A comparison of culture and cooling for the short term preservation of in vivo derived dromedary camel embryos of varying morphological quality.

Despite recent advancements in the cryopreservation of dromedary camel embryos, widespread application of the technique is still limited by the need for specialised vitrification equipment and supplies. Temporary, liquid-phase embryo storage methods provide a useful tool for short-term preservation of camel embryos. In the current study, we compared the use of in vitro embryo culture with cold liquid storage in order to maintain both high- (Grade 1- Excellent and 2-Good) and low- (Grade 3- Moderate and 4-Poor) morphological grade Day-7 dromedary camel embryos in vitro for up to 3 days. Embryos were either cooled and placed in Hams-F10 medium supplemented with HEPES and 10% FBS and then kept at 4 °C; or placed in Hams-F10 supplemented with sodium bicarbonate and 10% FBS and then cultured in a humidified atmosphere of 6% CO2 at 37 °C before being assessed for viability at 24 h. In high-morphological grade embryos, both cold storage and culture supported 100% viability (maintenance of normal morphology) over this period (Cooled n = 22, Cultured n = 20). In low-morphological grade embryos, culture supported higher viability (16/18, 88.9%) than did cooling (4/18, 22.2%). We then evaluated the effect of up to 3 days of cold storage or culture on embryo morphological grade, diameter, and developmental competence following embryo transfer. High-grade embryos were divided between culture and cold storage; low-grade embryos were evaluated only after culture. Over 3 days of culture, both high- and low-grade embryos tended to either maintain or improve upon their initial morphological score (P < 0.05) and increased in diameter (P < 0.001). Embryos subjected to cooling tended to have reduced morphological scores by 48 h of storage and decreased in diameter by 72 h (P < 0.05). No significant influence of storage method (cooling vs. culture), duration (24-72 h), or embryo grade (high vs low) was observed on pregnancy establishment at Day-60 (22.2%-57.2% pregnancy rates for all treatments). Overall, rates of pregnancy establishment were similar for transferred cultured (n = 45) and cooled (n = 45) embryos (pregnancy rates at Day 18, 48% vs 51.1%; at Day 60, 37.7% vs 37.7%). Rates of embryonic loss also were similar (22.7% vs 26%). In conclusion, whilst similar rates of pregnancy and pregnancy loss were observed following the transfer of both cooled and cultured embryos held in vitro for up to 3 days, amongst the two methods, only embryo culture appears to provide a means of effectively preserving Day- 7 dromedary camel embryos with reduced morphological values in vitro. Considering these embryos appear to show poor tolerance to the cooling procedure and are unlikely candidates for vitrification, embryo culture may provide an effective method for deriving pregnancies from low-morphological grade embryos when immediate transfer is not possible on the day of flushing.

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 the spermatozoa of the dromedary camel (Camelus dromedarius) by chilling and freezing: The effects of cooling time, extender composition and catalase supplementation.

This study sought to determine the characteristics of dromedary camel sperm following 24 h chilling and cryopreservation, testing two different buffers and cryoprotectants and the presence of catalase (500 IU/mL). Ejaculates were liquefied in Tris-Citric acid-Fructose buffer, and centrifuged through a colloid. For Experiment 1 (n = 5) sperm were cooled 24 h in Green Buffer or INRA-96® containing 0 or 3% glycerol or ethylene glycol. Experiment 2 (n = 5) used the same six treatments to evaluate sperm cryopreserved after 24 h cooling. A test of fertility was run (n = 12 recipients) with split ejaculates of fresh semen cooled 24 h in Green Buffer with and without glycerol. Experiment 3 (n = 7) cryopreserved sperm cooled 2 and 24 h in Green Buffer without cryoprotectant and with and without catalase. Sperm parameters measured before and after treatments included motility, viability and acrosome integrity. Experiment 1 showed no reduction in all sperm parameters after 24 h and no differences between buffers or presence or not of either cryoprotectant. Experiment 2 showed Green Buffer to be better than INRA for supporting sperm frozen after 24 h cooling while, for both buffers, there were few differences in sperm parameters if cryoprotectant was present or absent. Pregnancies were confirmed in 4/6 animals (67%) while no recipients receiving sperm chilled with glycerol were pregnant. In Experiment 3, catalase-supplemented sperm had maintained better motility 2 h post thaw; there were no differences between 2 or 24 h cooled sperm parameters for presence or absence of catalase. There was neither advantage nor disadvantage to coooling sperm 24 h prior to cryopreservation. We concluded that dromedary sperm can be chilled (24 h) and then either inseminated or cryopreserved. While glycerol presence in Green Buffer during chilling did not interfere with cryosurvival it may be toxic to the fertility of fresh chilled sperm. Catalase supplementation during cooling helps maintain sperm motility post thaw.

