Kinetics of nuclear maturation and effect of holding ovaries at room temperature on in vitro maturation of camel (Camelus dromedarius) oocytes

Experiments were conducted to investigate kinetics of in vitro nuclear maturation and the effect of storing ovaries at room temperature on initial chromatin configuration and in vitro maturation of dromedary camel oocytes. Cumulus oocyte complexes (COCs) were collected from slaughterhouse ovaries and matured in vitro for 4-48h. At every 4h interval (starting from 0 to 48 h), groups of oocytes were fixed, stained and evaluated for the status of nuclear chromatin. Oocytes were categorized as germinal vesicle (GV), diakinesis (DK), metaphase-I (M-I), anaphase-I (A-I), metaphase-II (M-II) stage and those with degenerated, fragmented, activated or without a visible chromatin as others. At the start of culture, 74% (66/89) oocytes were at GV stage, 13% (12/89) at DK and 12% (11/89) were classified as others. Germinal vesicle breakdown started spontaneously in culture and at 20 h of culture 97% oocytes had already completed this process. After 8 and 16 h of maturation the highest proportion of oocytes (42%, 48/114 and 41%, 51/123) were at DK and M-I stage, respectively. The proportions of oocytes reaching M-II stage at 32 (42%, 50/118), 36 (45%, 47/104), 40 (49%, 57/117), 44 (52%, 103/198) and 48 h (46%, 55/120) of culture were not different from each other (P>0.05). The proportion of oocytes categorized as others, however, increased after 40 h of culture and was higher (P<0.05) at 48 h compared with other maturation periods. There was no difference (P>0.05) in the proportion of oocytes reaching M-II stage from the ovaries collected and stored in normal saline solution (NSS) at room temperature for 12h (43%, 64/148) and those collected in warm NSS (37 degrees C) and processed immediately after arrival in laboratory (49%, 57/117). However, low number of oocytes reached M-II stage from ovaries collected in warm NSS but stored at room temperature (29%, 37/128) compared with other two groups (P<0.05). It may be concluded that dromedary oocytes require 32-44h of in vitro culture to have an optimum number of oocytes in M-II stage. However, further studies are required to find out the most appropriate maturation period, which will result in the further development of these oocytes after IVF, ICSI, parthenogenetic activation or nuclear transfer. Ovaries can be collected and stored in normal saline solution at room temperature for 12h without any appreciable effect on the nuclear maturation of the oocytes.     

Spermatogenesis in the camel (Camelus dromedarius)

Spermatogenesis was studied with the aid of the light and electron microscopes in fourteen sexually mature camels slaughtered at different times of the year. The testes were fixed by vascular perfusion with glutaraldehyde. Spermatogenesis in the camel was generally similar to that of most mammalian species, although some features specific for the camel were observed. Spermatogenesis was found to be continuous throughout the year.     

Chemical activation of in vitro matured dromedary camel (Camelus dromedarius) oocytes: optimization of protocols

