Chromosomal Aberrations, Sister Chromatid Exchanges and Nuclear Status of Immature Oocytes in Relation to Age of Dromedary Camels

Karima Gh. M. Mahmoud1,*, T. H. Scholkamy2, A. Farghaly3 and M. F. Nawito1

1 Department of Animal Reproduction & A.I, National Research Center, Dokki, Tahrir Street, 12622 Giza, Egypt 2 Department Field Investigation, Animal Reproduction Research Institute, Al-Ahram, Giza, Egypt 3 Department Genetic and Cytology, National Research Center, Dokki, Tahrir Street, 12622 Giza

Received May 27, 2005; accepted July 1, 2005

Summary The present study was conducted to analyse the chromosomes of blood culture and oocyte chromatin quality at the time of recovery in dromedary camels in relation to age. Twelve young (about one year) and twelve adult (4–10 years old) female camels were used. These animals with unknown reproductive history were slaughtered in Kerdasa abattoir (Giza province, Egypt). Blood samples were collected via sterile syringes from camels before slaughtering for chromosomal analysis. Oocytes from ovaries of both ages were aspirated from small antral follicles 1 to 5 mm in diameter and classified according to their quality into four categories. Nuclear status of cumulus oocytes complexes (COCs) were evaluated directly after collection. The results indicated that, the frequencies of chromosomal abnormalities and sister chromatid exchanges (SCE’S) were increased significantly (p0.05) with age. An increase in structural aberrations could be observed. There were no significant differences between young and adult camels in total number and quality of oocytes. Statistically significant (p0.01) differences were between percentages of germinal vesicle breakdown (GVBD) and Germinal vesicle (GV) in young and adult camels. It is concluded that, the increase of age may have significant effects on structural chromosomal aberrations, SCE’S and meiotic stages of immature camel oocytes but not on the number and quality of oocytes.

Key words Cytogenetics, Immature oocytes, Age, Dromedary camels.

There are two genera within the Camelidae family, Camelus and Lama. The genus Camelus consists of Camelus dromedarius, dromedary camel (one hump) and C. bactrianus, Bactrian camel (two humps). Both species are also known as Old World camelides. The genus Lama constitutes of four species: two domesticated, Lama glama, llama; L. pacos, alpaca; and two wild species, L. guanicoe, guanaco, and L. vicugna, vicuna. They are collectively known as South American or New World camelids. All of them have 74 chromosomes (Taylor et al. 1968, Hassanane and Omar 2001). The adverse effect of age on reproduction of dromedary camels was reported (Bezrukov and Shmidt 1970, Abdel-Raouf et al. 1975, Al-Qarawi et al. 2000, Kidd et al. 2001). But little is known about age effect on mitotic and meiotic chromosomes, inspite of several studies on other animals which have been conducted on chromosomes in relation to age (Golbus 1981, Tease and Fisher 1986, Robbins et al. 1995, Farag and El-Nahass 1996, Tucker et al. 1999, Sloter et al. 2004). The extra follicular maturation and in vitro fertilization in camelus and lama oocytes showed low success rate (Del Campo et al. 1994, Ghoneim et al. 1999). Quality of oocytes is one of the important factors affecting successful rates of these techniques (Mahmoud et al. 2003). Indeed, most previous studies of oocyte evaluation in camelidae depending on the nuclear status were determined after in vitro maturation (Mahmoud 2003, Ratto et al. 2004, Rezk 2005). Selection of immature

* Corresponding author, e-mail: [email protected] 296 Karima Gh. M. Mahmoud et al. Cytologia 70(3) oocytes by evaluation of chromatin quality before maturation will help in maximization of the em- bryo production in this species. Age may be another factor that affects the oocytes in vitro maturation and fertilization in bovine (Chian et al. 1992, Kuzmina et al. 2003, Chohan and Hunter 2004) and equine (Brinsko et al. 1994). Due to shortage in literature of this aspects in camels, research is needed to improve in vitro maturation and consequently in vitro fertilization by selection of the source and quality of oocytes. The aim of the present study was to analyse the chromosomes of blood culture, sister chromatid exchanges and oocyte chromatin quality at the time of recovery in dromedary camels particu- larly in relation to age progress.

