**Cryopreservation of Cattle, Pig, Inobuta Sperm and Oocyte after the Fukushima Nuclear Plant Accident**

Hideaki Yamashiro, Yasuyuki Abe, Yoshikazu Kuwahara, Tomokazu Fukuda, Yasushi Kino, Kazuya Inoue, Shintaro Takahashi, Motoi Fukumoto, Jin Kobayashi, Bin Tong, Sachio Takino, Takahisa Yamada, Tsutomu Sekine, Emiko Isogai and Manabu Fukumoto

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/58629

**1. Introduction**

After the Great East Japan Earthquake on 11 March 2011, the Fukushima Daiichi Nuclear Power Plant (FNPP) accident led to a discharge of a tremendous amount of radioactive substances [1, 2]. On 22 April 2011, the evacuation zone was set to a 20-km radius surrounding the FNPP, leaving approximately 3,400 cows, 31,500 pigs, and 630,000 chickens behind within the zone. On 12 May 2011, the Government of Japan ordered Fukushima prefectural govern‐ ment to euthanize unleashed livestock within the evacuation zone. Abandoned animals now have formed an invaluable model for studying the effects of chronic radionuclide intake. A comprehensive assessment of the effect of long-term exposure to internally deposited radio‐ nuclides on surviving domestic animals in the evacuation area is therefore urgently needed for the benefit of the livestock industry, as well as for human health. Radiobiological data from the FNPP accident could help to develop a set of internationally harmonized measures to protect domestic animals in the event of a future nuclear or radiological emergency.

Exposure to a large dose of ionizing radiation can cause irreparable damages to multiple organ systems, particularly those with highly proliferative cells, such as the skin, the hematopoietic and gastrointestinal system [3]. The testis and ovary are relatively radiosensitive organs [4], composed of a series of spermatogenic cells such as stem cells, spermatogonia, spermatids,

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

spermatocytes, sperm, and oogonium, primary oocyte, secondery oocyte and ovum, respec‐ tively. These different types of germ cells differ remarkably in their susceptibility to radiationinduced effects according to their level of reproductive activity [5]. The effect on reproductive organs and behaviour by chronic exposure to radionuclides is one of major concerns. Further‐ more, radiation-induced genomic changes, occurring in germ cells may have hereditary effects, including carcinogenesis, congenital malformation and growth retardation in offsprings. A germ cell is the only cell that can produce next-generation. Therefore, greater use of cryopre‐ servation of germ cells provide an essential resource to preserve their genetics and foetuses obtained by fertilization using those of the freezing sperm and oocytes for further studies on the effect of ionizing radiation on the next generations.

**3. Cryopreservation of cattle, pig, inobuta sperm**

Hygiene Service Center of Fukushima prefecture.

dose rate was 20 µSv/h.

C: Inobuta

The Japanese government ordered Fukushima prefecture to euthanize cattle in the evacuation zone on 12 May 2011 to prevent radio-contaminated livestock products from entering the human food chain. We obtained testes and ovaries from the euthanized cattle, pigs and inobutas collected by the combined unit of veterinary doctors belonging to the Livestock

Cryopreservation of Cattle, Pig, Inobuta Sperm and Oocyte after the Fukushima Nuclear Plant Accident

http://dx.doi.org/10.5772/58629

75

Almost bulls and boars were castrated. Therefore, we could only collect testes from 11 euthanized Japanese black beef bulls, 3 boars, and 1 male inobuta between 29 August 2011 and 28 February 2013 (Figure 1). Testes were collected in Kawauchi village located 15 km southwest of FNPP: the air dose rate was 0.5 µSv/h, Naraha town located 17 km south of FNPP: the air dose rate was 2 µSv/h, Tomioka town located 7 km south of FNPP: the air

**Figure 1.** Animals in the evacuation zone of the Fukushima Daiichi Nuclear Plant. A: Japanease black beef cattle B: Pig

In bull, sperm from two caudae epididymides were collected. Immediately after collection, sperm were diluted with a Triladyl freezing extender containing egg yolk at natural temper‐ ature (Mini Tube, Germany). The tubes containing sperm were transferred to the Niigata University within 6-8 h after collection. Semen samples were cooled to 5-10°C during trans‐ ferring. Aliquots of 0.5 ml of sperm suspension were individually placed in straws and ends were sealed. The straws were then placed in liquid nitrogen vapor for 10 min and then plunged directly into liquid nitrogen. In boars and male inobuta, freezing protocol was performed as described above. The semen extender, Modena extender containing egg yolk was used for freezing epididymal sperm. Total number of frozen sperm was 507 straws from bulls, 160

straws from boars and 83 straws from inobutas (Table 1).