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.

Single layer centrifugation of fresh dromedary camel semen improves sperm quality and in vitro fertilization capacity compared with simple sperm washing.

Single layer centrifugation (SLC) through a colloid is a tool for selecting viable mammalian spermatozoa but has not been used previously for fresh dromedary camel sperm. Semen from six camels (2 ejaculates/male) was diluted 1:5 (v:v) or 1:10 (v:v) in a Tris-citrate-fructose buffer for mechanical liquefaction by gentle pipetting. Following liquefaction, semen was processed either by SLC or by centrifugation without a colloid (control). Total and progressive motilities, CASA kinematics, vitality and acrosome integrity (eosin-nigrosin) and plasma membrane integrity (Hypo-osmotic swelling test; HOST), and fertilizing ability in a heterologous assay (zona-free goat oocytes) were evaluated. Both total (p = .003) and progressive motilities (p = .003) were higher in SLC-processed than in control semen samples, irrespective of dilution. Positive HOST values increased when using colloid in 1:5 (p = .001) and 1:10 dilution (p = .010). Colloid-selected sperm had higher penetration rates than controls (p < .001 and p = .02 for 1:5 and 1:10 dilutions, respectively). However, only the SLC sperm at 1:5 dilution showed higher percentages of pronuclear formation (p = .02) than controls. Dilution effect was only significant for total motility before in vitro fertilization, with higher values for the 1:5 dilution (p = .033). The recovery rates of motile sperm between dilutions were similar (26.1% vs 35.4%; p = .226). In conclusion, SLC is a promising tool for selecting functional dromedary camel sperm and warrants more research.

Current status and future direction of cryopreservation of camelid embryos.

Over the past 3 decades, and similar to the horse industry, fresh embryo transfer has been widely practiced on large commercial scales in different camelid species, especially the dromedary camel and alpaca. However, the inability to cryopreserve embryos significantly reduces its broader application, and as such limits the capacity to utilize elite genetic resources internationally. In addition, cryopreservation of the semen of camelids is also difficult, suggesting an extreme sensitivity of the germplasm to cooling and freezing. As a result, genetic resources of camelids must continue to be maintained as living collections of animals. Due to concerns over disease outbreaks such as that of the highly pathogenic Middle East Respiratory Syndrome in the Middle East and Asia, there is an urgent need to establish an effective gene banking system for camelid species, especially the camel. The current review compares and summarizes recent progress in the field of camelid embryo cryopreservation, identifying four possible reasons for the slow development of an effective protocol and describing eight future directions to improve the current protocols. At the same time, the results of a recent dromedary camel embryo transfer study which produced a high morphologic integrity and survival rate of Open Pulled Straw-vitrified embryos are also discussed.

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.

Artificial insemination in dromedary camels.

