A Method for Cryopreserving Semen from Yakido Roosters using. N-Methylacetamide as a Cryoprotective Agent

Transcription

1 Research Note A Method for Cryopreserving Semen from Yakido Roosters using N-Methylacetamide as a Cryoprotective Agent Kenji Sasaki 1), Toshiaki Tatsumi 1), Mariko Tsutsui 2), Tatsuya Niinomi 2), Takayuki Imai 2), Mitsuru Naito 3), Atsushi Tajima 4) and Yasuhiro Nishi 1). 1) Mie Prefecture Livestock Research Institute, Ureshino Cho , Matsuzaka City, Mie , Japan. 2) National Livestock Breeding Center Okazaki Station, OoyanagiCho Kurisawa 1, Okazaki City, Aichi , Japan. 3) National Institute of Agrobiological Sciences, Tsukuba City, Ibaraki , Japan 4) Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba city, Ibaraki , Japan. Running title: Cryopreservation of chicken semen using N-methylacetamide Correspondence: K. Sasaki, Mie Prefecture Livestock Research Institute, Ureshino Cho , Matsuzaka City, Mie , Japan. ( sasakk01@pref.mie.jp)

2 A method of freezing semen using N-methylacetamide (MA) as a cryoprotective agent was applied to Yakido, a chicken breed designated as a Natural Monument under the Law for the Protection of Cultural Property (Law No. 214, May 30, 1950) in Japan. Semen was collected from Yakido roosters, and pooled semen was diluted 1:1 using semen diluent containing no cryoprotective agent. After equilibration at 5 C for 30 min, diluted semen was further diluted 1:1 using semen diluent containing MA at a final concentration of 9%. Diluted semen was packaged into 0.5-mL plastic straws and frozen by placing the straws in liquid nitrogen vapor followed by plunging into liquid nitrogen at -196 C. After storage in liquid nitrogen for about one month, frozen semen was thawed in ice water. Intravaginal artificial insemination (AI) was performed immediately after thawing semen without removing the cryoprotective agent. In Fertility Trial 1, AI was performed once a week for four consecutive weeks. Weekly fertility rates using frozen semen were 83.8 ± 3.8%, 77.1 ± 8.8%, 88.6 ± 5.1%, and 77.3 ± 5.7% for weeks 1, 2, 3, and 4, respectively. Hatchability was over 90% throughout the experimental period. In Fertility Trial 2, the duration of fertility was examined after intravaginal insemination for 2 consecutive days. Daily fertility rates using frozen-thawed semen were 90.0%, 90.0%, 91.7%, 88.9%, 100.0%, 100.0%, 100.0%, 55.6%, 60.0%, 25.0%, 30.0%, 30.0%, and 9.1% for days 1 through 13 after AI, respectively. Overall hatchability was 89.5%. These results indicated that freezing chicken spermatozoa using MA as a cryoprotective agent is a practical method of preserving chicken semen collected from genetically valuable chicken breeds. Key words: Chicken, Cryopreservation, Genetic resources, N-Methylacetamide, Semen.

3 Introduction The importance of securing poultry genetic resources, such as local breeds, research resources, and grandparent stocks from unexpected accidents or disease outbreaks cannot be overemphasized, particularly after the recent worldwide epidemic of avian influenza. A systematic approach is necessary to reduce the risk of losing valuable poultry genetic resources. Recent technological developments in the production of germline chimeras allow the recall of poultry genetic resources, provided that the primordial germ cells (PGCs) have been collected and cryopreserved beforehand (Naito et al., 1994; Tajima et al., 1998). However, the efficiency of retrieving poultry genetic resources is expected to be low when male and female germline chimeras are mated directly because of the difficulty of complete replacement of germ cells in the gonads of PGC recipients. It has been suggested that the efficiency of retrieving poultry genetic resources can be improved significantly when semen from a genetically valuable poultry genetic resource is cryopreserved beforehand and used to inseminate female germline chimeras (Tajima, 2002). In this respect, semen cryopreservation technology plays a key role in the efficient retrieval of poultry genetic resources through germline chimeras. A number of studies have been performed to freeze chicken semen since Polge (1951) first reported successful freezing of chicken spermatozoa using glycerol as a cryoprotective agent (Sexton, 1979; Lake et al., 1981; Donoghue and Wishart, 2000; Long, 2006; Blesbois, 2007). Three cryoprotective agents, i.e., glycerol, dimethylsulfoxide (DMSO), and dimethylacetamide, have since been found to be effective in protecting domestic chicken sperm to varying degrees of freezing damage (Tajima et al., 1990). Among these three cryoprotective agents, glycerol has been most widely used for freezing chicken semen because it has the highest post-thaw fertility

