Developmental characteristics of aggregated lymphoid nodules area in the abomasum of fetal Bactrian camels (Camelus bactrianus).

BACKGROUND: Bactrian camel is one of the important economic animals in northwest China. They live in arid desert, and their gestation period is about 13 months, which is longer than other ruminants (such as cattle and sheep). The harsh living conditions have made its unique histological characteristics a research focus. Aggregated lymphoid nodules area (ALNA) in the abomasum of Bactrian camels, as one of the most important sites for the induction of the immune response, provide a comprehensive and effective protective role for the organism, and their lack of information will affect the feeding management, reproduction and epidemic prevention of Bactrian camels. In this study, the histological characteristics of the fetal ALNA in the abomasum of Bactrian camels at different developmental gestation have been described by using light microscopy and histology . RESULTS: The ALNA in the abomasum of the Chinese Alashan Bactrian camel is a special immune structure that was first discovered and reported by Wen-hui Wang. To further establish the developmental characteristics of this special structure in the embryonic stage, the abomasum ALNA of 8 fetuses of Alashan Bactrian camels with different gestational ages (5~13 months) were observed and studied by anatomy and histology. The results showed that the aggregation of reticular epithelial cells (RECs) surrounded by a very small number of lymphoid cells was detected for the first time in the abomasum of fetal camel at 5 months gestation, which was presumed to be primitive ALNA. At 7 months gestation, the reticular mucosal folds region (RMFR) appeared, but the longitudinal mucosal folds region (LMFR) was not significant, and histological observations showed that there were diffusely distributed lymphocytes around the RECs. At 10months gestation, RMFR and LMFR were clearly visible, lymphoid follicles appeared in histological observation, lymphocytes proliferated vigorously. By 13 months, the volume of lymphoid follicles increased, forming the subepithelial dome (SED), and there was a primitive interfollicular area between the lymphoid follicles, which contained high endothelial vein (HEV), but no germinal center (GC) was found. In summary, ALNA of Bactrian camels is not fully mature before birth. CONCLUSIONS: Generally, the small intestine PPs of ruminants (such as cattle and sheep) is already mature before birth, while the ALNA in the abomasum of Bactrian camels is not yet mature in the fetal period. During the development of ALNA in Bactrian camel, the development of lymphoid follicles extends from submucosa to Lamina propria. Interestingly, the deformation of FAE changes with age from simple columnar epithelium at the beginning of pregnancy to Simple cuboidal epithelium, which is opposite to the FAE deformation characteristics of PPs in the small intestine of fetal cattle and sheep. These results are the basis of further research on the specificity of ALNA in the abomasum of Bactrian camels.

Successful vitrification of dromedary camel embryos using a novel embryo vitrification kit.

Embryos (n = 87) collected 8 days after mating and 7 days after ovulation were vitrified using a camel-specific vitrification kit. Vitrification solutions (VS) containing 20% foetal calf serum, with or without 2% bovine serum albumin (BSA) were used to cryopreserve embryos, in three steps VS1 (5 min), VS2 (5 min) and VS3 (30-35 s) at room temperature (RT) before being loaded into open pulled straws and immediately frozen in liquid nitrogen. Embryos were subsequently thawed in warming solutions (WS) in three steps: WS1 at 37 °C (1 min), WS2 at RT (5 min) then into holding media at RT (5-60 min) prior to transfer, in pairs, into recipient camels 6 days after ovulation. There were 42 of 43 embryos viable after vitrification in media without BSA and these were subsequently transferred into 21 recipient females which resulted in ten pregnancies 60 days after transfer (48% pregnancy rate). There were 38 of 44 viable embryos vitrified in media containing BSA that were transferred in pairs into 19 recipient females which resulted in five pregnancies 60 days after transfer (26% pregnancy rate; P > 0.05). Of the total 15 foetuses that developed to 60 days of gestation after vitrification, 11 resulted from embryos of 200-499 µm diameter and four from embryos of 500-700 µm diameter (P > 0.05). There were encouraging results with use of this novel vitrification kit for the commercial application of cryopreservation of camel embryos with a diameter of 300-550 µm.