Experiments were conducted to study the efficiency of sequential treatments of ionomycine and ethanol combined with phosphorylation inhibitor (6-dimethylaminopurine) or the specific maturation promoting factor inhibitor (roscovitine) in inducing artificial activation in dromedary M-II oocytes. Cumulus oocyte complexes (COCs), collected from slaughterhouse ovaries were cultured at 38.5 degrees C in an atmosphere of 5% CO2 in air for 24-48 h. In experiment 1, the COCs were either fertilized in vitro or activated with 5 microM ionomycine for 5 min or 7% ethanol for 7 min, both followed by exposure to 6-diethylaminopurine or roscovitine for 4h. After 14-15 h of in vitro culture, the oocytes were fixed and stained with 1% aceto-orcein to evaluate their nuclear status. In experiment 2, the oocytes were activated in the same manner as in experiment 1 but were cultured for 7 days to evaluate their post-parthenogenetic development. In experiment 3, oocytes were exposed to the ionomycine for 2, 3, 4 or 5 min to evaluate the better exposure time while as in experiment 4, the oocytes matured for 28-48 h were activated to see the effect of aging on post-parthenogenetic development. Higher proportion (P<0.01) of oocytes was activated in ionomycine/6-DMAP and ionomycine/roscovitine groups when compared with ethanol/6-DMAP, ethanol/roscovitine and in vitro fertilized groups. However, there was no difference (P>0.05) in the proportion of oocytes activated with ethanol when compared with in vitro fertilized group. No significant difference was seen on the proportion of morula on day 7 of culture, however the development to blastocyst stage was higher (P<0.01) in ionomycine/6-DMAP and ionomycine/roscovitine when compared with ethanol/6-DMAP and ethanol/roscovitine treated oocytes. A higher proportion of oocytes reached blastocyst stage when they were exposed to ionomycine for 3 min but they were not significantly different from the others (P>0.05). The proportion of blastocysts obtained was higher (P<0.05) in oocytes activated after 28 h of maturation when compared with oocytes activated after 32, 36, 40, 44 and 48 h of maturation. In conclusion, a protocol for chemical activation of dromedary camel oocytes with ionomycine/6-DMAP is demonstrated and optimized in the present study for further use in the development of assisted reproductive techniques in this species.     

Forestomach motility in the camel (Camelus dromedarius)

There is a cyclical pattern of motility in compartments 1 and 2 of the forestomach of the camel which can be categorized into A- and B-contractions. An average motility cycle is composed of 7 A- and 5 B-contractions and lasts 5 min, including a pause of 2.3 min. The glandular sacs within the caudal sac of compartment 1 contract 1.7 sec earlier than the caudal sac. The proximal part of the canal between compartment 2 and 3 contracts 1.2 sec prior to the distal part. Forestomach motility is stimulated by distention of the cranial sac of compartment 1 and inhibited by distention of the tubiform portion of compartment 3.     

The locomotion of the camel (Camelus dromedarius)

The feet and gaits of many camels Camelus dromedarius were studied and filmed in Mauritania, Africa. The camel has a digitigrade stance, large feet to support the animal in soft sand, and soles of flexible pads that step readily onto small stones where necessary. The walking stride is long and slow, with the body supported for much of each stride on the two right or two left legs. The pattern of supporting legs was significantly different in slow compared to fast walking camels, and in young compared to adult camels and compared to adults pulling water at the wells. There was no difference in pattern in one individual's walk, when it was either loaded or unloaded. The angles that the leg bones made with each other and with the horizon are depicted for the walk and the pace. The camel is the only animal which paces often and never trots. The pace is an unstable gait only suitable for flat terrain such as that in deserts. It may have evolved from the pace-like walk which is by far the dominant gait in this animal, which spends most of each day walking from plant to plant browsing or grazing. The pace is not used by all camelids, as one author has claimed. The pace and the gallop were only used by the camels at wells, when the animals were chased from the water by men.     

Induction of oestrus in the camel (Camelus dromedarius) during seasonal anoestrus

Injection of 7000 i.u. PMSG induced oestrus in 7 camels during the last part of seasonal anoestrus. Mature follicles developed and a CL was formed after fertile mating. However, pregnancy was not maintained by Day 60 in the 3 females detected as pregnant by rectal palpation and increased progesterone concentrations at Day 50. A single male camel mated with 4 of the females 2-16 days after the PMSG injection, and 2 or 3 matings occurred. The failure of pregnancy after induction of oestrus and mating during seasonal anoestrus was probably due to inadequate luteal function.     