4 rates (Tajima et al., 1990). However, it has been reported that the glycerol concentration must be decreased to below M prior to insemination to obtain fertile eggs due to the contraceptive effect of glycerol (Lake et al., 1981). On the other hand, post-thaw fertility tends to be lower in both DMSO and dimethylacetamide methods compared with the glycerol method even though it is not necessary to remove these two cryoprotective agents prior to artificial insemination (Tajima et al., 1990). It is, therefore, necessary to develop a method of cryopreserving chicken semen with high post-thaw fertility by direct insemination of thawed semen that contains the cryoprotective agent. Yakido is a local variety of mid-sized Japanese game bird (Shamo) that was first produced in Mie Prefecture, Japan, at the end of the 19 th century. Currently, Yakido is one of seventeen chicken breeds designated as Natural Monuments under the Law for the Protection of Cultural Property (Law No. 214, May 30, 1950) in Japan. Average body weights of male and female Yakido are 2.6 kg and 2.1 kg, respectively. It has greenish black feathers and a pea comb with red earlobes, and color of the beak and the shank are yellow or black/yellow. It is important to preserve the germ plasm of Yakido, as the number of the purebred birds is estimated to be fewer than 100 according to the census conducted by Mie Prefecture in 2009 (unpublished data). In the present study, a new method of cryopreserving chicken semen using N-methylacetamide (MA) developed by Hanzawa et al. (2010) was applied to Yakido as an example of a genetically valuable chicken breed.

5 Materials and Methods Semen was collected randomly from Yakido roosters (8 9 months old), maintained at Mie Prefecture Animal Research Center, Japan, using the abdominal massage method described by Burrows and Quinn (1937). Collected semen was aspirated into 1-mL syringes (SP-01P; Terumo, Tokyo, Japan). Special care was taken to avoid contamination with transparent fluid and feces during semen collection. Semen from individual roosters was placed immediately into 15-mL test tubes ( ; AGC Techno Glass, Funabashi, Chiba, Japan), which were precooled to 5 C in ice water. Primary semen dilution was performed by mixing equal volumes of semen and semen diluent, which was also precooled to 5 C. The chemical composition of the semen diluent used in the present study was as follows (per 100 ml): D(+)-glucose 0.2 g ( ; Wako, Osaka, Japan), D(+)-trehalose dihydrate 3.8 g ( ; Wako); L-glutamic acid, monosodium salt 1.2 g ( ; Nacalai Tesque, Kyoto, Japan); potassium acetate 0.3 g ( ; Nacalai Tesque); magnesium acetate tetrahydrate 0.08 g ( ; Wako); potassium citrate monohydrate 0.05 g ( ; Wako); BES 0.4 g ( ; Wako); Bis-Tris 0.4 g ( ; Wako); gentamicin sulfate g ( ; Wako) at ph 6.8. Osmotic Pressure = 360 mosm/kgh 2 O. The second semen dilution was conducted 30 min after primary semen dilution by mixing an equal volume of diluted semen with semen diluent containing 18% MA ( ; Wako). Therefore, the final concentration of MA in the diluted semen was 9%. Diluted semen containing MA was packaged into 0.5-mL plastic straws ( (NFA101); Fujihira, Tokyo, Japan) and frozen for 30 min by exposure to liquid nitrogen vapor 4 to 4.5 cm above the surface of liquid nitrogen, followed by plunging into liquid nitrogen at 196 C. After storing straws containing semen in liquid nitrogen for approximately one month, frozen semen was thawed in water at 5 C for 1 min 40 s. The