Preservation of dromedary camel embryos at 4 °C for up to 5 days: Factors affecting the pregnancy and pregnancy loss rates.

The aims of the present study were to evaluate the effect of cooling of the dromedary camel embryos on the pregnancy and pregnancy loss rates, and to investigate the factors which might affect the outcomes of the transfer of cooled embryos. After the donors (n = 56) had been super-ovulated and mated, they were flushed at Day 8 or 9 post-mating. Of 487 collected embryos, 110 were refrigerated at 4°C for up to 5 days in holding medium (HM), flushing medium supplemented with 10% fetal calf serum (FM + FCS) or TCM199 supplemented with 50% FCS and HEPES (TCM + FCS + HEPES). Both fresh (n = 377) and cooled embryos were transferred individually into synchronized recipients. Pregnancy diagnoses were carried out at Days 18-19, 30 and 60 post-mating of the donors. Transferring of fresh embryos into the recipients resulted in significantly higher pregnancy rates at Days 18-19 (53.1% vs. 38.2%, P < 0.01), Day 30 (46.4% vs. 31.8%, P < 0.01) and Day 60 (42.4% vs. 26.4%, P < 0.005) compared with those of cooled embryos, respectively. Pregnancy rates after transferring cooled embryos progressively decreased with the prolongation of the storage period. A significant difference in the pregnancy rate (56% vs. 13%, respectively, P < 0.05) was recorded only at Days 18-19 between cooled embryos held for one day and those held for 5 days. The pregnancy rates at Days 18-19, Day 30 and Day 60 were non-significantly higher when TCM + HEPES and FCS medium used for cooling of embryos compared to those of FM + FCS or HM medium. Cooling of spherical embryos resulted in significantly higher pregnancy rates at Days 30 (45.6% vs. 17.0%, respectively, P < 0.005) and 60 (42.1 vs. 9.4%, respectively, P < 0.005) and a significantly lower pregnancy loss rate (11.1% vs. 66.6%, respectively, P < 0.005) compared to those resulting from cooling of folded embryos. Neither the size of embryo nor the day of flushing had a significant effect on the pregnancy and pregnancy loss rates after the transfer of cooled embryos. In conclusion, pregnancy could be obtained after the transfer of camel embryos refrigerated for up to 5 days. In addition, higher pregnancy rates could be obtained if only spherical embryos are selected for cooling.

Molecular cloning and cellular expression of the cholesterol synthesizing enzymes during the prenatal development of the optic nerve in the dromedary camel (Camelus Dromedarius).

The Cholesterol-synthesizing proteins (HMGCS1 and HMGCS2) are mitochondrial enzymes that believed to catalyze the first reaction of ketogenesis, the process by which energy is provided from fats in the absence of carbohydrates. Typically, astrocytes developed from its progenitor cells in the embryonic optic nerve and enriched with HMGCS1 and 2. However, the detailed histomorphology of camel HMGCS1 and 2 remains to be clearly defined. Here, we investigated the changes that associate with astrocytes differentiation within the developing camel optic nerve. Firstly, we isolated cDNAs encoding HMGCS1 and 2 from the optic nerve. Then, we found that HMGCS1 shared high similarity to human, while HMGCS2 showed a lower similarity and was more diverse. Immunohistochemical studies revealed that distinct correlation of astrocytes differentiation with HMGCS1 and 2 expressions in the developing camel optic nerve. Both encoded proteins were localized throughout the cytoplasm, as well as the nuclei of the astrocytes. In addition, semi-quantitative PCR analysis and western analysis confirmed that both HMGCS1 and 2 were highly expressed in camel optic nerve as well as other tissue, but they were lower in both skeletal and heart muscles. Moreover, various stains such as Sudan black and florescence filipin stains were used to visualize the free cholesterol in the astrocytes, indicating the enzymatic activity of HMGCS1 and 2. Together, our study reported the first comprehensive investigation of the molecular cloning and cellular expression of HMGCS1 and 2 in the optic nerve of dromedary camel.

