Research Note. Kenji Sasaki, Toshiaki Tatsumi, Mariko Tsutsui, Tatsuya Niinomi,, -. + Takayuki Imai, Mitsuru Naito, Atsushi Tajima and Yasuhiro Nishi

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1 doi: +*.,+. + /jpsa.**3+++ Copyright,*+* Poultry Science Association. Research Note, + +,, Kenji Sasaki, Toshiaki Tatsumi, Mariko Tsutsui, Tatsuya Niinomi,, -. + Takayuki Imai, Mitsuru Naito, Atsushi Tajima and Yasuhiro Nishi + Mie Prefecture Livestock Research Institute, Ureshino Cho , Matsusaka City, Mie /+/,-,. National Livestock Breeding Center Okazaki Station, OoyanagiCho Kurisawa +, Okazaki City, Aichi National Institute of Agrobiological Sciences, Tsukuba City, Ibaraki -*/ 20*, Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba city, Ibaraki -*/ 2/1,. 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.,+., May -*, +3/* ) in Japan. Semen was collected from Yakido roosters, and pooled semen was diluted + : + using semen diluent containing no cryoprotective agent. After equilibration at / for -* min, diluted semen was further diluted + : + using semen diluent containing MA at a final concentration of 3. Diluted semen was packaged into *./-ml plastic straws and frozen by placing the straws in liquid nitrogen vapor followed by plunging into liquid nitrogen at +30. 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 +, AI was performed once a week for four consecutive weeks. Weekly fertility rates using frozen semen were , , 220. /+., and 11-. /. 1 for weeks +,,, -, and., respectively. Hatchability was over 3* throughout the experimental period. In Fertility Trial,, the duration of fertility was examined after intravaginal insemination for, consecutive days. Daily fertility rates using frozen-thawed semen were 3*.*, 3*.*, 3+. 1, 22. 3, +**.*, +**.*, +**.*, //. 0, 0*.*,,/.*, -*.*, -*.*, and 3. + for days + through +- after AI, respectively. Overall hatchability was 23./. 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 J. Poult. Sci.,.1:,31 -*+,,*+* Introduction female germline chimeras are mated directly because of the di$ culty of complete replacement of germ cells in the The importance of securing poultry genetic resources, gonads of PGC recipients. It has been suggested that the such as local breeds, research resources, and grandparent e$ ciency of retrieving poultry genetic resources can be stocks from unexpected accidents or disease outbreaks improved significantly when semen from a genetically cannot be overemphasized, particularly after the recent valuable poultry genetic resource is cryopreserved beforeworldwide epidemic of avian influenza. A systematic ap- hand and used to inseminate female germline chimeras proach is necessary to reduce the risk of losing valuable (Tajima,,**,). In this respect, semen cryopreservation poultry genetic resources. Recent technological develop- technology plays a key role in the e$ cient retrieval of ments in the production of germline chimeras allow the poultry genetic resources through germline chimeras. recall of poultry genetic resources, provided that the pri- A number of studies have been performed to freeze mordial germ cells (PGCs) have been collected and chicken semen since Polge ( +3/+ ) first reported successful cryopreserved beforehand (Naito et al., +33. ; Tajima et freezing of chicken spermatozoa using glycerol as a cryoal., +332 ). However, the e$ ciency of retrieving poultry protective agent (Sexton, +313 ; Lake et al., +32+ ; Donoghue genetic resources is expected to be low when male and and Wishart,,***; Long,,**0; Blesbois,,**1). Three cryoprotective agents, i.e., glycerol, dimethylsulfoxide Received: November +3,,**3, Accepted: July +3,,*+* Released Online Advance Publication: September,/,,*+* (DMSO), and dimethylacetamide, have since been found Correspondence: K. Sasaki, Mie Prefecture Livestock Research Institute, to be e# ective in protecting domestic chicken sperm to Ureshino Cho , Matsusaka City, Mie /+/,-,.. varying degrees of freezing damage (Tajima et al., +33* ). ( sasakk *+@pref.mie.jp) Among these three cryoprotective agents, glycerol has