Materials and methods Animals Twelve young (about one year old) and twelve adult (4–10 years old) female camels were used. These animals of unknown reproductive history were slaughtered in Kerdasa abattoir (Giza province, Egypt) during months of September, October and November 2003.

Chromosomal aberrations and sister chromatid exchange Blood samples were collected via sterile syringes from camels before slaughtering. Each sam- ple was divided into two halves, one for detection of chromosome aberrations and the other for SCE’S frequency. Blood cells were cultured for 72 h at 38°C in 5 ml TCM-199, 1 ml fetal calf serum and 0.1 ml phytohaemagglutinin (PHA). After incubation, cells were treated with colchicine (0.05%) for 2 h, then with a hypotonic (0.075 M KCL) for 30 min. After fixation in acetic acid : ethanol (1 : 3) solution, the cells suspension were dropped on wet slides then flammed to dry. The slides were stained with Giemsa stain and covered with DPX mounting media for chromosomal analysis. Chromosomal abnormalities were recorded in at least 50 metaphase spreads for each animal. For sister chromatid exchanges, 5-Bromodeoxyuridine (BrdU, Sigma) was added 48 h before harvesting. Then after hypotonic treatment and fixation, differential staining of sister chromatids was performed with fluorescence plus Giemsa method of Goto et al. (1978).

Collection and classiﬁcation of oocytes Ovaries were collected from both young and adult camels. Within 2 h of slaughter, the ovaries were transported in physiological normal saline (0.9%, w/v, NaCl) with antibiotic maintained at 30°C to the lab. The ovaries were washed three times in phosphate fuffered saline (PBS). The oocytes were aspirated from small antral follicles 1 to 5 mm in diameter using an 20-gauge needle attached to a 5 ml syringe containing PBS with 3% bovine serum albumin (BSA) and antibiotics (100 mg/ml streptomycin and 100 IU/ml penicillin). Oocytes were counted and classiﬁed according to their quality into four categories: class A, COC (Cumulus oocytes complex), class B, POC (Par- tial oocytes complex), class C, DO (Denuded oocytes) and class D, degenerated (fragmented denuded oocytes).

Evaluation of nuclear status Nuclear status of cumulus oocytes complexes (COCs) were evaluated using a modification of the air-drying technique as described by Tarkowski (1966). Briefly, cumulus cells were removed by vortexing for 5 min. Each oocyte was transferred to 1% hypotonic sodium citrate solution for 10 min and then placed on a microscope slide with a minimal amount of hypotonic solution. Three drops of fixative (methanol : acetic acid, 3 : 1) were dropped onto the oocytes. Subsequently, the fixed material was stained with 1% orcin stain. COCs were at germinal vesicle (GV) when the nu- 2005 Chromosomal Aberrations, Sister Chromatid 297 cleus was very distinct with an intact nucleolus. Germinal vesicle breakdown (GVBD) was consid- ered to have taken place, when the chromatin material started condensing and was observed as iso- lated shrunken bodies.

Statistical analysis Data were subjected to statistical analysis according to Snedecor and Cochran (1982).

Results The diploid chromosome number of dromedarius camel raised in Egypt was found to be 74. Results in Table 1 showed that, the frequencies of chromosomal abnormalities increased signiﬁcant- ly (p0.05) with age. The percentage reached 6.830.68 in adult compared with 4.00.53 for the young. An increase in structural aberrations can be observed in the form of fragments, gaps, breaks and deletions (Figs. 1 and 2E, F). Table 2 and Fig. 2G presents the frequency of SCE’S/cell in camel lymphocytes in relation to

Table 1. Chromosomal aberrations in camel lymphocytes in relation to age

Chromosome aberrations Number Number of Number of metaphase with Number of (meanS.E.) Animals of abnormal metaphases animals metaphases Including gaps Excluding gaps Gaps Fragment Break Deletion

Young 12 600 24 4.00.53 3.00.58 6 8 5 5 Adult 12 600 41 6.830.68* 5.340.45 9 14 10 8

* p0.05 (t-test).