We have collected and cryopreserved the sperm and oocytes from three spices of domestic animals in the FNPP evacuation zone between 27 September 2011 and 31 March 2013. In this chapter, we introduce approaches to cryopreserve germ cells from the cattle, the pig, and the inobuta which is a mongrel of the wild boar and the pig for further research of radiobiology.

#### **2. Reporting studies of Chernobyl for domestic animals and human**

Data used for estimating the risk associated with exposure to ionizing radiation have been primarily obtained from epidemiological studies of survivors of the atomic bombing of Hiroshima and Nagasaki [6], the Chernobyl nuclear accident [7], and some complementary animal experiments [8–10]. However, reports of the effect of chronic low-dose radiation on livestock animals are limited.

Direct radiation injury to animals was reported only in local areas within the 30-km exclusion zone in Chernobyl nuclear power plant [11]. In some cases, chronic dose rates may have reduced the fertility of some animal species inside the zone. Recently, review of Russian language studies on radionuclide behavior in agricultural animals has been published [12, 13]. There are several important animal pathways for radionuclide transfer to the diet of humans. The important for many contamination scenarios for radiologically important radionuclides (90Sr, 131I and 137Cs) is muscle (meat) consumption. The information presented this review has reported values are for Cs due to the Chernobyl accident in cattle, sheep, goat, rabbits, and chicken.

On the other hand, irradiation damages the ovaries and testes as direct effects of radiation, and indirectly affect through hormonal disruption. In human and wild animals, several studies of the functional changes in the reproductive tract have been made as a result of Chernobyl accident, abnormalities in spermatozoa and reproductive failures have been described [7]. Additionally, Weinberg et al. reported some genetic changes of unclear importance in offspring of Chernobyl accident liquidators [14, 15]. Although there are some claim that its changes is caused by the psychological factors (stressful conditions), it does not yet have enough information to explain all of the serious changes.

## **3. Cryopreservation of cattle, pig, inobuta sperm**

spermatocytes, sperm, and oogonium, primary oocyte, secondery oocyte and ovum, respec‐ tively. These different types of germ cells differ remarkably in their susceptibility to radiationinduced effects according to their level of reproductive activity [5]. The effect on reproductive organs and behaviour by chronic exposure to radionuclides is one of major concerns. Further‐ more, radiation-induced genomic changes, occurring in germ cells may have hereditary effects, including carcinogenesis, congenital malformation and growth retardation in offsprings. A germ cell is the only cell that can produce next-generation. Therefore, greater use of cryopre‐ servation of germ cells provide an essential resource to preserve their genetics and foetuses obtained by fertilization using those of the freezing sperm and oocytes for further studies on

We have collected and cryopreserved the sperm and oocytes from three spices of domestic animals in the FNPP evacuation zone between 27 September 2011 and 31 March 2013. In this chapter, we introduce approaches to cryopreserve germ cells from the cattle, the pig, and the inobuta which is a mongrel of the wild boar and the pig for further research of radiobiology.