Control of luteolysis in the one-humped camel (Camelus dromedarius)

Blood plasma concentrations of 13,14-dihydro-15-keto PGF2 alpha (PGFM) were measured in groups of mature non-pregnant and pregnant camels to study PGF2 alpha release patterns around the time of luteolysis and the timing of the signal for pregnancy recognition. Injection of each of four camels with 10 and 50 mg of PGF2 alpha showed clearly that five times the dose of exogenous hormone produced five times the amount of PGFM in peripheral plasma, thereby indicating that, as in other animal species, PGFM is the principal metabolite of PGF2 alpha in the camel. Serial sampling of three non-pregnant camels on each of days 8, 10 and 12, and three pregnant camels on day 10, after ovulation for 8 h showed a significant (P < 0.05) rise in mean plasma PGFM concentrations only on day 10 in the non-pregnant, but not the pregnant, animals. A single intravenous injection of 20, 50 or 100 iu oxytocin given to three groups of three non-pregnant camels on day 10 after ovulation did not increase their basal serum PGFM concentrations. However, daily treatment of six non-pregnant camels between days 6 and 15 (n = 3) or 20 (n = 3) after ovulation with 1-2 g of the prostaglandin synthetase inhibitor, meclofenamic acid, inhibited PGF2 alpha release and thereby resulted in continued progesterone secretion throughout the period of meclofenamic acid administration. These results showed that, as in other large domestic animal species, release of PGF2 alpha from, presumably, the endometrium controls luteolysis in the dromedary camel. Furthermore, reduction in the amount of PGF2 alpha released is associated with luteal maintenance and the embryonic signal for maternal recognition of pregnancy must be transmitted before day 10 after ovulation if luteostasis is to be achieved. However, the results also indicate that, in contrast to ruminants, the release of endometrial PGF2 alpha in the non-pregnant camel may not be controlled by the release of oxytocin.     

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.     

In vitro embryo production in camel (Camelus dromedarius) from in vitro matured oocytes fertilized with epididymal spermatozoa stored at 4 degrees C

Experiments were conducted to study the effect of storing epididymal spermatozoa, in tris-tes- and tris-lactose egg yolk extenders, on their fertilizing ability and subsequent in vitro embryo development. Ovaries and testes were collected from a local slaughterhouse in normal saline solution (NSS) at 37 degrees C and on ice (0-1 degrees C), respectively. Cumulus oocyte complexes (COCs) aspirated from the follicles were randomly distributed to 4-well culture plates (20-25COCs/well) containing 500 microL of maturation medium and cultured at 38.5 degrees C in an atmosphere of 5% CO(2) in air for 36 h. Spermatozoa were collected from the cauda epididymides in syringes containing 2-3 mL of either tris-tes- or tris-lactose egg yolk extender. They were cooled down slowly and stored at refrigeration (4 degrees C) temperature. The spermatozoa were evaluated for motility and used for IVF of IVM oocytes on the day of collection and after 2, 4, 6 and 8 days of storage. On the day of IVF, spermatozoa were prepared by the swim up technique and both spermatozoa and oocytes were co-incubated at 38.5 degrees C in a humidified atmosphere of 5% CO(2) in air for 15-16 h. Presumptive zygotes were either fixed and stained with Hoechst 33342 for evaluation of fertilization or were cultured in 500 microL of the culture medium at 38.5 degrees C in an atmosphere of 5% CO(2), 5% O(2) and 90% N(2) in air. There was no significant difference (P>0.05) in the proportion of oocytes fertilized with spermatozoa stored in either of the two extenders for up to 8 days. The proportion of oocytes that cleaved (43-60%) and those that developed to blastocysts (14-21%) did not show any difference (P>0.05) either, when spermatozoa from different days of storage were used. First cleavage was observed as early as 16 h after IVF, early blastocysts had developed by day 4, expanded blastocysts after day 5 and hatching of blastocysts started after day 6 of culture. It may be concluded that dromedary epididymal spermatozoa survive in storage for at least 8 days in tris-lactose- and tris-tes egg yolk diluents at 4 degrees C. These spermatozoa maintain fertilizing ability and may be suitable for use in IVF and other assisted reproductive procedures.     