Fig. 1. Metaphases spread from blood cultured cells of female camel showing A normal metaphase, B chromatid deletion, C chromatid gap, and D fragment.

Fig. 2. Metaphases spread from blood cultured cells of female camel showing E break, F fragment and G sister chromatid exchange. 298 Karima Gh. M. Mahmoud et al. Cytologia 70(3)

Table 2. Frequency of sister chromatid exchange (SCE’S) in camel lymphocytes in relation to age

Number of metaphases Animals Number of animals Number of SCE’S SCE’S/cell meanS.E. with SCE’S

Young 12 300 869 2.890.47 Adult 12 300 1552 5.170.51*

* p0.05 (t-test).

Table 3. Recovery and quality of camel oocytes by aspiration method in relation to age

Classiﬁcation of oocytes No. of No. of Average No. of Animals ovaries oocytes oocytes per ovary Class AB Class CD No. % No. %

Young 24 164 6.610.38 104 65.743.47 60 34.253.47 Adult 24 139 5.770.43 93 68.814.05 46 31.184.05

The results are expressed in meanS.E.M. of three replicates.

Table 4. Nuclear stages of immature camel oocytes after collection from ovarian follicles in relation to age

Germinal vesicle Germinal vesicle Total No. Total No. breakdown and Animals stage of oocytes of ﬁxed oocytes condensed stage No. % No. %

Young 98 78 58 74.290.38 20 25.700.38** Adult 82 52 41 78.690.91** 11 21.30.91

The results are expressed in meanS.E.M. of three replicates. ** p0.01. age. The results showed a significant (p0.05) increased in SCE’S frequency in adult than young animals. A total of 164 and 135 oocytes were collected after aspiration from young and adult camel’s ovaries respectively. The average number of recovered oocytes per ovary was 6.610.38 in young and 5.770.43 in adult camels. Perusal of Table 3 indicated that, about one third of collected oocytes in young and adult camels was denuded and fragmented. The percentage of compact and partially denuded cumulus cells was about two third in both ages. There are no significantly differences between young and adult camels in total number of oocytes and different classes of oocyte. In order to evaluate the nuclear stage before culture (Table 4), COCs were fixed direct- Fig. 3. Showing a camel oocyte at germinal vesicle ly after collection and aspiration from camel break down with a condensation of chromatin. 2005 Chromosomal Aberrations, Sister Chromatid 299 ovaries. We noted that 25.700.38% and 21.30.91% of oocytes had already undergone some nuclear maturation and were at GVBD stage or early condensed stage (Fig. 3) in young and adult camels respectively. Only 74.290.38% of oocytes in young and 78.690.915% of oocytes in adult camels were in GV stage. There are statistically significant (p0.01) differences between percentages of GVBD and GV in young and adult camels.