Effect of macromolecule supplement on nuclear and cytoplasmic maturation, cryosurvival and in vitro embryo development of dromedary camel oocytes.

The current evaluation of oocyte vitro maturation (IVM) media has progressed toward more defined conditions in human and livestock. In this study, the replacement of fetal calf serum (FCS) with bovine serum albumin (BSA) and polyvinyl alcohol (PVA) was evaluated during IVM in dromedary camel. Nuclear maturation rates in presence of FCS and PVA were comparable (81.6 ±â€¯1 and 75.5 ±â€¯5%, respectively). BSA, whether used alone or in combination with FCS, significantly reduced nuclear maturation (51.6 ±â€¯3.9 and 54.6 ±â€¯1.1%, respectively), compared to FCS and PVA. BSA also increased the rates of chromosome aberrations compared to FCS and PVA (25.7 ±â€¯7.4, 8.8 ±â€¯2.3 and 6.0 ±â€¯2.0%, respectively). IVM macromolecule differentially affected morphological aspects of cumulus expansion and FCS promoted the highest dissociation of cumulus cells, compared to all the other groups. FCS significantly increased mean lipid intensity of oocytes compared to BSA, FCS-BSA and PVA which could explain the lower cryo-survival of oocytes matured in presence of FCS compared to BSA and PVA (56.1 ±â€¯5.2, 91.0 ±â€¯19.5, and 87.8 ±â€¯6.7%, respectively). Mitochondrial activity was not affected by macromolecules, but oocytes cultured with PVA had the best redox status, compared to other IVM groups. Cleavage was not affected by IVM macromolecule, but FCS promoted significantly higher rate of morula development (51.6 ±â€¯5.2 vs. 33.6 ±â€¯2.9% for PVA) and blastocyst development (36.8 ±â€¯1.4 vs. 20.5 ±â€¯2.0% for BSA). Although adding FCS during IVM supported highest hatching rate of the resulting blastocysts, differential cell number showed no long lasting effect of IVM macromolecules on blastocyst quality. Obtained results suggest the possibility to switch from undefined to more defined IVM systems for efficient in vitro maturation and subsequent vitrification of dromedary camel oocytes. Keywords: camel, oocyte maturation, protein supplement, cryosurvival.

Development of a modified method of handmade cloning in dromedary camel.

In this study, a modified method of handmade cloning (m-HMC), which had been originally developed in sheep, was used for somatic cell nuclear transfer (SCNT) in the dromedary camel. The unique feature of m-HMC over current SCNT methods lies in the use of a simple device (a finely drawn micropipette made of Pasteur pipette) for chemically-assisted enucleation of oocytes under a stereomicroscope with improved efficiency and ease of operation. Using this system, the throughput of cloned embryo reconstitution was increased over 2-fold compared to the control SCNT method (c-NT). Stepwise measurement of reactive oxygen species (ROS) revealed that method, steps, and duration of SCNT all influenced oxidative activity of oocytes, but their impact were not similar. Specifically, UV-assisted oocyte enucleation was identified as the major source of ROS production, which explained significantly higher total ROS of reconstituted embryos in c-NT compared to m-HMC. Fusion efficiency (95.3±3.3 vs. 75.4±7.6%) and total efficiency of blastocyst development (22.5±3.0 vs. 14.1±4.3%) were significantly higher in m-HMC compared to c-NT, respectively, and blastocysts of transferable quality were obtained in similar rates (41.9±8.2 vs. 48.0±15.2%, respectively). Significance differences were observed in total cell number (155.3±13.6 vs. 123.6±19.5) and trophectoderm (145±9.5 vs. 114.3±15.2), but not inner cell mass (10.3±4.1 vs. 9.3±5.3) counts between blastocysts developed in c-NT compared to m-HMC, respectively. However, expression of key developmental genes (POU5F1, KLF4, SOX2, MYC, and CDX2) was comparable between blastocysts of both groups. The introduced m-HMC method might be a viable approach for efficient production of dromedary camel clones for research and commercial utilization.