The intratesticular excurrent ducts of the camel (Camelus dromedarius)

The histology and fine structure of the epithelial cells of the intratesticular excurrent ducts were studied in material collected from fourteen adult camels and fixed by perfusion. The intratesticular excurrent ducts consisted of a terminal segment of the seminiferous tubule, a tubulus rectus, and a rete testis. The terminal segment was lined with modified Sertoli cells which formed a plug-like structure in the receptacle. The tubulus rectus was subdivided into the receptacle, the narrow main part, and the wider distal part, and these parts were lined with different types of epithelium. The rete testis occupied an axial mediastinum testis, and the height of its epithelium varied quite considerably. Degenerated spermatozoa were seen engulfed by the epithelial cells of the entire intratesticular duct system. Light cells, lymphocytes, and macrophages were observed. The fine structure of the epithelium of the intratesticular ducts is discussed in relation to its possible functions.     

Levels of glycosylated haemoglobin in the Arabian camel (Camelus dromedarius)

Proportions of glycosylated haemoglobin (Hb) were determined in 10 Arabian camels (Camelus dromedarius) and were compared with normal controls (n = 59) and diabetic patients (n = 47) using the thiobarbituric acid (TBA) method. The level of glycosylated haemoglobin (5.5%) in camels is significantly different from that of normal healthy humans (4.9%) (P less than 0.001). Whereas the glucose levels were comparable, this difference in percentages of glycosylated haemoglobin may be explained by the difference in survival time between human and camel red blood cells.     

Fine structure of epididymal spermatozoa in the camel (Camelus dromedarius)

The epididymides from five sexually mature camels were fixed by vascular perfusion with glutaraldehyde, and the spermatozoa from four different regions were studied. The fine structure of the camel spermatozoon is similar to that of related species such as bulls and boars. It is relatively short and has a short middle piece. The main morphological changes during epididymal transit are confined to the acrosome and to the position and structure of the cytoplasmic droplet.     

Fine structure of sertoli cells in the camel (Camelus dromedarius)

The testes of fourteen sexually mature camels were fixed by perfusion with glutaraldehyde. The fine structure of Sertoli cells and of their surface specializations towards other Sertoli cells and towards germ cells is described. Sertoli cell nucleolar vacuoles, like those described in several ruminant species, were not found. The general appearance of the camel Sertoli cell suggests that its function is similar to that of other mammals.     

Clinical and anatomical studies of the camel (Camelus dromedarius) genitalia

Clinical and anatomical studies of the genitalia of 294 camel (197 males and 97 females) were carried out between May 1984 and October 1985. Preslaughter (clinical) examination was followed by detailed post-mortem (anatomical) examination and dissection of genitalia at the abattoir. Measurements and weights of the various segments of genitalia were made to establish the baseline data for breeding soundness evaluation in the dromedary in Northern Nigeria.     

A review of reproduction in the female camel (Camelus dromedarius)

The Arabian camel (Camelus dromedarius), the one-humped camel, is a primary inhabitant of the northern half of Africa, the Middle East and Pakistan and India. The dromedary is a multipurpose animal used for transportation, the production of milk and meat, as well as such by-products as wool, hair and hides. Dromedaries are extremely well adapted to their hot, arid environment. Consequently, Arabian camels are of considerable economic importance. Relatively little attention has been paid to the breeding of dromedaries. In the literature, particularly that in the English language, information on reproduction in the dromedary tends to be fragmentary. This review consolidates several widely scattered reports as projected against the author's own experience. It is hoped that the information is of particular help for those in charge of the breeding of dromedaries in zoological gardens and animal parks in countries where the one-humped camel is a rare species.     