Discussion It is important to study the age effect on chromosomes abnormalities. Our data demonstrate that, there is a significant increased in structural chromosomal aberrations and no increase in nu- merical aberrations. Plas et al. (2000) recorded the same result in chromosomes of spermatozoa from ageing males. On the contrary, Farag and El-Nahass (1996) found that the effect of age on nu- merical aberrations was greater than on those of structural aberrations in goats. The common structural abnormalities in this study is single break which includes fragments and chromatid deletions. In this respect, Boerjan and de Boer (1988) found more chromatid types of damage in metaphases from aged zygotes compared to damage in metaphases from unaged zygotes. In addition to age, abnormalities recorded in this work, were previously recorded to be associated with other infertility problems (Mahmoud 1997, Mahmoud et al. 2004) and environmental pollution (Farghaly 2003a). Sloter et al. (2004) cited that, the adverse effect of age on chromosomes may include accumu- lation of environmental damage, reduced efficiency of DNA repair, increased genomic instability, genetic factors, hormonal influences, suppressed apoptosis, or decreased effectiveness of antioxi- dants and micronutrients. Moreover, it has been previously demonstrated that a shortening of telomeres (chromosomal ends) which occurred with ageing (Harley et al. 1990, Mariani et al. 2003). In- deed, DNA double-strand breaks was statistically significantly higher in men aged 36–57 years than in those aged 20–35 years (Singh et al. 2003). Also, the incidence of nondisjunction of paternal sex chromosome in meiosis I was higher in older men with idiopathic infertility (Asada et al. 2000). On the contrary, the absence of maternal age effect on the first meiotic division of oocytes was recorded by Golbus (1981). A significant increase in the frequency of SCE’S was observed more in adult than in young camel. These data coincide with the result of chromosome aberrations obtained in the present work. Moreover, SCE’S has high resolving statistical power and greater sensitivity than chromosome aberrations (Farghaly and Ibrahim 2003). SCE is a well known biomarker for exposure to muta- gens/carcinogens (Abe and Sasaki 1982) due to environmental pollution by chemicals (Farghaly 2003b). The increase frequency of SCE in this study may be due to increase the period of exposure to environmental pollution. With respect to total number and quality of oocytes, there are no significantly differences between young and adult camels. Inspite of no age effect in the current work, the number and the quality of oocytes are influenced by the method of oocyte recovery (Alm et al. 1997, Abdoon 2001) and season (Abdoon 2001, Hamam et al. 2001). In our study, the lower number of oocytes recovered could, perhaps, be attributed to the beginning of the breeding season (October; marked with lower follicular growth). This is also reported by Torner et al. (2003). In the present study, there are no significant difference in the quality of oocytes between young and adult camel. These results dis- agree with Palma et al. (1993) who found that the quality of calf oocytes was significantly lower than that of cow oocytes. The percentages of good quality oocytes (class A and B) was about two third in both ages. Similar percentage was reported in camel (Mahmoud et al. 2003) and buffalo (Mahmoud 2001). In this respect, Abdoon et al. (2001) reported that good quality buffalo oocytes surrounded by multi-layers of compact investment with a homogenous ooplasm had a significantly higher cleavage, and developmental rates up to the morula stage compared with oocytes of fair or poor quality. 300 Karima Gh. M. Mahmoud et al. Cytologia 70(3)

Cytogenetic analysis of immature camel oocytes revealed that 25.700.38% and 21.30.91% were at GVBD and condensed stage in young and adult camels respectively. Ghoneim et al. (1999) found that 54.9% of pregnant and 52.6% of non pregnant camel were in resumption of meiosis at the time of recovery. Le Beux et al. (2003) recorded lower percentage (13%) of pig oocytes at GVBD before culture. The fact that some of the oocytes were already in the state of meiotic progression just after collection were indicative that these oocytes were collected from follicles where maturation process was already in progress in vivo. The observation that 74.290.38% of oocytes in young and 78.690.915% of oocytes in adult at GV just after collection confirmed the suitability of such oocytes for culture. However, Kafi et al. (2002) in camel and Datta and Goswami (1999) in buffalo reported higher percentage of oocytes at GV stage just after collection. Lower percentage of GV stages oocytes found in the present study was probably due to difference in criteria used for selection of oocytes. So more proper criteria are needed for the selection and evaluation of camel oocytes before and after culture. In this respect, de Loos and Kruip (1988) atributed the variation in success of in vitro fertilization to the heterogenesity of bovine cumulus oocyte complexes before onset of culture. With regard to age, the GV stage was significantly higher in adult than young camels at the time of collection. Our data may explain the results of previous studies about in vitro fertilization in relation to age. Many authors concluded that the use of calf ovaries is less efficient than the use of adult ovaries in the in vitro fertilization programme (Palma et al. 1993, Revel et al. 1995, Martino et al. 1995). Moreover, the poor in vitro maturation (IVM), fertilization and embryonic development of fetal oocytes may be due to higher incidence of blockage at GV and metaphases-1 stage after IVM (Chohan and Hunter 2004). In contrast, Jainudeen et al. (1993) reported that higher rate of in vitro maturation in prepubertal than adult buffaloes. In conclusion, ages may have significant effects on structural chromosomal aberrations, SCE’S and meiotic stage of immature camel oocyte but no effect on t he number and quality of oocytes.