Artificial insemination (AI) is an important technique in all domestic species to ensure rapid genetic progress. The use of AI has been reported in camelids although insemination trials are rare. This could be because of the difficulties involved in collecting as well as handling the semen due to the gelatinous nature of the seminal plasma. In addition, as all camelids are induced ovulators, the females need to be induced to ovulate before being inseminated. This paper discusses the different methods for collection of camel semen and describes how the semen concentration and morphology are analyzed. It also examines the use of different buffers for liquid storage of fresh and chilled semen, the ideal number of live sperm to inseminate and whether pregnancy rates are improved if the animal is inseminated at the tip of the uterine horn verses in the uterine body. Various methods to induce ovulation in the female camels are also described as well as the timing of insemination in relation to ovulation. Results show that collection of semen is best achieved using an artificial vagina, and the highest pregnancy rates are obtained if a minimum of 150×10(6) live spermatozoa (diluted in Green Buffer, lactose (11%), or I.N.R.A. 96) are inseminated into the body of the uterus 24h after the GnRH injection, given to the female camel to induce ovulation. Deep freezing of camel semen is proving to be a great challenge but the use of various freezing protocols, different diluents and different packaging methods (straws verses pellets) will be discussed. Preliminary results indicate that Green and Clear Buffer for Camel Semen is the best diluent to use for freezing dromedary semen and that freezing in pellets rather than straws result in higher post-thaw motility. Preservation of semen by deep-freezing is very important in camelids as it prevents the need to transport animals between farms and it extends the reproductive life span of the male, therefore further work needs to be carried out to improve the fertility of frozen/thawed camel spermatozoa.

Use of assisted reproduction for the improvement of milk production in dairy camels (Camelus dromedarius).

Despite their production potential and ability to survive on marginal resources in extreme conditions, dromedaries have not been exploited as an important food source. Camels have not been specifically selected for milk production, and genetic improvement has been negligible. High individual variation in milk production both within the population and within breeds provides a good base for selection and genetic progress. In this paper, we discuss the possibilities and constraints of selective breeding for milk production in camels, and include a summary of the use of embryo transfer at the world's first camel dairy farm. Embryo transfer is an integral part of the breeding strategy at the camel dairy farm because it increases selection intensity and decreases the generation interval. Using high milk-producing camels as donors and low producing camels as recipients, 146 embryos were recovered (6.1±1.0embryos/donor; range: 0-18). Embryos were transferred non-surgically into 111 recipients (83 single and 28 twin embryo transfers). Pregnancy rate at 21 days and 5 months was 55% (61/111) and 45% (50/111), respectively. Finally, a total of 46 recipients delivered a live calf. These results document the utility of embryo transfer using high milk producing dromedaries as donors.

Effect of green buffer storage on the fertility of fresh camel semen after artificial insemination.

Artificial insemination (AI) is one of the most widely used reproductive technologies, and there is considerably interest in commercializing this technology in camels. Storage of semen extender frozen (at -20 °C) is of considerable interest to scientists working with camels, as transportation of diluents at refrigeration temperature is not always possible given the hot, arid and remote conditions that dromedary camels exist in. Therefore, this study was conducted to compare the fertility of fresh camel semen, after dilution in fresh or frozen-thawed green buffer (GB), after AI into single and multiple ovulating female camels. No differences were observed in any sperm characteristics (motility, membrane integrity, acrosome integrity or morphology) when semen was diluted in fresh or frozen-thawed GB (p>0.05). Sperm motility was increased by dilution (fresh: 70.7 ± 4.9% and frozen: 68.8 ± 3.1%) compared with the motility of sperm in neat semen (35 ± 2.85%; p<0.05), and sperm motility changed from oscillatory to forward progressive after dilution. Pregnancy rates were higher (p<0.05) for single ovulating camels inseminated with semen diluted in fresh (72.7%) compared with frozen-thawed GB (27.3%), and fertilization rates were also higher (p<0.05) for multiple ovulating camels inseminated with semen diluted in fresh (83.3%) compared with frozen-thawed GB (11.1%). These results clearly demonstrate the detrimental effect of freezing and thawing semen diluent on the fertility of fresh camel semen. However, further studies are required to elucidate the mechanism responsible for this reduction in fertility. Moreover, these results demonstrate that the fertility of fresh camel semen diluted in fresh GB is high enough to be considered commercially viable.