2 298 Journal of Poultry Science,.1 (.) been most widely used for freezing chicken semen because ph Osmotic Pressure -0* mosm/kgh, O. it has the highest post-thaw fertility rates (Tajima et al., The second semen dilution was conducted -* min after +33* ). However, it has been reported that the glycerol primary semen dilution by mixing an equal volume of concentration must be decreased to below *. +0- M prior diluted semen with semen diluent containing +2 MA to insemination to obtain fertile eggs due to the contracep- ( /+ ; Wako). Therefore, the final concentration of tive e# ect of glycerol (Lake et al., +32+ ). On the other MA in the diluted semen was 3. Diluted semen containhand, post-thaw fertility tends to be lower in both DMSO ing MA was packaged into *./-ml plastic straws (*++,-+** and dimethylacetamide methods compared with the gly- (NFA +*+ ); Fujihira, Tokyo) and frozen for -* cerol method even though it is not necessary to remove min by exposure to liquid nitrogen vapor. to../ cm above these two cryoprotective agents prior to artificial insemi- the surface of liquid nitrogen, followed by plunging into nation (Tajima et al., +33* ). It is, therefore, necessary to liquid nitrogen at +30. After storing straws containdevelop a method of cryopreserving chicken semen with ing semen in liquid nitrogen for approximately one month, high post-thaw fertility by direct insemination of thawed frozen semen was thawed in water at / for + min.* s. semen that contains the cryoprotective agent. The average number of sperm in the thawed semen was Yakido is a local variety of mid-sized Japanese game 0 00* +* /ml. In Fertility Trial +, artificial insemination bird (Shamo) that was first produced in Mie Prefecture, was performed once a week for. consecutive weeks, th Japan, at the end of the +3 century. Currently, Yakido is except that insemination was performed on, consecutive one of seventeen chicken breeds designated as Natural days in the first week. Monuments under the Law for the Protection of Cul- Ten White Leghorn hens were used in the fertility trial, tural Property (Law No.,+., May -*, +3/* ) in Japan. and intravaginal insemination was performed by injecting Average body weights of male and female Yakido are,. 0 -** ml of thawed semen directly into the vagina within, kg and,+. kg, respectively. It has greenish black feathers min after thawing. The same volume of non-frozen diland a pea comb with red earlobes, and color of the beak uted semen (without containing MA) was inseminated and the shank are yellow or black/yellow. It is important into +* White Leghorn hens as a control. Eggs produced to preserve the germ plasm of Yakido, as the number of between days, and 2 after each insemination were stored the purebred birds is estimated to be fewer than +** in the refrigerator maintained at +,,, followed by according to the census conducted by Mie Prefecture in incubation at for,+ days in a model P-**2 incuba-,**3 (unpublished data). tor (Showa Furanki, Saitama). Fertility data were In the present study, a new method of cryopreserving subjected to analysis of variance using the Generalized chicken semen using N-methylacetamide (MA) developed Linear Model using SAS/STAT (SAS, +333) after arcsine by Hanzawa et al. (,*+*) was applied to Yakido as an square root transformation. example of a genetically valuable chicken breed. In Fertility Trial,, semen collection, dilution, freezing, Materials and Methods and thawing were performed as described in Fertility Trial +. Twenty hens were randomly divided to two groups of Semen was collected randomly from Yakido roosters ( 2 3 months old), maintained at Mie Prefecture Livestock Research Institute, using the abdominal massage 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 method described by Burrows and Quinn ( +3-1). Collected