References

Abdel-Raouf, M., El-Bab, M. R. and Owaida, M. M. 1975. Studies on reproduction in t he camel (Camelus dromedarius) V. morphology of the testis in relation to age and season. J. Reprod. Fertil. 43: 109–116. Abdoon, A. S. 2001. Factors affecting follicular population, oocytes yield and quality in camels (Camelus dromedarius) ovary with special reference to maturation time in vitro. Anim. Reprod. Sci. 66: 71–79. —, Kandil Omaima, M., Otoi, T. and Susuki, T. 2001. Inﬂuence of oocyte quality, culture media and gonadotropins on cleavage rate and development of in vitro fertilized buffalo embryos. Anim. Reprod. Sci. 65: 215–223. Abe, S. and Sasaki, M. 1982. SCE as an index of mutagenesis and/or carcinogenesis. In: Sandberg, A. A. (ed.). Sister chromatid exchange. Alan Liss, New York. pp. 461–514. Alm, H., Torner, H., Kanitz, W., Becker, F. and Hinrichs, K. 1997. Comparison of different methods for recovery of horse oocytes. Equine Vet. J., Suppl. 25: 47–50. Al-Qarawi, A. A., Abdel-Rahman, H. A., El-Belely, M. S. and El-Mougy, S. A. 2000. Age-related changes in plasma testos- terone concentrations and genital organs content of bulk and trace elements in the male dromedary camel. Anim. Reprod. Sci. 62: 297–307. Asada, H., Sueoka, K., Hashiba, T., Kuroshima, M., Kobayashi, N. and Yoshimura, Y. 2000. The effects of age and abnormal sperm count on the nondisjunction of spermatozoa. J. Assist Reprod. Genet. 17: 51–59. Bezrukov, N. I. and Shmidt, G. A. 1970. Age and functional changes in the ovaries and uterus of the two-humped camel. Arkh Anat Gistol Embriol. 58: 64–73. Boerjan, M. L. and de Boer, P. 1988. Post-ovulatory changes in mouse oocytes, a study of fertilization kinetics and DNA-repair capacity. 11th International Congress on Animal Reproduction and Artiﬁcial Insemination. Dublin, Ireland. pp. 321–323. Brinsko, S. P., Ball, B. A., Miller, P. G. and Thomas, P. G. A. 1994. In vitro development of day two embryos obtained from young, fertile mares or aged, subfertile mares. Theriogenology 41: 169. Chian, R. C., Nakahara, H., Niwa, K. and Funahashi, H. 1992. Fertilization and early cleavage in vitro of aging bovine oocytes after maturation in culture. Theriogenology 37: 665–672. Chohan, K. R. and Hunter, A. G. 2004. In vitro maturation, fertilization and cleavage rate of bovine fetal oocytes. Theri- 2005 Chromosomal Aberrations, Sister Chromatid 301