**2. Reporting studies of Chernobyl for domestic animals and human**

Data used for estimating the risk associated with exposure to ionizing radiation have been primarily obtained from epidemiological studies of survivors of the atomic bombing of Hiroshima and Nagasaki [6], the Chernobyl nuclear accident [7], and some complementary animal experiments [8–10]. However, reports of the effect of chronic low-dose radiation on

Direct radiation injury to animals was reported only in local areas within the 30-km exclusion zone in Chernobyl nuclear power plant [11]. In some cases, chronic dose rates may have reduced the fertility of some animal species inside the zone. Recently, review of Russian language studies on radionuclide behavior in agricultural animals has been published [12, 13]. There are several important animal pathways for radionuclide transfer to the diet of humans. The important for many contamination scenarios for radiologically important radionuclides (90Sr, 131I and 137Cs) is muscle (meat) consumption. The information presented this review has reported values are for Cs due to the Chernobyl accident in cattle, sheep, goat,

On the other hand, irradiation damages the ovaries and testes as direct effects of radiation, and indirectly affect through hormonal disruption. In human and wild animals, several studies of the functional changes in the reproductive tract have been made as a result of Chernobyl accident, abnormalities in spermatozoa and reproductive failures have been described [7]. Additionally, Weinberg et al. reported some genetic changes of unclear importance in offspring of Chernobyl accident liquidators [14, 15]. Although there are some claim that its changes is caused by the psychological factors (stressful conditions), it does not yet have

the effect of ionizing radiation on the next generations.

enough information to explain all of the serious changes.

livestock animals are limited.

74 Recent Advances in Cryopreservation

rabbits, and chicken.

The Japanese government ordered Fukushima prefecture to euthanize cattle in the evacuation zone on 12 May 2011 to prevent radio-contaminated livestock products from entering the human food chain. We obtained testes and ovaries from the euthanized cattle, pigs and inobutas collected by the combined unit of veterinary doctors belonging to the Livestock Hygiene Service Center of Fukushima prefecture.

Almost bulls and boars were castrated. Therefore, we could only collect testes from 11 euthanized Japanese black beef bulls, 3 boars, and 1 male inobuta between 29 August 2011 and 28 February 2013 (Figure 1). Testes were collected in Kawauchi village located 15 km southwest of FNPP: the air dose rate was 0.5 µSv/h, Naraha town located 17 km south of FNPP: the air dose rate was 2 µSv/h, Tomioka town located 7 km south of FNPP: the air dose rate was 20 µSv/h.

**Figure 1.** Animals in the evacuation zone of the Fukushima Daiichi Nuclear Plant. A: Japanease black beef cattle B: Pig C: Inobuta

In bull, sperm from two caudae epididymides were collected. Immediately after collection, sperm were diluted with a Triladyl freezing extender containing egg yolk at natural temper‐ ature (Mini Tube, Germany). The tubes containing sperm were transferred to the Niigata University within 6-8 h after collection. Semen samples were cooled to 5-10°C during trans‐ ferring. Aliquots of 0.5 ml of sperm suspension were individually placed in straws and ends were sealed. The straws were then placed in liquid nitrogen vapor for 10 min and then plunged directly into liquid nitrogen. In boars and male inobuta, freezing protocol was performed as described above. The semen extender, Modena extender containing egg yolk was used for freezing epididymal sperm. Total number of frozen sperm was 507 straws from bulls, 160 straws from boars and 83 straws from inobutas (Table 1).


aspirated from small ovarian follicles and incubated in *in vitro* maturation (IVM) medium for 22 h. After maturation, the COCs were fertilized by co-incubation with thawed sperm. Then presumptive zygotes were cultured for *in vitro* development to the blastocyst stage. As a result, total 493 of morphologically normal COCs were recovered, and 40 blastocysts were yielded from 9 donors following *in vitro* embryo production. The bovine blastocysts produced were cryopreserved by vitrification using a nylon mesh method described in the previous report [19] (Table 2). On the other hand, pig and inobuta ovaries were transported to the laboratory at 37°C. Pig and inobuta COCs aspirated were carried out IVM for 44 h, and the presumptive matured oocytes were vitrified by similar method above. Total number of vitrified oocytes

Cryopreservation of Cattle, Pig, Inobuta Sperm and Oocyte after the Fukushima Nuclear Plant Accident