were collected for +- days from the nd, day after the semen was aspirated into + -ml syringes (SP-*+ P; Terumo, second insemination. In both fertility trials, hatchability Tokyo). Special care was taken to avoid contamination was examined on day,, after starting incubation. Un- with transparent fluid and feces during semen hatched eggs were broken to examine embryonic death collection. Semen from individual roosters was placed immediately during incubation. Eggs with embryonic death were regarded into +/ -ml test tubes ( ++ *-3 **. ; AGC Techno Glass, Funabashi, Chiba), which were precooled to as fertilized eggs. using chi-square test. Fertility data were analyzed / in ice water. Primary semen dilution was performed by mixing equal volumes of semen and semen diluent, which was also precooled to /. The chemical composition of the semen diluent used in the present study was as follows (per +** ml): D ( )-glucose *., g (*.1 **/3, ; The experimental animals were treated in accordance with accepted standards for the humane treatment of animals. Results Wako, Osaka), D( )-trehalose dihydrate -2. g Fertility Trial + (,*- *,,/, ; Wako); L-glutamic acid, monosodium salt +., g ( */ ; Nacalai Tesque, Kyoto); potassium acetate *.- g (,2.*/ */ ; Nacalai Tesque); magnesium Fertility and Hatchability After Repeated Weekly AI As shown in Fig. +, weekly fertility rates using frozen semen were , , /. +, and acetate tetrahydrate *.*2 g ( +-* ***3/ ; Wako); potassium /. 1 for weeks +,,, -, and., respectively. citrate monohydrate *.*/ g ( +0+ *-/2/ ; Wako); BES No significant di# erences in fertility were observed *.. g (-.+ **,0, ; Wako); Bis-Tris *.. g (-.- *.1., ; nd among the four weeks ( P **/. ), except for the, week, Wako); gentamicin sulfate *.**+ g (*1- *,31+ ; Wako) at during which fertility using frozen semen was significantly

3 Sasaki et al. : Cryopreservation of Chicken Semen Using N-Methylacetamide 299 Fig. +. Fertility of frozen-thawed Yakido semen cryopreserved in the presence of N-methylacetamide in Fertility Trial +. Asterisk indicates significant di# erence compared with non-frozen control at P **/.. Vertical bars indicate standard error associated with the means. Fig.,. Duration of fertility after initial artificial insemination with frozen/thawed Yakido semen using N-methylacetamide as a cryoprotective agent in Fertility Trial,. Arrows indicate the date of artificial insemination. Asterisks indicate significant differences compared with non-frozen control at P **/.. lower compared with non-frozen semen ( P **/. ). Hatchability was over 3* throughout the experimental period. Fertility Trial, Duration of Fertility After Initial Artificial Insemination No significant di# erences ( P *.*/) were observed in of Frozen/thawed Yakido Semen Using MA as a Cryoprotective hatchability between frozen and non-frozen semen (data Agent not shown). As shown in Fig.,, fertility rates after initial artificial insemination were 3*.*, 3*.*, 3+. 1, 22. 3, +**.*

4 300 Journal of Poultry Science,.1 (.), +**.*, +**.*, //. 0, 0*.*,,/.*, -*.*, spermatozoa are stored in the sperm storage tubules (SST) -*.*, and 3. + for days + to +- after AI, respectively. for days or weeks in hens (Bakst et al., +33. ). Although A rapid decrease in fertility was observed from days 2 the SST are located in the uterovaginal junction (UVJ) after artificial insemination compared with non-frozen and infundibulum, the primary site of sperm storage is the control. Overall hatchability was 23./ in Fertility Trial SST in the UVJ (Frieß et al., +312). The duration of,. No significant di# erences ( P *.