ogenology 61: 373–380. Datta, T. K. and Goswami, S. L. 1999. Time dynamics and chronology of meiotic progression of buffalo (Bubalus bubalis) oocytes during in vitro maturation. Buffalo J. 1: 53–60. Del Campo, M. R., Del Campo, C. H., Donoso, M. X. and Berland, M. 1994. In vitro fertilization of Llama (Lama glama) follicular oocytes. Theriogenology 41: 187 (Abstr.). de Loos, F. and Kruip, Th. A. M. 1988. Ultrastructure of immature bovine oocytes. 11th International Congress on Animal Reproduction and Artificial Insemination. Dublin, Ireland. pp. 323–325. Farag, I. M. and El-Nahass, E. 1996. The effect of age on chromosomes of goats. Bull. NRC, Egypt 21: 191–198. Farghaly, A. A. 2003a. Mutagenic evaluation of paracetamol in somatic cells and germ cells of mice. Cytologia 68: 133–139. — 2003b. Anticytogenetic effect of vitamin C against cytogenicity of aluminium sulphate in mice. Bull. NRC, Egypt 28: 671–683. — and Ibrahim, A. A. 2003. The protective role of folic acid on the mutagenicity induced by sodium sulfite in different tis- sues of male mice. Bull. NRC, Egypt 28: 749–760. Ghoneim, I. M., Torner, H., Heleil, B., Srsen, V., Fattouh, E. M. and Alm, H. 1999. In vitro maturation of cumulus oocyte complexes of pregnant and non-pregnant Camelus dromaderius.J. Egypt. Vet. Med. Ass. 59: 1649–1659. Golbus, M. S. 1981. The influence of strain, maternal age, and method of maturation on mouse oocytes aneuploidy. Cyto- genet. Cell Genet. 31: 84–90. Goto, K., Maeda, S., Kano, Y. and Sugiyama, T. 1978. Factors involved in differential Giemsa staining of sister chromatids. Chromosoma 66: 351–359. Hamam, A. M., Mahmoud, Karima, Gh. M., Nawito, M. F., Seida, A. A. M. and Nawar, S. M. A. 2001. Effect of the season- al changes on recovery, quality and maturation of buffalo oocytes in vitro. Egypt. J. Vet. Sci. 35: 123–133. Harley, C. B., Futcher, A. B. and Greider, C. W. 1990. Telomeres shorten during ageing of human fibroblasts. Nature 345: 458–460. Hassanane, M. S. and Omer, M. A. 2001. Chromosomal studies on the dromedarius camels raised in Egypt. The New Egyptian Journal of Medicine 24: 57–62. Jainudeen, M. R., Takahashi, Y., Nihayah, M. and Kanagawa, H. 1993. In vitro maturation and fertilization of swamp buffalo (Bubalus bubalis) oocytes. Anim. Reprod. Sci. 31: 205–212. Kafi, M., Nilli, H. and Mesbah, F. 2002. Changes in ultrastructure/timing of in vitro maturation of camel oocytes. Adv. Re- prod. 6: 68. Khatir, H., Anouassi, A. and Tibary, A. 2004. Production of dromedary (Camelus dromedaries) embryos by IVM and IVF and co-culture with oviductal or granulosa cells. Theriogenology 62: 1175–1185. Kidd, S. A., Eskenazi, B. and Wyrobek, A. J. 2001. Effects of male age on semen quality and fertility: A review of the literature. Fertil Steril. 75: 237–248. Kuzmina, T. I., Torner, H., Alm, H. and Kanitz, W. 2003. Effect of doner’s age on bovine oocyte maturation and early embryonic development in vitro. EAAP-54th Annual Meeting, Rome. pp. 225. Le Beux, G., Richard, F. J. and Sirard, M. 2003. Effect of cycloheximide, 6-DMAP, roscovitine and butyrolactone I on resumption of meiosis in porcine oocytes. Theriogenology 60: 1049–1058. Mahmoud, K. Gh. M. 1997. Some studies on infertilty of buffaloes with reference to chromosomal aberrations. M. V. Sc. Thesis (Theriogenology), Faculty Veterinary Medicine Cairo University. — 2001. Cytogenetic studies on in vitro fertilization in buffaloes. Ph. D. Thesis, (Theriogenology) Fac. Vet. Med., Cairo University. —, El-Shahat, K. H. and El-Nattat, W. S. 2003. Chromosome configuration during in vitro maturation of dromedary camel oocytes. Vet. Med. J. Giza 51: 411–420. —, Ziada, Maha, S. and Scholkamy, T. H. 2004. Freezability of buffalo semen in relation to its in vitro fertilizing capacity and chromosomal picture. J. Egypt. Vet. Med. Assoc. 64: 239–251. Mariani, E., Meneghetti, A., Formentini, I., Neri, S., Cattini, L., Ravaglia, G., Forti, P. and Facchini, A. 2003. Telomere length and telomerase activity: effect of ageing on human NK cells. Mechanisms of Ageing and Development 124: 403–408. Martino, A., Morgas, T., Palomo, M. J. and Paramio, M. T. 1995. In vitro maturation and fertilization of prepubertal goat oocytes. Theriogenology 43: 473–485. Palma, G. A., Clement-Sengewald, A. and Krefft, H. 1993. In vitro production of cattle embryos from calf oocytes. Theri- ogenology 39: 278. Plas, E., Berger, P., Hermann, M. and Pfluger, H. 2000. Effects of aging on male fertility. Exp. Gerontol. 35: 543–551. Ratto, M., Berland, M., Huanca, W., Singh, J. and Adams, G. P. 2004. In vitro and in vivo maturation of llama oocytes. The- riogenology, in press. Revel, F., Mermillod, P., Peynot, N., Renard, J. P. and Heyman, Y. 1995. Low developmental capacity of in vitro matured and fertilized oocytes from calves compared with that of cows. J. Reprod. Fertil. 103: 115–120. 302 Karima Gh. M. Mahmoud et al. Cytologia 70(3)