Embryo transfer in the dromedary camel (Camelus dromedarius) using non-ovulated and ovulated, asynchronous progesterone-treated recipients.

The aim of the present study was to investigate the use of exogenous progesterone and equine chorionic gonadotrophin (eCG) in non-ovulated and ovulated, asynchronous dromedary camel recipients being prepared for an embryo transfer programme. The uteri of 12 mated donor camels were flushed non-surgically 7 days after ovulation and 42 embryos were recovered. In Experiment 1, 16 embryos were transferred non-surgically to recipients on Day 3 or 4 after ovulation (ov+3 and ov+4, respectively). Each recipient received a daily dose of 75 mg, i.m., progesterone-in-oil from 2 days before embryo transfer until 6 days after ovulation. Thereafter, the progesterone dose was reduced to 50 mg on Day 7 and finally to 25 mg day(-1) on Days 8 and 9. Nine of 16 recipients (56%; ov+3, n=4; ov+4, n=5) became pregnant compared with none of eight non-progesterone treated controls, into which embryos were transferred on Day 4 after ovulation. In Experiment 2, 18 non-ovulated recipients received 75 mg, i.m., progesterone-in-oil daily from 3 days before until 12 days after non-surgical transfer of a Day 7 blastocyst, at which time pregnancy was diagnosed by ultrasonography. All pregnant recipients continued to receive 75 mg progesterone-in-oil daily for a further 6 days, when each camel received 2000 IU, i.m., eCG. Progesterone treatment was then reduced to 50 mg day(-1) and, when a follicle(s) ≥1.3 cm in diameter were present in the ovaries, each animal received 20 µg buserelin to induce ovulation. Once the corpora lutea had developed, progesterone treatment was reduced to 25 mg day(-1) for a final 3 days. Fourteen of 18 recipients (78%) became pregnant and seven of these (50%) remained pregnant after eCG treatment. Of the seven pregnancies that were lost, two were lost before eCG treatment, two did not respond to eCG treatment and three responded to eCG treatment and ovulated, but lost their pregnancies 6-8 days after the last progesterone injection.

Ultrasonographic-guided retrieval of cumulus oocyte complexes after super-stimulation in dromedary camel (Camelus dromedarius).

In Experiment 1, studies were conducted to apply the transvaginal ultrasound guided ovum pick-up (OPU) technique in dromedary camels after their ovarian super-stimulation and in vivo oocyte maturation. In Experiment 2, the developmental potential of two commonly used oocyte types, i.e., in vivo matured oocytes collected by OPU and abattoir derived in vitro-matured oocytes was compared after their chemical activation. In Experiment 3, developmental competence of oocytes collected from super-stimulated camels by OPU, matured either in vivo or in vitro, was compared after their chemical activation. Mature female dromedary camels super-stimulated with a combination of eCG and pFSH were given an injection of 20 microg of the GnRH analogue, buserelin 24, 26, or 28 h before the scheduled OPU. For collection of cumulus oocyte complexes (COCs) the transducer was guided through the vulva into the cranial most portion of the vagina and 17-gauge, 55 cm single-lumen needle was placed in the needle guide of the ultrasound probe and advanced through the vaginal fornix and into the follicle. Follicular fluid was aspirated using a regulated vacuum pump into tubes containing embryo-flushing media. Aspirates were searched for COCs using a stereomicroscope, and they were then denuded of cumulus cells by hyaluronidase and repeated pipetting. The oocytes were classified as mature (with a visible polar body), immature (with no visible polar body), activated (with divided or fragmented ooplasm) and others (degenerated and abnormal). Overall an average of 12.12 +/- 7.9 COCs were aspirated per animal with an oocyte recovery rate from the aspirated follicles of about 77%. The majority (> 90%) of the collected COCs by OPU were with loose and expanded cumulus cells. The proportion of matured oocytes obtained at 28-29 h (91.2 +/- 4.1) and 26-27 h (82.1 +/- 3.4) were higher (P < 0.005) when compared with those obtained at 24-25 h (40.4 +/- 16.3) after GnRH administration. In Experiment 2, a higher proportion (P < 0.05) of in vivo matured oocytes cleaved (84.6 +/- 2.1 vs. 60.9 +/- 6.6) and developed to blastocyst stages (52.4 +/- 4.1 vs. 30.5 +/- 3.3) when compared with in vitro matured oocytes collected from slaughterhouse ovaries. In Experiment 3, no difference was observed between the developmental competences of oocytes, collected from super stimulated camels, matured in vitro with those collected after their in vivo maturation. In conclusion, about 80-90% mature oocytes can be collected by ultrasound guided transvaginal ovum pick-up from super-stimulated dromedary camels 26-28 h after GnRH administration. The developmental response, to chemical activation, of in vivo matured oocytes collected by ultrasound guided transvaginal OPU is better than in vitro matured oocytes obtained from slaughterhouse ovaries. However, no difference was observed in the developmental competence of oocytes collected by OPU whether they were matured in vivo or in vitro.