**cryopreservation**

**Number of cryopreserved egg**

http://dx.doi.org/10.5772/58629

77

**Animal Number of animals Developmental stage for**

Caw 9 Blastocyst 40 Sow 12 Metaphase II 371 Inobuta 2 Metaphase II 64

Germ cells in the testis show one of the highest mitotic activities of any tissue in the body, so that in the human adult about 100 million new cells are produced each day [20]. Spermato‐ genesis is highly regulated, starting with spermatogonial stem cells and ending with differ‐ entiated, motile spermatozoa. The testis is one of the most radiosensitive tissues, with very low doses of radiation causing significant impairment of function. It is well known that immature cells are more radiosensitive to doses as low as 0.1 Gy, causing morphological and quantitative changes to the spermatogonia in human testis. Doses of 2–3 Gy result in overt damage to spermatocytes, leading to a reduction in the spermatid numbers. At doses of 4–6 Gy, the numbers of spermatozoa significantly decrease, implying damage to the spermatids. A recent study in mice showed that low-dose-rate radiation exposure (3.49 mGy/h) did not cause adverse effects at dose levels of ≤2 Gy, but the testis weight, sperm count and motility

We needed to overcome a number of obstacles while working in the evacuation area, that is, dissections under the sun in the summer and snow in the winter, gathering the dissectors, as well as drive 400 miles a day. We also had restrictions on the time that we were allowed to stay in the area and the radiocontaminated materials to bring out of the area. We recently reported radionuclide deposition in organs of abandoned cattle following the FNPP accident. The deposition occurred in an individual radionuclide and in an organ-specific manner, and

**Table 2.** Total number of cryopresearved oocytes and embryos from caws, sows, and female inobutas in the

was 371 from sows and 64 from inobutas.

evacuation zone of the Fukushima Daiichi Nuclear Plant.

**5. Concluding remarks**

decreased at a dose of 2 Gy [21].

**Table 1.** Total number of cryopreserved straws contained sperm from bulls, boars, and male inobuta in the evacuation zone of the Fukushima Daiichi Nuclear Plant.

#### **4. Cryopreservation of cattle, pig, inobuta oocytes**

Oogenesis is associated closely with folliculogenesis in mammals. Oogenesis begins in the fetal ovary when the primordial germ cells arrive in the gonad of a genetic female and become oogonia. These cells proliferate via mitosis during fetal development. When proliferation ceases and the cells enter meiosis either before birth (human, cows, sow) or shortly thereafter (mice, rats, hamster) [16, 17], they are defined as primary oocytes arrested in the first meiotic prophase. Primordial follicles are formed in which the primary oocytes are surrounded by single layer of flattened granulose cells. Although the primordial follicles remain in this state of suspended animation for long time, oocytes and follicle resume the development near the time of ovulation. When follicles enter the development phase, they develop into primary and subsequent secondly follicles, along with proliferation of granulose cells and the oocytes growth. Primary and secondly follicle have cuboidal single layer and multiple layers of granulose cells, respectively. During the next phase, a fluid-filled cavity is formed adjacent to the oocyte in the follicle defined as antral follicle. Finally, one follicle growth rapidly and become the ovulatory follicle (maturation). In most mammals, the oocytes resume and complete the first meiotic division at ovulation.

Ionizing radiation may affect infertility or genetic disorders in subsequent generation induced by DNA damage in the germ cells and follicular cells. Many experiments have shown that radiosensitivity of follicle/oocyte varies widely according to the developmental stage of them and species [18]. In mice, the genetic sensitivity of oocyte in early stages of follicle development is a relatively high, and which decreases during the last week before ovulation. However, the sensitivity increases around the time of ovulation again. In contrast, oocytes in primordial follicle show a very low genetic sensitivity, and which increase with follicle development thereafter in guinea pigs. However, the knowledge in livestock is limited. In order to reveal the affect of exposure to low-dose of radiation on germ cells, it is necessary to study carefully at long term, including the influences on subsequent generations.