*/) in hatchability fertility is correlated with the number of sperm in the SST were observed throughout the experimental period be- at the UVJ (Pierson et al., +322). The results of the tween frozen and non-frozen semen (data not shown). present study suggested earlier degradation of frozen/ Discussion thawed spermatozoa in the SST of the oviduct compared with non-frozen controls. In the present study, chicken semen collected from Semen collected from Yakido was used in the present genetically valuable Yakido roosters was cryopreserved study. The freezability of chicken semen has been sugusing MA as a cryoprotective agent. Artificial insemina- gested to vary among breeds or lines (Tajima et al., +33* ; tion was performed by depositing thawed semen directly Alexander et al., +33-). Furthermore, the relative fertilizinto the vagina of hens within, min after thawing without ing ability was +./-fold higher for Rhode Island Red than removing the cryoprotective agent. White Leghorn (Tajima et al., +323). The results of these It has been reported that more than 2* fertility was observed for one week after single intravaginal inseminastudies indicated the necessity of evaluating the ability of MA to protect against cryoinjury using semen collected tion of frozen/thawed semen using DMSO as a cryopro- from chicken breeds other than Yakido. Modification of tective agent (Van Voorst and Leenstra, +33/ ). Tselutin the freezing/thawing method may be required to adjust et al. ( +333 ) used dimethylacetamide as a cryoprotective for the di# erences in freezability as well as relative fertilizagent, and reported more than 2* fertility for. days ing ability of each breed. after insemination with frozen/thawed semen. However, From the theoretical viewpoint, damage to the sperm the fertility after repeated insemination of frozen/thawed membrane must be compared between chicken semen semen for more than one week was not examined in these frozen under di# erent cryoprotective agents. Potential studies. Furthermore, a constant weekly decrease in fertil- points of interaction of glycerol with the chicken sperm ity was observed after the weekly insemination of frozen/ cell are motility, osmotic damage, lectin binding, morphthawed chicken semen for - weeks using semen frozen in ological damage, and cation loss (Hammerstedt and the presence of glycerol, DMSO, or dimethylacetamide Graham, +33, ). Comparison of the sites of cryoinjury (Tajima et al., +33* ). In this respect, the quality of post- against sperm cells in the presence of di# erent cryoprotecthaw spermatozoa, such as fertilizing ability, needs to be tive agents will contribute to the improvement of semen considered in addition to short-term fertility. As weekly cryopreservation. fertility rate remained over 2* for. consecutive weeks One of the unusual characteristics of freezing chicken after weekly insemination of frozen/thawed semen in the semen using MA as a cryoprotective agent is the slow present study, it was assumed that the fertilizing ability of recovery of post-thaw motility compared with when glycerol spermatozoa had been well restored during freeze/thaw was used. Motility was recovered gradually only processes when MA was used as a cryoprotective agent. when thawed semen on glass slides was warmed on a Wishart ( +32/ ) reported that the fertilizing ability of warm plate maintained at -2 for an extended period frozen/thawed chicken semen is +0. compared with (data not shown). A special precaution, therefore, is 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 of the non-frozen semen using sperm competition assay (Tajima et al., +323). 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. In Fertility Trial,, although the fertility rate with frozen/thawed semen for, consecutive days remained over 3* for the first 1 days after insemination, an earlier decrease in fertility was observed from day 2 compared with non-frozen controls. The durations of fertility using non-frozen semen have been estimated in broiler roosters (Kirby et al., +332 ), chicken hens (Liu et al.,,**2), and Japanese quail (Reddish et al., +330). However, the duration of fertility using frozen/thawed semen was not examined in these studies. It has been reported that the 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. Acknowledgments 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. References Alexander A, Graham J, Hammerstedt RH and Barbato GF. E# ects of genotype and cryopreservation of avian semen on

