and further assesses the fertility of microencapsulated spermatozoa in vivo.
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2. Materials and methods 2.1. Experimental animals, semen collection and examination Ejaculated semen from sexually mature boars was collected by the gloved-hand technique (Hancock and Hovell, 1959), after which the sperm concentration was determined via the haemacytometer method. Sperm motility was visually assessed using a phase contrast microscope equipped with a 37 ◦ C heating plate (Huang et al., 2000) by the same welltrained technician to reduce variation. Briefly, fresh semen was dropped onto a pre-warmed (37 ◦ C) slide, on which a coverslip was overlaid. The motility was examined at 100–200× magnification, as described by Niwa (1961). Only ejaculate samples with sperm motility higher than 70% were further used for microencapsulation. Seven pure bred Landrace sows were used for inseminating encapsulated and nonencapsulated semen under natural estrus status. 2.2. Preparation of the concentrated semen The sperm-rich fraction of the semen was diluted with an equal volume of Modena extender (Swine Genetics International, Cambridge, IA, USA). The diluted semen was stored at 17 ◦ C for 4 h, then centrifuged at 15 ◦ C with 800 × g for 10 min. The supernatants were discarded and the sperm fraction was diluted with an equal volume of extender containing 20% egg yolk. The concentrated semen was used for microencapsulation. 2.3. Microencapsulation of porcine spermatozoa Sperm microcapsule was prepared as described in a previous report (Torre et al., 1998) with some modifications. Briefly, the concentrated semen was mixed with an equal volume of 15% alginic acid (Sigma Chemical Co., St. Louis, MO, USA). The sperm suspension was then forced through a 21-gauge hypodermic needle using a peristaltic pump (Tokyo Rikakikai Co., Ltd., Tokyo, Japan) into a solution containing 100 mM CaCl2 and 50 nM HEPES. The microencapsulated sperm were kept in the same solution for 15 min, and then were rinsed three times with 0.9% NaCl. The microcapsules were transferred into 0.1% poly-l-lysine (Sigma), stirred for 15 min and then rinsed three times with 0.9% NaCl. The prepared microcapsules were stored at 5 ◦ C in Semengra extender (China Chemical & Pharmaceutical Co., Ltd., Taipei, Taiwan). 2.4. Evaluation of sperm releasing rate of microencapsulated semen The sperm release rate from the capsule was assessed by an in vitro release test (Torre et al., 2000). Following storage for 1, 3 and 7 days, the microcapsules were taken out and incubated at 37 ◦ C with or without 110 rpm stirring. The sperm release rate was determined by calculating the number of sperm in the medium at 2, 4, 6, 8 and 24 h after incubation. The sperm number was calculated using the haemacytometer method developed by Herrick and Self (1962).
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2.5. Evaluation of motility of microencapsulated sperm For evaluating the motility of microencapsulated sperm, the capsules were removed after 1–9 days of storage. The microcapsules were dissolved by repeat pipetting before sperm motility assessment. The motility was evaluated after 10 min, 8, 24, 32, 48, 56 and 72 h under 37 ◦ C incubation, as described above. The motility of the concentrated (free) semen was also evaluated. 2.6. Assessment of the fertility of microencapsulated semen Since the motility of the microencapsulated and non-encapsulated semen differed after 3 days of storage, the semen were further used to evaluate fertility after 4 days of storage. To evaluate the fertility of microencapsulated sperm, the microcapsules or free semen containing approximately 5 × 109 sperms were used for insemination. Both semen types were initially inseminated to sows at 24–28 h following the detection of standing heat. The second insemination was performed 8–12 h after the first one. Finally, the conception rate, farrowing rate and litter size were recorded. 2.7. Statistical analysis The percentage sperm release, and the effects of treatments on sperm motility and litter size, were analyzed using the ANOVA procedure of SAS (SAS, 1989). The significance of differences (P < 0.05) among treatments was determined by the Duncan multiple range test.