Production of blastocysts following in vitro maturation and fertilization of dromedary camel oocytes vitrified at the germinal vesicle stage.

Cryopreservation of oocytes would serve as an alternative to overcome the limited availability of dromedary camel oocytes and facilitate improvements in IVP techniques in this species. Our goal was to develop a protocol for the vitrification of camel oocytes at the germinal vesicle (GV) stage using different cryoprotectant combinations: 20% EG and 20% DMSO (VS1), 25% EG plus 25% DMSO (VS2) or 25% EG and 25% glycerol (VS3) and various cryo-carriers; straws or open pulled-straw (OPS) or solid surface vitrification (SSV); and Cryotop. Viable oocytes were cultured in vitro for 30 h. Matured oocytes were fertilized with epididymal spermatozoa and then cultured in vitro in modified KSOMaa medium for 7 days. Survival and nuclear maturation rates were significantly lower (P ≤ 0.05) in oocytes exposed to VS3 (44.8% and 34.0%, respectively) than those exposed to VS1 (68.2% and 48.0%, respectively) and VS2 (79.3% and 56.9%, respectively). Although recovery rates were significantly lower (P ≤ 0.05) in SSV and Cryotop vitrified oocytes (66.9% to 71.1%) than those vitrified by straws with VS1 or VS2 solutions (86.3% to 91.0%), survival rates were higher in the SSV and Cryotop groups (90.7% to 94.8%) than in the straw and OPS groups (68.2% to 86.5%). Among vitrified groups, maturation and fertilization rates were the highest in the Cryotop-VS2 group (51.8% and 39.2%, respectively). These values were comparable to those seen in the controls (59.2% and 44.6%, respectively). Cleavage (22.5% to 27.9%), morula (13.2% to 14.5%), and blastocyst (6.4% to 8.5%) rates were significantly higher (P ≤ 0.05) in SSV and Cryotop groups than in straws. No significant differences were observed in these parameters between the Cryotop and control groups. We report for the first time that dromedary oocytes vitrified at the GV-stage have the ability to be matured, fertilized and subsequently develop in vitro to produce blastocysts at frequencies comparable to those obtained using fresh oocytes.

Source, treatment and type of nuclear donor cells influences in vitro and in vivo development of embryos cloned by somatic cell nuclear transfer in camel (Camelus dromedarius).

Experiments were conducted to evaluate the effect of source, treatment and type of nuclear donor cells on embryonic and fetal development of somatic cell nuclear-transfer reconstructs in dromedary camel. In experiment 1, actively growing, serum starved or confluent skin fibroblast cells were used as nuclear donors. In experiment 2, skin fibroblasts from 4 different animals while in experiment 3, skin fibroblasts and cumulus cells from the same animal were used as nuclear donors. In all the three experiments, mature oocytes collected by transvaginal ovum pick up were used as recipient cytoplasts. All the blastocysts obtained were transferred to synchronized recipients on Day 5-6 after ovulation. In experiment 1, pregnancies were achieved from the embryos reconstructed with all the groups of cells, however, only 1 full term calf was delivered from the embryos reconstructed with serum-starved cells. In experiment 2, significant differences were observed in embryo development and establishment of pregnancies among the donor cell lines from different animals. Five cloned calves were delivered from the embryos reconstructed with skin fibroblast cells of 3 animals, while the sole pregnancy from fourth animal aborted on Day 224 of gestation. Three full term calves were delivered from pregnancies achieved by the embryos reconstructed with cumulus cells in experiment 3, while a single pregnancy achieved from skin fibroblast cells was lost on Day 296 of gestation. In conclusion, we observed that cell donor, cell type and their treatment affect the outcome of cloning by somatic cell nuclear transfer in camels.