Mating-induced luteinizing hormone surge and ovulation in the female camel (Camelus dromedarius)

Blood samples were collected after mating from four female one-humped camels every 10 min for 9-12 h. Luteinizing hormone (LH) was quantified in plasma by radioimmunoassay using antibovine LH. Of the seven observed matings, five were followed by a release of LH, and three by an ovulation (indicated by a subsequent secretion of progesterone). LH levels at the time of mating ranged from 0.7 to 3 ng/ml. When an LH response occurred, the levels increased 1 h after mating and reached a maximum in 2-3 h (ranging from 2.9 to 19.1 ng/ml). A decrease in LH was observed starting 6 h after mating and lasting for 6 h. These results are in agreement with a coitus-induced mechanism of ovulation in the one-humped camel (Camelus dromedarius). They confirm and extend the observations reported in the bactrian camel (Camelus bactrianus).     

Synchronisation of ovarian follicular waves in the dromedary camel (Camelus dromedarius)

This study was designed to compare the efficacy of various treatments intended to synchronise follicular wave cycles in dromedary camels by removing the existing follicle of unknown size and replacing it with a follicle capable of ovulating at a known time. Camels were randomly assigned to one of five groups and treated with either (1) 5mg oestradiol benzoate (i.m.) and 100mg progesterone (i.m.; E/P, n=15), (2) 20 icrog GnRH analogue, buserelin (i.m.; GnRH, n=15), (3) 20 microg buserelin (i.m.) on Day 0 (T=0) and 500 microg prostaglandin on Day T+7 (GnRH/PG n=15), (4) transvaginal ultrasound-guided follicle ablation of all follicles > or =0.5 cm (ABL, n=15) or (5) 5 ml saline (i.m; Controls n=15). All camels were subsequently injected with 20 microg buserelin 14 days after the first treatment was given. The ovarian response was monitored daily by transrectal ultrasonography and the intervals from treatment to follicular wave emergence and also the day on which the new dominant follicle reached 1.3 cm was recorded. Amongst the treatment groups the mean interval from treatment to new follicle wave emergence and treatment to time taken for the new dominant follicle to reach 1.3 cm in diameter was shortest in the ABL group (2.3+/-0.5 days and 8.8+/-1.1 days respectively, P=0.044) and longest in the E/P group (6.4+/-0.8 days and 12.2+/-1.0 days respectively, P<0.001) whereas the GnRH and GnRH/PG groups were intermediate (3.0+/-0.5 days and 11.1+/-0.8 days GnRH; and 4.5+/-0.5 days and 10.7+/-0.7 days GnRH/PG). A total of 11/15 camels in both the GnRH and GnRH/PG groups had dominant follicles between 1.3 and 1.9 cm 14 days post treatment, of which 21 of the 22 follicles ovulated after GnRH injection on T+14. The ABL, E/P and control groups however, showed greater variability in follicle size with less camels having dominant follicles between 1.3 and 1.9 cm than the GnRH and GnRH/PG groups and more in the > or =2.0 cm or follicle regressing groups, therefore fewer of these camels ovulated (ABL n=7; E/P n=9; Control n=6) after GnRH injection on Day T+14. In conclusion, two GnRH injections 14 days apart or two GnRH injections 14 days apart and PG on Day 7 after the first GnRH were the most effective methods to synchronise ovulation rate in dromedary camels at a fixed time interval of 14 days after treatment.     

Offspring resulting from transfer of cryopreserved embryos in camel (Camelus dromedarius)

The dromedary embryos, collected at hatched blastocyst stage, survived freezing and thawing in the presence of a high concentration of ethylene glycol (7.0 mol/L) with sucrose (0.5 mol/L) and direct plunging in liquid nitrogen. The rate of survival, as judged by the morphological appearance of the embryos after thawing, was high (92%). The transfer of frozen-thawed embryos into the recipients during the breeding (n=20) and non-breeding season (n=25) resulted in two and one pregnancy, respectively. One of the two pregnant recipients, with embryos transferred during the breeding season, delivered a normal healthy male calf at term. To our knowledge, this offspring is the first camelid produced following transfer of a frozen-thawed embryo.