3. Results 3.1. Releasing of the microencapsulated boar spermatozoa To evaluate the release of alginate microencapsulated spermatozoa, the sperm-rich fraction was microencapsulated and stored at 5 ◦ C. Following storage for 1, 3 and 7 days, the microcapsule was removed to assess sperm release. Table 1 lists the measurement results for sperm release kinetics. The results indicated whether, with or without stirring, the sperm release increased with the 37 ◦ C incubation time. Significant differences in sperm release existed after 2 h incubation on days 3 and 7. Although the difference was not significant, the sperm release of microcapsules in the stirring group following 24 h incubation was higher than for microcapsules in the non-stirring group (81 versus 60%). 3.2. Motility of the microencapsulated boar spermatozoa To examine whether alginate microencapsulization process can extend the longevity of boar semen, the concentrated and microencapsulated semen were removed every day to assess sperm motility. Table 2 lists the measurement results for sperm motility. Little difference in motility existed between the free and encapsulated semen after 10 min incubation at 37 ◦ C on every sampling day. The motility of encapsulated spermatozoa was significantly
Days of storage
Incubation without stirring (h) 2
4
Incubation with 110 rpm stirring 6
8
24
2
4
1 20.4 ± 6.9 40.9 ± 8.9 46.1 ± 10.1 48.3 ± 13.2 49.5 ± 13.5 15.2 ± 5.7 51.3 ± 4.3 3 40.8 ± 5.1a 57.7 ± 10.6 61.9 ± 10.2 67.2 ± 10.7 71.4 ± 10.0 11.9 ± 4.8b 39.1 ± 8.5 7 28.0 ± 3.3a 49.5 ± 8.4 53.0 ± 10.1 56.5 ± 9.4 60.9 ± 8.8 9.4 ± 1.7b 26.8 ± 4.6 a, b Sperm release differ significantly among treatments given the same storage and incubation time (P < 0.05).
6
8
24
59.2 ± 2.7 54.2 ± 8.5 47.6 ± 3.5
67.3 ± 4.8 62.4 ± 9.0 52.1 ± 5.5
83.5 ± 7.1 83.3 ± 12.9 76.5 ± 7.5
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Table 1 The spermatozoa release (%) of encapsulated semen cultured at 37 ◦ C after storage at 5 ◦ C for different periods (n = 3)
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Storage time (Day)
Treatment of semen
Duration of 37 ◦ C incubation 10 min
8h
24 h
Encapsulated 72.9 ± 5.2 60.0 ± 7.4 51.4 ± Free 82.1 ± 2.4 56.4 ± 5.2 37.1 ± 2 Encapsulated 67.1 ± 3.1b 57.9 ± 3.8 35.7 ± Free 76.4 ± 2.1a 47.1 ± 5.8 32.9 ± 3 Encapsulated 70.0 ± 6.9 55.7 ± 6.8 45.0 ± Free 78.6 ± 3.4 37.9 ± 6.4 25.0 ± 4 Encapsulated 70.0 ± 6.8 51.4 ± 6.5a 37.9 ± Free 72.1 ± 3.4 30.0 ± 4.4b 15.7 ± 5 Encapsulated 61.4 ± 6.1 45.7 ± 5.6a 34.3 ± Free 68.6 ± 2.6 23.6 ± 3.9b 14.7 ± 6 Encapsulated 60.0 ± 4.1 44.3 ± 4.4a 33.6 ± Concentrate 67.1 ± 1.8 21.4 ± 3.6b 10.9 ± 7 Encapsulated 55.7 ± 4.4 47.9 ± 4.6a 44.3 ± Free 62.1 ± 3.2 14.3 ± 3.2b 9.3 ± 8 Encapsulated 55.7 ± 6.8 48.6 ± 7.1a 42.9 ± Free 55.0 ± 4.8 14.4 ± 4.7b 9.4 ± 9 Encapsulated 51.4 ± 6.3 49.3 ± 7.0a 33.9 ± Free 48.6 ± 3.7 13.6 ± 3.7b 5.6 ± a,b Motility differ significantly among treatments given the same storage time (P < 0.05). 1
32 h 8.7 5.8 6.3 6.3 5.6a 6.2b 4.7a 4.7b 4.4a 4.6b 4.7a 3.9b 6.5a 3.6b 7.5a 3.5b 7.2a 2.1b
45.0 30.0 32.9 20.7 30.0 16.1 25.0 10.0 24.3 10.9 27.9 5.9 39.3 8.1 37.9 5.4 22.1 3.0
48 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
9.3 6.1 5.2 3.5 3.6a 3.4b 3.8a 3.6b 5.5a 2.7b 4.6a 2.2b 6.9a 4.0b 7.3a 2.8b 4.1a 1.3b
20.1 17.1 26.6 13.6 18.0 9.0 13.6 1.9 17.1 3.0 23.6 5.4 27.9 3.3 21.1 2.1 11.0 2.1
56 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
5.3 4.3 5.3 3.0 5.1 2.1 4.8a 0.9b 3.4a 1.3b 6.3a 2.8b 7.3a 2.1b 6.6a 1.5b 2.9a 0.8b
12.9 10.0 19.3 2.9 9.1 4.3 8.6 1.4 6.0 1.0 20.4 2.3 16.4 2.1 7.6 1.1 7.6 1.0
72 h ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
6.0 3.6 5.1a 1.5b 3.9 1.2 3.6 0.7 2.2 0.7 6.2a 1.4b 6.5 1.5 2.9 0.8 2.5a 0.7b
5.9 3.0 7.9 0.9 6.4 1.3 7.1 0.7 3.9 0.3 11.6 0.9 7.6 1.1 2.3 0.7 2.6 0.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
2.9 1.4 3.2 0.7 4.5 0.7 3.4 0.5 1.9 0.3 4.8a 0.7b 4.2 1.1 1.3 0.7 1.0a 0.1b