5 Sasaki et al. : Cryopreservation of Chicken Semen Using N-Methylacetamide 301 fertility and number of perivitelline spermatozoa. British Reproduction and Fertility, +*, : -,+ -,/ Poultry Science, -.: 1/ Pierson EE, McDaniel GR and Krista LM. Relationship between Bakst MR, Wishart G and Brillard JP. Oviducal sperm selection, fertility duration and in vivo sperm storage in broiler breeder transport, and storage in poultry. Poultry Science Reviews, hens. British Poultry Science,,3: +33,* /: Polge C. Functional survival of fowl spermatozoa after freezing Blesbois E. Current status in avian semen cryopreservation. at 13. Nature, +01: 3.3 3/*. +3/+. World s Poultry Science Journal, 0- :,+-,,,.,**1. Reddish JM, Kirby JD and Anthony NB. Analysis of poultry Burrows WH and Quinn JP. The collection of spermatozoa from fertility data. -. Analysis of the duration of fertility in the domestic fowl and turkey. Poultry Science, +0 : +3,.. naturally mating Japanese quail. Poultry Science, 1/ : +-/ Donoghue AM and Wishart GJ. Storage of poultry semen. SAS/STAT SAS User s guide Version 2. SAS Institute, Cary, NC Animal Reproduction Science, 0, :,+-,-,.,***.,1/++, USA Frieß AE, Sinowatz F, Wrobel K-H and Scklek-Winnisch R. The Sexton TJ. Preservation of Poultry Semen - A Review. Animal uterovaginal sperm host glands of the quail ( Coturnix Reproduction. BARC. Symposium Number -, Harold Coturnix japonica): An ultrastructural and ultracytochem- Hawk (ed) +/3 +1* ical study. Cell and Tissue Research, +3+ : +* Tajima A, Graham EF and Hawkins DM. Estimation of the Hammerstedt RH and Graham JK. Cryopreservation of poultry relative fertilizing ability of frozen chicken spermatozoa sperm: The enigma of glycerol. Cryobiology,,3:,0-2. using a heterospermic competition method. Journal of Re- +33,. production and Fertility, 2/ : + / Hanzawa S, Niinomi T, Miyata T, Tsutsui M and Tajima A. Tajima A, Graham EF, Sho# ner RN, Otis JS and Hawkins DM. Cryopreservation of chicken semen using N-methylacet- Cryopreservation of semen from unique lines of chicken amide as cryoprotective agent. Japanese Journal of Poultry germ plasm lines. Poultry Science. 03: 333 +**,. +33*. Science..1: J,1 J -,.,*+*. Tajima A, Naito M, Yasuda Y and Kuwana T. Production of Kirby JD, Tressler CJ and Kirby YK. Evaluation of the duration germ-line chimeras by transfer of cryopreserved gonadal of sperm fertilizing ability in five lines of commercial broiler primordial germ cells (gpgcs) in chicken. The Journal of breeder and Delaware cross males. Poultry Science, 11 : +022 Experimental Zoology,,2*:,0/, Tajima A. Production of germ-line chimeras and their applica- Lake PE, Ravie O and McAdam J. Preservation of fowl semen in tion in domestic chicken. Avian and Poultry Biology Reliquid nitrogen: Application to breeding programmes. Brit- views, +- : +/ -*.,**,. ish Poultry Science,,,: Tselutin K, Seigneurin F and Blesbois E. Comparison of cryo- Liu a GQ, Zhu JJ, Wang ZY, Jiang XP and Dafalla MM. protectants and methods of cryopreservation of fowl sper- Analysis of sperm storage ability using duration of fertility matozoa. Poultry Science, 12 : /20 /3* in hens. British Poultry Science,.3: 11* 11/.,**2. Van Voorst A and Leenstra FR. Fertility rate of daily collected Long JA. Avian semen cryopreservation: What are the biological and cryopreserved fowl semen. Poultry Science, 1. : +-0 challenges? Poultry Science, 2/ :,-,,-0.,**0. +.*. +33/. Naito M, Tajima A, Tagami T, Yasuda Y and Kuwana T. Pre- Wishart GJ. Quantitation of the fertilising ability of fresh comservation of chick primordial germ cells in liquid nitrogen pared with frozen and thawed fowl spermatozoa. British and subsequent production of viable o# spring. Journal of Poultry Science,,0: -1/ -2*. +32/.