The aim of our studies is to examine of development of female germ cells in livestock within the evacuation zone and to preserve of female gametes for future studies. We collected ovaries from 36 cows, 12 sows, and 2 female inobutas. In cows, collected ovaries were washed and stored at 20°C in physiological saline containing antibiotics, and were transported to the laboratory within 7 h after the collection. Cattle cumulus-oocyte complexes (COCs) were aspirated from small ovarian follicles and incubated in *in vitro* maturation (IVM) medium for 22 h. After maturation, the COCs were fertilized by co-incubation with thawed sperm. Then presumptive zygotes were cultured for *in vitro* development to the blastocyst stage. As a result, total 493 of morphologically normal COCs were recovered, and 40 blastocysts were yielded from 9 donors following *in vitro* embryo production. The bovine blastocysts produced were cryopreserved by vitrification using a nylon mesh method described in the previous report [19] (Table 2). On the other hand, pig and inobuta ovaries were transported to the laboratory at 37°C. Pig and inobuta COCs aspirated were carried out IVM for 44 h, and the presumptive matured oocytes were vitrified by similar method above. Total number of vitrified oocytes was 371 from sows and 64 from inobutas.


**Table 2.** Total number of cryopresearved oocytes and embryos from caws, sows, and female inobutas in the evacuation zone of the Fukushima Daiichi Nuclear Plant.

#### **5. Concluding remarks**

**Animal Number of animals Number of cryopreserved sperm**

**Bull** 11 507 **Boar** 3 160 **Inobuta** 1 83

**Table 1.** Total number of cryopreserved straws contained sperm from bulls, boars, and male inobuta in the evacuation

Oogenesis is associated closely with folliculogenesis in mammals. Oogenesis begins in the fetal ovary when the primordial germ cells arrive in the gonad of a genetic female and become oogonia. These cells proliferate via mitosis during fetal development. When proliferation ceases and the cells enter meiosis either before birth (human, cows, sow) or shortly thereafter (mice, rats, hamster) [16, 17], they are defined as primary oocytes arrested in the first meiotic prophase. Primordial follicles are formed in which the primary oocytes are surrounded by single layer of flattened granulose cells. Although the primordial follicles remain in this state of suspended animation for long time, oocytes and follicle resume the development near the time of ovulation. When follicles enter the development phase, they develop into primary and subsequent secondly follicles, along with proliferation of granulose cells and the oocytes growth. Primary and secondly follicle have cuboidal single layer and multiple layers of granulose cells, respectively. During the next phase, a fluid-filled cavity is formed adjacent to the oocyte in the follicle defined as antral follicle. Finally, one follicle growth rapidly and become the ovulatory follicle (maturation). In most mammals, the oocytes resume and

Ionizing radiation may affect infertility or genetic disorders in subsequent generation induced by DNA damage in the germ cells and follicular cells. Many experiments have shown that radiosensitivity of follicle/oocyte varies widely according to the developmental stage of them and species [18]. In mice, the genetic sensitivity of oocyte in early stages of follicle development is a relatively high, and which decreases during the last week before ovulation. However, the sensitivity increases around the time of ovulation again. In contrast, oocytes in primordial follicle show a very low genetic sensitivity, and which increase with follicle development thereafter in guinea pigs. However, the knowledge in livestock is limited. In order to reveal the affect of exposure to low-dose of radiation on germ cells, it is necessary to study carefully

The aim of our studies is to examine of development of female germ cells in livestock within the evacuation zone and to preserve of female gametes for future studies. We collected ovaries from 36 cows, 12 sows, and 2 female inobutas. In cows, collected ovaries were washed and stored at 20°C in physiological saline containing antibiotics, and were transported to the laboratory within 7 h after the collection. Cattle cumulus-oocyte complexes (COCs) were

zone of the Fukushima Daiichi Nuclear Plant.

76 Recent Advances in Cryopreservation

**4. Cryopreservation of cattle, pig, inobuta oocytes**

complete the first meiotic division at ovulation.

at long term, including the influences on subsequent generations.