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Table 2 Sperm motility (%) of microencapsulated and free semen cultured at 37 ◦ C after stored at 5 ◦ C for different duration (n = 5)
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Table 3 Reproductive performance of the sows inseminated with microencapsulated or free porcine semen Treatment
No. of sows inseminated (heads)
Conception rate (%)
Farrowing rate (%)
Average litter size (heads)
Encapsulated Free
4 3
75 100
75 100
12.3 ± 1.7 12.0 ± 0.6
higher than that of free semen after 8 h incubation at 37 ◦ C following storage for more than 3 days (P < 0.05). This effect continued until 48 h of incubation. When the 37 ◦ C incubation time exceeds 48 h, sperm motility decreased to a low level and the difference became non-significant. 3.3. Fertility of the microencapsulated boar spermatozoa For further evaluating the fertility of microencapsulated sperm, the concentrated and microencapsulated semen containing 5 × 109 sperm were inseminated to sows with natural estrus after 4 days of storage. The reproductive performance of the sows after insemination was shown in Table 3. No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and concentrated free semen.
4. Discussion Semen left-over is a common problem in the AI center practice. Microencapsulation of semen has been proposed as a method of choice for prolonged storage or sustained release of sperm within the female reproductive tract (Nebel and Saacke, 1996). However, this method of sperm storage appears more successful in bovines than that in pigs. This study evaluated the effect of alginate microencapsulization on extending the longevity of boar spermatozoa and on the fertility of microencapsulated spermatozoa in vivo. The results suggested that microencapsulation could prolong the storage time of boar spermatozoa and also sustain the fertility of microencapsulated sperm under common AI practice. The microencapsulation of bovine spermatozoa with calcium alginate has been successful and can achieve prolonged release (Nebel et al., 1985, 1993; Nebel and Saacke, 1994, 1996). However, the method used in bovines is not suitable for encapsulation of boar spermatozoa, and viability cannot be maintained for more than 16 h after encapsulation using this method (Nebel and Saacke, 1996). Consequently, a new encapsulation method specifically for swine has been developed (Torre et al., 1998, 2000). Torre et al. (2000) has assessed the release profile of microencapsulated spermatozoa under stirring. Torre et al. (2000) found that 0.12 M calcium concentration released approximately 40% cells in 6 h, and the lower concentration (0.07 M) released almost 100% cells at the same time. Using barium alginate and protamine–barium alginate for microencapsulation, Torre et al. (2002) found that encapsulation enhances semen preservation, providing protection against membrane damage on dilution. The in vitro release test of spermatozoa revealed massive cell delivery from barium alginate microcapsules within 6 h, and slow
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release from protamine–barium alginate capsules. This study evaluated the release of spermatozoa of capsules prepared with 0.1 M calcium under both static and stirring status. The result suggested that stirring can enhance sperm release (Table 1). However, the release rate was lower than reported in Torre et al. (2000). This inconsistency may result from the different equipment used for stirring the capsules and the method used for assessing the release. Previous studies have demonstrated that sodium alginate-poly-l-lysine cause minimal injury to the microencapsulated spermatozoa (Esbenshade and Nebel, 1990; Nebel et al., 1993, 1996). Nebel et al. (1996) showed that microcapsule wall thickness did not influence sperm motility, and no apparent cellular injury was noted following encapsulation. In boar spermatozoa, Torre et al. (2000) reported that sperm motility reduced to 