Morphogenesis of lingual papillae of one-humped camel (Camelus dromedarius) during prenatal life: A light and scanning electron microscopic study.

This study was made on 24 camel fetuses of crown-rump vertebral length (CVRL) ranging from 10.5 cm to 105 cm CVRL (94-352 days old). These camel fetuses were classified into three groups representing the three trimesters of prenatal life. During the first trimester (94-142 days), lingual papillae (circumvallate and lentiform papillae) were demonstrated on the lingual root, but lingual body and the apex were almost free of papillae except for some scattered epithelial projections especially near the lateral borders of the body. In the second trimester (152-229 days), the lentiform papillae covered the entire root of the tongue except for areas occupied by the circumvallate papillae. Taste buds with clear pores were observed for the first time in areas between the circumvallate gustatory furrow and surface epithelium of the tongue. In addition, short numerous filiform papillae were observed on the rostral part of the lingual body and the lateral parts of the apex. Fungiform papillae, however, were demonstrated amidst the filiform papillae. In this trimester, taste buds were also seen on the top of the fungiform papillae. In the third trimester (256-352 days), all lingual papillae were clearly demonstrated on the dorsum of the root, body and apex of the tongue. Both types of gustatory papillae (circumvallate and fungiform) had well-developed taste buds. Mechanical papillae (filiform and lentiform) were well developed. Lentiform papillae occupied most of the dorsal aspect of the Torus linguae; they were larger in size with semicircular apices. Filiform papillae, however, were numerous and demonstrated heavily on the lateral and rostral parts of the body as well as on the apex of the tongue.

Successful pregnancies from vitrified embryos in the dromedary camel: Avoidance of a possible toxic effect of sucrose on embryos.

Successful embryo cryopreservation facilitates the wider application of assisted reproduction technologies and also provides a useful method for gene banking of valuable genetics. Unfortunately attempts to establish an effective cryopreservation protocol for camelid embryos have been unsuccessful. In the current study, a modified vitrification protocol with three steps was investigated, whereby embryos were exposed to solutions containing increasing amounts of glycerol and ethylene glycol for fixed time periods. Embryos were then loaded into an Open Pull Straw (OPS) and plunged directly into liquid nitrogen for storage. Three experiments were designed to investigate the effect of 1) artificial shrinkage (AS) of embryos, 2) the addition of sucrose to the vitrification solutions, and 3) the replacement of sucrose by galactose in the warming solution, on the outcome of vitrification. The results showed that neither AS of hatched embryos prior to vitrification, nor the addition of sucrose into vitrification solutions improves the outcome of vitrification, while replacement of sucrose with galactose in warming solution increases the survival and developmental rates of vitrified embryos in culture. Transfer of vitrified embryos that were warmed in galactose resulted in a pregnancy rate of 42.8% per embryo or 46.1% per recipient. Collectively, these results suggest a possible species-specific toxic effect of sucrose on camel embryos, and that avoiding its use either in vitrification or warming solution is critical for establishing an effective protocol. This study may also be applicable to the vitrification of embryos of other camelid species including alpaca and llamas.

Isolation and characterization of the trophectoderm from the Arabian camel (Camelus dromedarius).

We isolated and characterized trophoblast from in vivo-derived camel embryos and compared with embryonic stem-like cells. Camel embryos were flushed on day 8 post-insemination and used to derive trophectoderm and embryonic stem-like cells under feeder-free culture conditions using a basement membrane matrix. Embryos were evaluated for the expression of POU5F1, MYC, KLF4, SOX2, CDX2, and KRT8 mRNA transcripts by relative quantitative polymerase chain reaction. Camel embryos grew and expanded to ∼4.5 mm and maintained their vesicular shape in vitro for 21 days post-insemination. Trophoblast and embryonic stem-like cell lines grew under feeder-free culture conditions and showed distinct morphological criteria and normal chromosomal counts. Embryonic stem-like cells showed positive staining in the alkaline phosphatase reaction. Trophoblast cells showed a significant increase in CDX2, KRT8, KLF4, and SOX2 expression compared with embryonic stem-like cells and whole embryos. Embryonic stem-like cells showed a significant decrease in CDX2 expression and increase in SOX2 and KRT8 expression compared to embryonic expression. POU5F1 and MYC expression showed no difference between embryos and both cell lines. We characterized embryo survival in vitro, particularly the derivation of trophectoderm and embryonic stem-like cells, providing a foundation for further analysis of early embryonic development and placentation in camels.