Germ cells in the testis show one of the highest mitotic activities of any tissue in the body, so that in the human adult about 100 million new cells are produced each day [20]. Spermato‐ genesis is highly regulated, starting with spermatogonial stem cells and ending with differ‐ entiated, motile spermatozoa. The testis is one of the most radiosensitive tissues, with very low doses of radiation causing significant impairment of function. It is well known that immature cells are more radiosensitive to doses as low as 0.1 Gy, causing morphological and quantitative changes to the spermatogonia in human testis. Doses of 2–3 Gy result in overt damage to spermatocytes, leading to a reduction in the spermatid numbers. At doses of 4–6 Gy, the numbers of spermatozoa significantly decrease, implying damage to the spermatids. A recent study in mice showed that low-dose-rate radiation exposure (3.49 mGy/h) did not cause adverse effects at dose levels of ≤2 Gy, but the testis weight, sperm count and motility decreased at a dose of 2 Gy [21].

We needed to overcome a number of obstacles while working in the evacuation area, that is, dissections under the sun in the summer and snow in the winter, gathering the dissectors, as well as drive 400 miles a day. We also had restrictions on the time that we were allowed to stay in the area and the radiocontaminated materials to bring out of the area. We recently reported radionuclide deposition in organs of abandoned cattle following the FNPP accident. The deposition occurred in an individual radionuclide and in an organ-specific manner, and radioactive Cs was detected in all the organs examined [22]. Discharge of 134Cs and 137Cs that emit γ-and β-rays is of primary concern, because they were released in a large amount and have a long half-lives [23]. Furthermore, we have investigated the effect of chronic radiation exposure on bull testes to 134Cs and 137Cs associated with the FNPP accident. Adverse radiationinduced effects, so far, have not been observed in bull testes following chronic exposure to the above levels of radiation for up to 10 months [24].

**Author details**

Hideaki Yamashiro1

Manabu Fukumoto3\*

Yasushi Kino5

**References**

1013-1014.

146, pp.1–27.

Bin Tong1

, Yasuyuki Abe2

\*Address all correspondence to: fukumoto@idac.tohoku.ac.jp

1 Faculty of Agriculture, Niigata University, Niigata, Japan

5 Department of Chemistry, Tohoku University, Sendai, Japan

, Kazuya Inoue3

, Sachio Takino1

, Yoshikazu Kuwahara3

, Shintaro Takahashi3

2 Graduate School of Science and Engineering, Yamagata University, Yamagata, Japan

6 School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai, Japan

[1] Kinoshita, N., Sueki, K., Sasa, K., Kitagawa, J., Ikarashi, S., Nishimura, T., Wong, Y.S., Satou, Y., Handa, K., Takahashi, T., Sato, M., & Yamagata, T. (2011) Assessment of individual radionuclide distributions from the Fukushima nuclear accident covering

[2] Zheng, J. Tagami, K., Watanabe, Y., Uchida, S., Aono, T., Ishii, N., Yoshida, S., Kubo‐ ta, Y., Fuma, S., & Ihara, S. (2012) Isotopic evidence of plutonium release into the en‐

[3] Chute, J.P. To survive radiation injury, remember your aPCs. (2012) *Nat Med.* 18, pp.

[4] Otala, M., Suomalainen. L., Pentikäinen, M.O., Kovanen, P., Tenhunen, M., Erkkilä, K., Toppari, J., & Dunkel, L. (2004) Protection from radiation-induced male germ cell

[5] Liu, G., Gong. P., Zhao, H., Wang, Z., Gong, S., & Cai, L. (2006) Effect of low-level

[6] Pierce, D.A., Shimizu, Y., Preston, D.L., Vaeth M., & Mabuchi, K. (1996) Studies of the mortality of atomic bomb survivors. Report 12, part 1. Cancer: 1950–1990. *Radiat Res.*

3 Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan

4 Graduate School of Agricultural Sciences, Tohoku University, Sendai, Japan

central-east Japan. *Proc Natl Acad Sci.* 108, pp.19526-19529.

vironment from the Fukushima DNPP accident. *Sci Rep*. 2, 304.

loss by sphingosine-1-phosphate. *Biol Reprod.* 70, pp.759-767.

radiation on the death of male germ cells. *Radiat Res.* 165, pp.379-389.