Unique strategies to control reproduction in camels.

The reproductive efficiency of camels is low under natural pastural conditions and so the use of artifical insemination and embryo transfer are becoming increasingly important to improve their breeding potential. Methods to control their reproductive cycle are therefore essential. This review describes characteristics of the ovarian follicular wave pattern in camels and exogenous hormonal control of ovulation. It also summarizes the difficulties involved with artifical insemination and analyzing the highly gelatinous semen, and reports on the latest methods used to try and reduce the viscosity and liquefy camel semen. In addition an account is given of different hormonal and physical methods used to synchronise follicular waves, and various hormone treatments used to broaden the availability of ovulated, asynchronous and non-ovulated recipients are discussed.

Production of the first cloned camel by somatic cell nuclear transfer.

In this study, we demonstrate the use of somatic cell nuclear transfer to produce the first cloned camelid, a dromedary camel (Camelus dromedarius) belonging to the family Camelidae. Donor karyoplasts were obtained from adult skin fibroblasts, cumulus cells, or fetal fibroblasts, and in vivo-matured oocytes, obtained from preovulatory follicles of superstimulated female camels by transvaginal ultrasound guided ovum pick-up, were used as cytoplasts. Reconstructed embryos were cultured in vitro for 7 days up to the hatching/hatched blastocyst stage before they were transferred to synchronized recipients on Day 6 after ovulation. Pregnancies were achieved from the embryos reconstructed from all cell types, and a healthy calf, named Injaz, was born from the pregnancy by an embryo reconstructed with cumulus cells. Genotype analyses, using 25 dromedary camel microsatellite markers, confirmed that the cloned calf was derived from the donor cell line and the ovarian tissue. In conclusion, the present study reports, for the first time, establishment of pregnancies and birth of the first cloned camelid, a dromedary camel (C. dromedarius), by use of somatic cell nuclear transfer. This has opened doors for the amelioration and preservation of genetically valuable animals like high milk producers, racing champions, and males of high genetic merit in camelids. We also demonstrated, for the first time, that adult and fetal fibroblasts can be cultured, expanded, and frozen without losing their ability to support the development of nuclear transfer embryos, a technology that may potentially be used to modify fibroblast genome by homologous recombination so as to generate genetically altered cloned animals.

Interspecies embryo transfer in camelids: the birth of the first Bactrian camel calves (Camelus bactrianus) from dromedary camels (Camelus dromedarius).