L-carnitine enhances oocyte maturation and improves in vitro development of embryos in dromedary camels (Camelus dromedaries).

The aim of the present study was to investigate the effect of L-carnitine (LC) addition during either IVM or IVC on the developmental potential of camel oocytes. In Experiment 1; camel oocytes were matured in the absence (control) or presence of different concentrations of LC (0.25 mg, 0.5 mg, 0.75 mg and 1 mg/ml) for 30 h followed by in vitro fertilization and culture up to blastocyst stage. Our results demonstrated that oocytes treated with 0.5 mg/ml LC showed higher (P < 0.05) rates of maturation (74.7%) and fertilization (62.2%) compared with control group, 0.25 and 1 mg/ml of LC (60.2, 63.9, 59.7; 46.2, 48.7, 47.6%, respectively). Addition of 0.5 mg/ml of LC to IVM medium improved the rates of cleavage and embryo development (morula and blastocyst) than those obtained in the control group, 0.25 and 1 mg/ml of LC. No significant differences were noticed between 0.5 and 0.75 mg/ml of LC supplemented groups in term of maturation, fertilization and culture. In Experiment 2; zygotes resulting from in vitro matured (without LC) and fertilized were cultured in embryo culture medium supplemented with different concentrations of LC (0.25 mg, 0.5 mg, 0.75 mg and 1 mg/ml) or without LC (control). Also, the results showed a higher developmental rates to morula and blastocyst stages while adding L-carnitine at a level of 0.5 or 0.75 mg/ml concentration in the culture medium during IVC when compared with other groups. In conclusion, the results demonstrated the usefulness of L-carnitine supplementation at the level of 0.5 mg/ml during IVM or IVC after on the developmental potential of camel oocytes.

In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals.

Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

Multiple Corpora Lutea in Dromedary Camels: Are They Associated with Multiple Pregnancies?

The aim of this study was to investigate whether multiple corpora lutea (CL) in dromedary camels were associated with multiple pregnancies. Three experiments were carried out. In the first experiment, reproductive tracts of 124 slaughtered female camels were examined for the incidence of multiple CL and the pregnancy status. In the second experiment, uteri and ovaries of 50 females were examined by ultrasound between the fourth and fifteenth weeks of pregnancy to estimate the number and location of the embryos/fetuses and the number of associated CL. In the third experiment, 20 females were mated and the pregnant animals were followed weekly to estimate the pattern of embryonic and fetal growth. At the slaughterhouse, multiple CL were detected in 44.4% of the pregnant animals. By ultrasonography, the incidence of twinning and triplets was 52%. The incidence significantly (P < 0.05) decreased from the fourth to the thirteenth week of pregnancy. Intrauterine growth retardation (IUGR) was observed in all animals of twins and triplets. Only one viable fetus could be detected after the ninth week of pregnancy in each pregnant animal. Both conceptuses died in one animal. In conclusion, multiple CL in dromedary camels are usually associated with multiple pregnancies. IUGR occurred in all animals of twins/triplets, with only one fetus surviving after the ninth week of pregnancy.

Optimization of a vitrification protocol for hatched blastocysts from the dromedary camel (Camelus dromedarius).