, Takahisa Yamada1

, Tomokazu Fukuda4

, Emiko Isogai4

, Motoi Fukumoto3

, Tsutomu Sekine5

Cryopreservation of Cattle, Pig, Inobuta Sperm and Oocyte after the Fukushima Nuclear Plant Accident

,

http://dx.doi.org/10.5772/58629

and

,

79

, Jin Kobayashi6

The paternal and maternal genomes are not equivalent and both are required for mammalian development. The difference between the parental genomes is believed to be due to gametespecific differential modification, a process known as genomic imprinting, suggesting that DNA methylation may play a role in genomic imprinting. Lie et al, have examined the expression of these three imprinted genes in mutant mice that are deficient in DNA methyl‐ transferase activity [25]. Results demonstrate that a normal level of DNA methylation is required for controlling differential expression of the paternal and maternal alleles of imprint‐ ed genes. Few animal studies have investigated the possible link between paternal exposures and effects on genomic imprinting. Chronic treatment of male rats with 5-azacytidine, a drug that alters DNA methylation resulted in abnormalities in male germ cells and early embryo development but no increase in the incidence of congenital malformations [26]. This is an important area with potential consequences for the offspring of exposed males, and warrants further study [27].

In addition, questions regarding the effect of log-term exposure to radiation on the genetic damage to next generation are now being raised, but no clear evidence for this has been reported, except laboratory animals. Genetic analysis of foetuses obtained by fertilization using cryopreserved sperm and oocytes from cattle in the evacuation zone, is underway in our laboratory.

In conclusion, cryopreservation of germ cells has potential applications not only for production of next generations of animals but also for general reproductive biology including the field of radiation biology.

#### **Acknowledgements**

We express our gratitude to the Iwaki Livestock Hygiene Service Centre in Fukushima Prefecture, especially to DVM. Yuji Kobayashi and livestock farmers in the 20-km FNPP evacuation zone. This work was partly supported by a grant for Manabu Fukumoto of the Japan Society for the Promotion of Science, and the Emergency Budget for the Reconstruction of Northeastern Japan, MEXT, Japan, Discretionary Expense of the President of Tohoku University and Nippon Life Insurance Foundation supported this study. This work was also supported by the Programme for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry.

### **Author details**

radioactive Cs was detected in all the organs examined [22]. Discharge of 134Cs and 137Cs that emit γ-and β-rays is of primary concern, because they were released in a large amount and have a long half-lives [23]. Furthermore, we have investigated the effect of chronic radiation exposure on bull testes to 134Cs and 137Cs associated with the FNPP accident. Adverse radiationinduced effects, so far, have not been observed in bull testes following chronic exposure to the

The paternal and maternal genomes are not equivalent and both are required for mammalian development. The difference between the parental genomes is believed to be due to gametespecific differential modification, a process known as genomic imprinting, suggesting that DNA methylation may play a role in genomic imprinting. Lie et al, have examined the expression of these three imprinted genes in mutant mice that are deficient in DNA methyl‐ transferase activity [25]. Results demonstrate that a normal level of DNA methylation is required for controlling differential expression of the paternal and maternal alleles of imprint‐ ed genes. Few animal studies have investigated the possible link between paternal exposures and effects on genomic imprinting. Chronic treatment of male rats with 5-azacytidine, a drug that alters DNA methylation resulted in abnormalities in male germ cells and early embryo development but no increase in the incidence of congenital malformations [26]. This is an important area with potential consequences for the offspring of exposed males, and warrants

In addition, questions regarding the effect of log-term exposure to radiation on the genetic damage to next generation are now being raised, but no clear evidence for this has been reported, except laboratory animals. Genetic analysis of foetuses obtained by fertilization using cryopreserved sperm and oocytes from cattle in the evacuation zone, is underway in our

In conclusion, cryopreservation of germ cells has potential applications not only for production of next generations of animals but also for general reproductive biology including the field of

We express our gratitude to the Iwaki Livestock Hygiene Service Centre in Fukushima Prefecture, especially to DVM. Yuji Kobayashi and livestock farmers in the 20-km FNPP evacuation zone. This work was partly supported by a grant for Manabu Fukumoto of the Japan Society for the Promotion of Science, and the Emergency Budget for the Reconstruction of Northeastern Japan, MEXT, Japan, Discretionary Expense of the President of Tohoku University and Nippon Life Insurance Foundation supported this study. This work was also supported by the Programme for Promotion of Basic and Applied Researches for Innovations

above levels of radiation for up to 10 months [24].

further study [27].

