Chromosomal Aberrations, Sister Chromatid Exchanges and Nuclear Status of Immature Oocytes in Relation to Age of Dromedary Camels
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© 2005 The Japan Mendel Society Cytologia 70(3): 295–301, 2005 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 (pϽ0.05) 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 (pϽ0.01) differences were between percentages of germinal vesicle break- down (GVBD) and Germinal vesicle (GV) in young and adult camels. It is concluded that, the in- crease 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 im- portant 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 chro- matid 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 chromoso- mal 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 be- fore harvesting. Then after hypotonic treatment and fixation, differential staining of sister chro- matids 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 denud- ed 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 (pϽ0.05) with age. The percentage reached 6.83Ϯ0.68 in adult compared with 4.0Ϯ0.53 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 (meanϮS.E.) Animals of abnormal metaphases animals metaphases Including gaps Excluding gaps Gaps Fragment Break Deletion Young 12 600 24 4.0Ϯ0.53 3.0Ϯ0.58 6 8 5 5 Adult 12 600 41 6.83Ϯ0.68* 5.34Ϯ0.45 9 14 10 8 * pϽ0.05 (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 meanϮS.E. with SCE’S Young 12 300 869 2.89Ϯ0.47 Adult 12 300 1552 5.17Ϯ0.51* * pϽ0.05 (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 AϩB Class CϩD No. % No. % Young 24 164 6.61Ϯ0.38 104 65.74Ϯ3.47 60 34.25Ϯ3.47 Adult 24 139 5.77Ϯ0.43 93 68.81Ϯ4.05 46 31.18Ϯ4.05 The results are expressed in meanϮS.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 74.29Ϯ0.38 20 25.70Ϯ0.38** Adult 82 52 41 78.69Ϯ0.91** 11 21.3Ϯ0.91 The results are expressed in meanϮS.E.M.