6 average number of sperm in the thawed semen was /ml. In Fertility Trial 1, artificial insemination was performed once a week for 4 consecutive weeks, except that insemination was performed on 2 consecutive days in the first week. Ten White Leghorn hens were used in the fertility trial, and intravaginal insemination was performed by injecting 300 μl of thawed semen directly into the vagina within 2 min after thawing. The same volume of non-frozen diluted semen (without containing MA) was inseminated into 10 White Leghorn hens as a control. Eggs produced between days 2 and 8 after each insemination were stored in the refrigerator maintained at 12 ± 2 C, followed by incubation at 37.8 C for 21 days in a model P-008 incubator (Showa Furanki, Saitama, Japan). Fertility data were subjected to analysis of variance using the Generalized Linear Model using SAS/STAT (SAS, 1999) after arcsine square root transformation. In Fertility Trial 2, semen collection, dilution, freezing, and thawing were performed as described in Fertility Trial 1. Twenty hens were randomly divided to two groups of ten hens, and assigned to inseminate either frozen-thawed semen or non-frozen semen as control. Artificial insemination was performed for two consecutive days, and eggs were collected for 13 days from the 2 nd day after the second insemination. In both fertility trials, hatchability was examined on day 22 after starting incubation. Unhatched eggs were broken to examine embryonic death during incubation. Eggs with embryonic death were regarded as fertilized eggs. Fertility data were analyzed using chi-square test. The experimental animals were treated in accordance with accepted standards for the humane treatment of animals.

7 Results Fertility Trial 1. Fertility and hatchability after repeated weekly AI. As shown in Figure 1, weekly fertility rates using frozen semen were 83.8±3.8%, 77.1 ± 8.8 %, 88.6 ± 5.1%, and 77.3 ± 5.7% for weeks 1, 2, 3, and 4, respectively. No significant differences in fertility were observed among the four weeks (P > 0.05), except for the 2 nd week, during which fertility using frozen semen was significantly lower compared with non-frozen semen (P < 0.05). Hatchability was over 90% throughout the experimental period. No significant differences (P > 0.05) were observed in hatchability between frozen and non-frozen semen (data not shown). Fertility Trial 2. Duration of fertility after initial artificial insemination of frozen/thawed Yakido semen using MA as a cryoprotective agent. As shown in Figure 2, fertility rates after initial artificial insemination were 90.0%, 90.0%, 91.7%, 88.9%, 100.0%, 100.0%, 100.0%, 55.6%, 60.0%, 25.0%, 30.0%, 30.0%, and 9.1% for days 1 to 13 after AI, respectively. A rapid decrease in fertility was observed from days 8 after artificial insemination compared with non-frozen control. Overall hatchability was 89.5% in Fertility Trial 2. No significant differences (P > 0.05) in hatchability were observed throughout the experimental period between frozen and non-frozen semen (data not shown).

8 Discussion In the present study, chicken semen collected from genetically valuable Yakido roosters was cryopreserved using MA as a cryoprotective agent. Artificial insemination was performed by depositing thawed semen directly into the vagina of hens within 2 min after thawing without removing the cryoprotective agent. It has been reported that more than 80% fertility was observed for one week after single intravaginal insemination of frozen/thawed semen using DMSO as a cryoprotective agent (Van Voorst and Leenstra, 1995). Tselutin et al. (1999) used dimethylacetamide as a cryoprotective agent, and reported more than 80% fertility for 4 days after insemination with frozen/thawed semen. However, the fertility after repeated insemination of frozen/thawed semen for more than one week was not examined in these studies. Furthermore, a constant weekly decrease in fertility was observed after the weekly insemination of frozen/thawed chicken semen for 3 weeks using semen frozen in the presence of glycerol, DMSO, or dimethylacetamide (Tajima et al., 1990). In this respect, the quality of post-thaw spermatozoa, such as fertilizing ability, needs to be considered in addition to short-term fertility. As weekly fertility rate remained over 80% for 4 consecutive weeks after weekly insemination of frozen/thawed semen in the present study, it was assumed that the fertilizing ability of spermatozoa had been well restored during freeze/thaw processes when MA was used as a cryoprotective agent. Wishart (1985) reported that the fertilizing ability of frozen/thawed chicken semen is 1.6% compared with non-frozen control by inseminating hens using a wide spermatozoa dose range. Alternatively, the relative fertilizing ability of frozen/thawed semen was estimated to be 19.7% of the non-frozen semen using sperm competition assay (Tajima et al., 1989). Studies to estimate the fertilizing ability and/or relative fertilizing ability of semen cryopreserved using MA are necessary, as these two experiments used glycerol as a cryoprotective agent.