The objective of this study was to modify and optimize a vitrification protocol (open pulled straw) that was originally designed for human oocytes and embryos, to make it suitable for the cryopreservation of camel hatched blastocysts. The original open pulled straw protocol was a complex process with 15-minute exposure of oocytes/embryos in 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (Me2SO) for equilibration, and cooling in 16% EG + 16% Me2SO + 1 M sucrose. Recognizing a need to better control the cryoprotectant (CPA) concentrations, while avoiding toxicity to the embryos, the effects on the survival rate and developmental potential of camel embryos in vitro were investigated using two different methods of loading the CPAs into the embryos (stepwise and semicontinuous increase in concentration), two different loading temperature/time (room temperature ∼24 °C/15 min and body 37 °C/3 min), and the replacement of Me2SO with EG alone or in combination with glycerol (Gly). A total of 145 in vivo-derived embryos were subjected to these processes, and after warming their morphological quality and integrity, and re-expansion was assessed after 0, 2, 24, 48, 72, and 96 hours of culture. Exposure of embryos in a stepwise method was more beneficial to the survival of embryos than was the semicontinuous process, and loading of CPAs at 37 °C with a short exposure time (3 minutes) resulted in an outcome comparable to the original processing at room temperature with a longer exposure time (15 minutes). The replacement of the Me2SO + EG mixture with EG only or a combination of EG + Gly in the vitrification medium significantly improved the outcome of all these evaluation criteria (P < 0.05). The modified protocol of loading EG at 37 °C for 3 minutes has increased the embryo survival of the original protocol from 67% to 91% and the developmental rate from 57% to 83% at 5-day culture. These results were comparable to or better than those reported in human or other species, indicating that this optimized method is well suited to any commercial embryo transfer program in the dromedary camel.

Immunohistochemical and lectin histochemical studies on the developing olfactory organs of fetal camel.

Little is known about the development of the olfactory organs of camel. In this study, prenatal development and neuronal differentiation of the vomeronasal organ (VNO) and the olfactory epithelium (OE) of the one-humped camel were studied by immunohistochemistry and lectin histochemistry. A neuronal marker, protein gene product (PGP) 9.5, but not a marker of fully differentiated olfactory receptor cells, olfactory marker protein, intensely labeled the olfactory receptor cells of the VNO and OE at 395 mm, 510 mm, and 530 mm fetal ages, indicating that the olfactory receptor cells are differentiated, but not fully matured both in the VNO and the OE. In 187 mm and 190 mm fetuses, PGP 9.5 yielded faint immunoreactive signals in the VNO, but not in the OE, although the presence of olfactory receptor cells were demonstrated in both tissues by intense WGA and LEL stainings. We conclude that the camel VNO and OE bear differentiated, but still immature receptor cells; in addition, the onset of neuronal differentiation seems to be somewhat earlier in the VNO than in the OE till half of the prenatal life.

Effect of pregnancy and embryonic mortality on milk production in dromedary camels (Camelus dromedarius).

The main objective of the present study was to compare milk production in pregnant versus nonpregnant dromedary camels. In addition, we described the effect of embryonic mortality on lactation and measured serum progesterone levels until d 60 to 90 of gestation. Twenty-five multiparous camels were selected in midlactation for 2 studies in consecutive years. Camels were mated naturally when the size of the dominant follicle reached 1.2 to 1.5cm. Pregnancy was diagnosed by ultrasonography and progesterone determination. In the first experiment (Exp 1), 8 of 11 animals conceived at 284±21.5d postpartum. Three pregnant dromedaries were given PGF2α to induce luteolysis and pregnancy loss on d 62 and spontaneous embryonic loss was detected in 2 camels (on d 27 and 60). Animals were allotted to 3 groups retrospectively: nonpregnant camels (group 1, n=4), pregnant camels (group 2; n=3), and camels with embryonic loss after d 55 (group 3; n=4). In the second study (Exp 2), 14 dromedaries were mated during midlactation. Seven of them failed to conceive (group 1) and 7 became pregnant (group 2). No embryonic loss was detected in Exp 2. Turning points in milk production were identified by change point analysis. In nonpregnant dromedaries (group 1), milk decreased slowly over time without significant change point. In pregnant camels (group 2), a gradual decline until 4 wk after mating was followed by a sudden drop, and the change point model resulted in one breakpoint at d 28±7 and 35±3 of gestation in Exp 1 and Exp 2, respectively. In camels with embryonic mortality (group 3, Exp 1), milk yield started to decline similarly as in pregnant animals, but milk production increased gradually after embryonic loss and reached similar levels as in their nonpregnant herdmates. Change point analysis for group 3 resulted in 2 turning points at 30±4 and 48±4d after conception. Mean length of lactation was shorter by 230 (34.2%) and by 249d (37.6%) and mean total lactation production was decreased by 1,532 (31.6%) and 2,151 kg (44.3%) in pregnant compared with nonpregnant camels in Exp 1 and Exp 2, respectively. We concluded that the calving interval can be shortened by mating during midlactation. However, pregnancy has a strong negative effect on milk production as dromedaries stop lactating by the fourth month of gestation. Following embryonic mortality within 3mo of conception, milk production is restored.