78 Recent Advances in Cryopreservation

laboratory.

radiation biology.

**Acknowledgements**

in Bio-oriented Industry.

Hideaki Yamashiro1 , Yasuyuki Abe2 , Yoshikazu Kuwahara3 , Tomokazu Fukuda4 , Yasushi Kino5 , Kazuya Inoue3 , Shintaro Takahashi3 , Motoi Fukumoto3 , Jin Kobayashi6 , Bin Tong1 , Sachio Takino1 , Takahisa Yamada1 , Tsutomu Sekine5 , Emiko Isogai4 and Manabu Fukumoto3\*

\*Address all correspondence to: fukumoto@idac.tohoku.ac.jp

1 Faculty of Agriculture, Niigata University, Niigata, Japan

2 Graduate School of Science and Engineering, Yamagata University, Yamagata, Japan

3 Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan

4 Graduate School of Agricultural Sciences, Tohoku University, Sendai, Japan

5 Department of Chemistry, Tohoku University, Sendai, Japan

6 School of Food, Agricultural and Environmental Sciences, Miyagi University, Sendai, Japan

#### **References**


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germinal vesicle oocytes in subsequent production of viable blastocysts. *Biol Reprod*.

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[20] Amann, R.P. (1981) A critical review of methods for evaluation of spermatogenesis

[21] Gong, E.J., Shin, I.S., Son, T.G., Yang, K., Heo, K., & Kim, J.S. Low-dose-rate radiation exposure leads to testicular damage with decreases in DNMT1 and HDAC1 in the

[22] Fukuda, T., Kino, Y., Abe, Y., Yamashiro, H., Kuwahara, Y., Nihei, H., Sano, Y., Irisa‐ wa, A., Shimura, T., Fukumoto, M., Shinoda, H., Obata, Y., Saigusa, S., Sekine, T., Iso‐ gai, E., & Fukumoto, M. (2013) Distribution of artificial radionuclides in the abandoned cattle in the evacuation zone of the Fukushima Daiichi Nuclear Power

[23] Calabrese, E. (2011) Improving the scientific foundations for estimating health risks

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80 Recent Advances in Cryopreservation

1001–1005.

pp.71-117.


**Chapter 6**

**The Maining of Cryopreservation for in-vitro**

The necessity to cryopreserve certain tissues and cell types for their use in assisted reproduc‐ tion has allowed technique developments that improve the quality of treatments and help both professionals and patients to perform these techniques. In this chapter we will talk about the importance that cryopreservation has meant to assisted reproduction techniques and about the benefit for patients of the advance and improvement of different cryopreservation techniques, for example in the recent and increasingly demanded technique Egg Vitrification,

From the cryopreservation point of view sperm, oocytes, embryos and ovarian tissue will be analyzed, by reviewing how different cryopreservation techniques have evolved up to reaching the techniques used nowadays giving, furthermore, a vision of how they will be in the future to optimize even further, the procedure increasing survival rates and viability of

The Development of cryopreservation techniques, the increase in demand for cryopreserved cells or tissues and the use of these techniques in cells or tissues from patients with infectious diseases, has forced us to reduce the risk of contamination during the freezing process and the risk of cross-contamination during the storage of this material. Recent publications that demonstrate the survival of pathogens at low temperatures and possible contamination of the cells or tissues stored have changed the laws of each country and the customs and protocols

To understand the problem of contamination in cryopreservation we need to have an overview of the current problem in which all researchers are concerned about, seeking a cryopreserva‐ tion protocol with good results but without contamination problems. Discussing the cryopre‐

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Enrique Criado Scholz and Cristina González Navas

Additional information is available at the end of the chapter

**Fertilization Patients**

http://dx.doi.org/10.5772/58746

**1. Introduction**

to preserve female fertility.

gametes, embryos and ovarian tissue to 100%.

used so far in the cryopreservation.