9 In Fertility Trial 2, although the fertility rate with frozen/thawed semen for 2 consecutive days remained over 90% for the first 7 days after insemination, an earlier decrease in fertility was observed from day 8 compared with non-frozen controls. The durations of fertility using non-frozen semen have been estimated in broiler roosters (Kirby et al., 1998), chicken hens (Liu et al., 2008), and Japanese quail (Reddish et al., 1996). However, the duration of fertility using frozen/thawed semen was not examined in these studies. It has been reported that the spermatozoa are stored in the sperm storage tubules (SST) for days or weeks in hens (Bakst et al., 1994). Although the SST are located in the uterovaginal junction (UVJ) and infundibulum, the primary site of sperm storage is the SST in the UVJ (Frieß et al., 1978). The duration of fertility is correlated with the number of sperm in the SST at the UVJ (Pierson et al., 1988). The results of the present study suggested earlier degradation of frozen/thawed spermatozoa in the SST of the oviduct compared with non-frozen controls. Semen collected from Yakido was used in the present study. The freezability of chicken semen has been suggested to vary among breeds or lines (Tajima et al., 1990; Alexander et al., 1993). Furthermore, the relative fertilizing ability was 1.5-fold higher for Rhode Island Red than White Leghorn (Tajima et al., 1989). The results of these studies indicated the necessity of evaluating the ability of MA to protect against cryoinjury using semen collected from chicken breeds other than Yakido. Modification of the freezing/thawing method may be required to adjust for the differences in freezability as well as relative fertilizing ability of each breed. From the theoretical viewpoint, damage to the sperm membrane must be compared between chicken semen frozen under different cryoprotective agents. Potential points of interaction of glycerol with the chicken sperm cell are motility, osmotic damage, lectin binding, morphological damage, and cation loss (Hammerstedt and Graham, 1992). Comparison of the sites of cryoinjury against sperm cells in the presence of different

10 cryoprotective agents will contribute to the improvement of semen cryopreservation. One of the unusual characteristics of freezing chicken semen using MA as a cryoprotective agent is the slow recovery of post-thaw motility compared with when glycerol was used. Motility was recovered gradually only when thawed semen on glass slides was warmed on a warm plate maintained at 38 C for an extended period (data not shown). A special precaution, therefore, is required to evaluate the post-thaw sperm motility when MA is used as a cryoprotective agent to cryopreserved chicken semen. In conclusion, the results of the present study indicated that freezing chicken spermatozoa using MA is a highly practical method for preserving spermatozoa collected from genetically valuable chicken breeds such as Yakido. Acknowledgment The present study was financially supported in part by Research and Development Projects for Application in Promoting New Policy of Agriculture Forestry and Fisheries from the Ministry of Agriculture, Forestry, and Fisheries, Japan.

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13 Journal of Experimental Zoology, 280: Tajima A. Production of germ-line chimeras and their application in domestic chicken. Avian and Poultry Biology Reviews, 13: Tselutin K, Seigneurin F and Blesbois E. Comparison of cryoprotectants and methods of cryopreservation of fowl spermatozoa. Poultry Science, 78: Van Voorst A and Leenstra FR. Fertility rate of daily collected and cryopreserved fowl semen. Poultry Science, 74: Wishart GJ. Quantitation of the fertilising ability of fresh compared with frozen and thawed fowl spermatozoa. British Poultry Science, 26:

14 Legends Fig. 1. Fertility of frozen-thawed Yakido semen cryopreserved in the presence of N-methylacetamide in Fertility Trial 1. Asterisk indicates significant difference compared with non-frozen control at P < Vertical bars indicate standard error associated with the means. Fig. 2. Duration of fertility after initial artificial insemination with frozen/thawed Yakido semen using N-methylacetamide as a cryoprotective agent in Fertility Trial 2. Arrows indicate the date of artificial insemination. Asterisks indicate significant differences compared with non-frozen control at P < 0.05.

15 Fig. 1

16 Fig. 2