35–40% at 4 h after semen microencapsulation. This work found that encapsulated spermatozoa motility continued to exceed 40% at 8 h incubation at 37 ◦ C, even after 9 days of storage (Table 2). Recently, barium alginate and protamine alginate were used to encapsulate swine semen by Vigo et al. (2002). Morphological and functional characteristics (acrosome integrity and motility) were not found to differ between the free and encapsulated semen. Faustini et al. (2004) further reported that encapsulated boar spermatozoa had significantly higher acrosome integrity and generally higher in situ enzymatic activity than those of unencapsulated spermatozoa stored for 72 h at 18 ◦ C. The observations in this study demonstrated that the motility of encapsulated spermatozoa was significantly higher than that of free concentrated semen following 48 h of incubation at 37 ◦ C after more than 3 days of storage (Table 2). The results indicated that the microencapsulated spermatozoa could maintain their motility at 37 ◦ C for longer than the non-encapsulated spermatozoa, thus enabling boar semen storage time to be extended. The result also suggested that microencapsulation can extend the survival of boar spermatozoa following insemination. Nebel et al. (1996) found that pregnancy rates were the same for heifers inseminated with semen either unencapsulated or microencapsulated with microcapsules of various wall thicknesses. The results presented by Nebel et al. showed that extended bovine spermatozoa can survive in the female reproductive tract at least 40 h prior to fertilization. Vigo et al. (2002) evaluated the in vitro fertilization potency of barium alginate and protamine alginate encapsulated swine semen. The results presented by Vigo et al. suggested that the technological encapsulation process did not compromise in vitro fertilization potency of the spermatozoa. Though this study contains a limited sample size, sows inseminated with non-encapsulated and microencapsulated semen after 4 days of storage displayed similar reproductive performances (Table 3). This preliminary finding indicated that encapsulated boar spermatozoa may maintain their ability to fertilize the ova in the sow reproductive tract. Vishwanath et al. (1997) indicated that the fertility of heifers with encapsulated semen increased with reducing interval between insemination and estrus. Consequently, the time window in which insemination could be conducted without reducing fertility was smaller with the encapsulated semen than with the control semen. Whether the same reproductive performance could be reached by inseminating the contact microcapsule into sows and also the optimal insemination time remained to be evaluated. This investigation found that some of the microcapsules ruptured during the insemination, and thus designing a suitable device for inseminating microcapsules into sows will be another issue that must be resolved for microencapsulation application.
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In conclusion, the findings of this study further confirmed that microencapsulation could prolong the storage time of boar spermatozoa and might also sustain the fertility of microencamsulated spermatozoa after 4 days of storage. However, due to limited number of sows inseminated, whether the microencapsulization could prolong spermatozoal retention time in the reproductive tract of sows and also fertilization ability requires further elucidation.
Acknowledgements The authors would like to thank the Council of Agriculture Executive Yuan, Republic of China, for financially supporting this research under Contract No. 88AST-1.5-AID-08. The ATIT transgenic team is also appreciated for its technical assistance related to artificial insemination using both microencapsulated and concentrated semen. Technical support from Dr. C.W. Liao and Miss E.C. Huang is also appreciated.
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