Patterning and post-patterning modes of evolutionary digit loss in mammals.

A reduction in the number of digits has evolved many times in tetrapods, particularly in cursorial mammals that travel over deserts and plains, yet the underlying developmental mechanisms have remained elusive. Here we show that digit loss can occur both during early limb patterning and at later post-patterning stages of chondrogenesis. In the 'odd-toed' jerboa (Dipus sagitta) and horse and the 'even-toed' camel, extensive cell death sculpts the tissue around the remaining toes. In contrast, digit loss in the pig is orchestrated by earlier limb patterning mechanisms including downregulation of Ptch1 expression but no increase in cell death. Together these data demonstrate remarkable plasticity in the mechanisms of vertebrate limb evolution and shed light on the complexity of morphological convergence, particularly within the artiodactyl lineage.

Prenatal metanephrogenesis of the camel: morphological evidence of epithelial-mesenchymal interaction.

The present investigation examined histogenesis of epithelial, stromal and angiogenic elements of the prenatal camel permanent or metanephric kidney. The primitive metanephros was first observed at the 13-mm crown vertebral rump length (CVRL) stage as an ovoid structure composed of a centrally located epithelial ureteric bud and peripheral circumscribed masses of undifferentiated mesenchymal cells. The first morphological evidence of glomerulogenesis was observed at the 28-mm CVRL stage. Developing renal corpuscles became obvious at the 35-mm CVRL stage. At the 60-mm CVRL stage, the epithelial renal pelvis gave rise to tubular branches that extended towards the cortical zone. These branches represented the presumptive collecting ducts. Differentiation of renal tubules into the proximal and distal convoluted tubules was observed at the 95-mm CVRL stage. At the 130-mm CVRL stage, the renal medulla was clearly delineated into medullary pyramids, which in association with the corresponding cortical caps formed the morphological basis of the renal lobar formation. A gradual nephrogenic decline was noticed from the 940-mm CVRL on; however, the process of nephrogenesis persisted throughout all the studied foetal stages.

Prenatal development of the eye tunics in the dromedary camel (Camelus dromedarius).

Studies of ocular development in the dromedary camel (Camelus dromedarius) have not been reported previously. The aim of the present investigation was therefore to document the major landmarks and the time course in the prenatal development of the eye tunics in dromedary camel and its accommodation with the surrounding hard environment of the desert. Serial histological sections of dromedary camel embryos and foetuses were used. Age estimation was made on the basis of gestational size, crown vertebral-rump length (CVRL), which ranged 1.2-110 cm. The eye of the dromedary camel developed in a similar manner to that of the human and domestic animals eyes; the principal differences were in the time of occurrence of certain developmental events, pigmented peripheral cornea near the limbus, a remarkably thickened Descemet's membrane and pigmentation in the corneo-scleral junction, which represent an adaptive modification in relation to a severe environment.