Rezk, W. A. Kh. 2005. Differences between farm animals in (IVM and IVF) in vitro maturation and in vitro fertilization. M. Agric. Sci (animal production), Faculty of Agriculture. Mansoura University. Robbins, W. A., Baulch, J. E., Moore, D., Weier, H. U., Blakey, D. and Wyrobek, A. J. 1995. Three-probe ﬂuorescence in situ hybridization to assess chromosome X, Y, and 8 aneuploidy in sperm of 14 men from two healthy groups: evi- dence for a paternal age effect on sperm aneuploidy. Reprod. Fertil. Dev. 7: 799–809. Singh, N. P., Muller, C. H. and Berger, R. E. 2003. Effects of age on DNA double-strand breaks and apoptosis in human sperm. Fertil Steril. 80: 1420–1430. Sloter, E., Nath, J., Eskenazi, B. and Wyrobek, A. J. 2004. Effects of male age on the frequencies of germinal and heritable chromosomal abnormalities in humans and rodents. Fertil Steril. 81: 925–943. Snedecor, G. W. and Cochran, W. G. 1982. Statistical Methods, 7th ed., Iowa Univ. Press, U.S.A. Tarkowski, A. K. 1966. An air-drying method for chromosome preparations from mouse eggs. Cytogenetic 5: 394–400. Taylor, K. M., Hungerford, D. A., Snyder, R. L. and Ulmer, F. A. 1968. Uniformity of karyotypes in the camelidae. Cytoge- netics 7: 7–15. Tease, C. and Fisher, G. 1986. Oocytes from young and old female mice respond differently to colchicine. Mut. Res. 173: 31–34. Torner, H., Heleil, B., Alm, H., Ghoneim, I. M., Srsen, V., Kanitz, W., Tuchscherer, A. and Fattouh, E. M. 2003. Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedaries) during maturation in vitro. Theriogenology 60: 977–987. Tucker, J. D., Spruill, M. D., Ramsey, M. J., Director, A. D. and Nath, J. 1999. Frequency of spontaneous chromosome aberrations in mice: Effects of age. Mutat. Res. 425: 135–141.