Interspecies embryo transfer is a possible approach that can be used to conserve endangered species. It could provide a useful technique to preserve the Iranian and wild Bactrian camels, both of which are threatened with extinction. In the present study, one Bactrian camel was superovulated using decreasing doses of FSH (60, 40, 30, 30, 20, 20 mg, b.i.d.; Folltropin-V; Bioniche, London, ON, Canada) for 6 days, followed by a single injection of FSH (20 mg, i.m.) on Day 7. Daily ovarian ultrasonography was performed until most of the growing follicles had reached a mature size of 13-17 mm, at which time the camel was mated twice, 24 h apart, with a fertile male Bactrian camel. At the time of first mating, female camels were given 20 microg, i.v., buserelin (Receptal; Intervet, Boxmeer, The Netherlands). One day after the donor camel had been mated, the dromedary recipients (n = 8) were injected with 25 mg, i.v., porcine LH (Lutropin-V; Bioniche) to induce ovulation. Embryos were recovered on Day 8.5 after the first mating and transferred non-surgically into recipients on Day 7.5 after LH injection. Pregnancy was diagnosed 25 days after embryo transfer. Healthy Bactrian camel calves (n = 4) were born without any particular complications at the time of parturition (e.g. dystocia and neonatal diseases). The present study is the first report of the birth of Bactrian camel calves from dromedary camels, as well as the first report of interspecies embryo transfer in old world camelids.

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.

Effect of different methods of cryopreservation on the cytoskeletal integrity of dromedary camel (Camelus dromedarius) embryos.

This study examined the effect of different methods of cryopreservation on the cytoskeletal integrity of camel embryos. A total of 32 embryos were recovered on Days 6 and 7 after ovulation and measured before being frozen using either a conventional slow-cooling technique (n=12: six Day 6 and six Day 7 embryos) or vitrification (n=12: four Day 6 and eight Day 7). The remaining 8 'control' embryos (four Day 6 and four Day 7) were not cryopreserved but instead incubated in holding medium for 30 min. After thawing, warming or incubation, the embryos were stained with 4,6-diamino-2-phenylindole dihydrochloride (DAPI) to identify dead cells. Subsequently, the embryos were fixed in 4% paraformaldehyde, permeabilized and labelled with Alexa Fluor 488-Phalloidin to enable assessment of cytoskeleton integrity. Vitrified-warmed embryos contained a significantly higher percentage of dead cells than either conventionally frozen embryos or controls (P<0.05). Although the proportion of dead cells in conventionally frozen embryos tended to be higher than in controls, the difference was not significant (P> or =0.07). Whereas embryo size did not affect the number of dead cells in conventionally frozen embryos, vitrified-warmed embryos >300 microm in diameter had a significantly higher percentage of dead cells than embryos < or =300 microm (P=0.01). Cytoskeleton integrity was also affected by both freezing method and embryo diameter. All 8 control embryos had a Grade I cytoskeleton, compared with only 2/24 (8.3%) frozen or vitrified embryos. Of the 8 slow-frozen or vitrified embryos with a Grade III cytoskeleton post-thaw, 7 had been vitrified and 6 were larger (Day 7) embryos. These results indicate that while both slow-freezing and vitrification of camel embryos lead to cytoskeleton disruption and cell death, embryo quality is better preserved by slow-freezing.

Synchronization of follicular wave emergence prior to superovulation in Bactrian camel (Camelus bactrianus).

This study was conducted to synchronize follicle wave emergence prior to superovulation using either GnRH or progestogen treatments, in Bactrian camels. GnRH group camels (n=5) received 20 microg of the GnRH analogue Buserelin on Days -18 and -4 of the experiment (initiation of superovulation=Day 0). Camels in the progestogen group (n=5) received two consecutive treatments of progestogens, 7 days apart, on Days -14 and -8 of the experiment. On each occasion, each female received three norgestomet implants and 200mg progesterone (i.m.) and all implants were removed 14 days after the first progestogen treatment coinciding with Day -1 of superovulation. A combination of eCG and FSH was used to induce superovulation and the growth of all subsequent follicles and CLs were monitored daily by ultrasonography. Following the first GnRH injection, mature follicles ovulated within 1-2 days, and a new follicle wave emerged after 3+/-0.77 days. At the time of the second GnRH injection, a mature follicle (15.6+/-0.97 mm) ovulated and a new follicular wave emerged between 1 and 2 days after GnRH injection. Growing follicles at the time of the first progestogen treatment became either atretic (n=1) or persistent (n=4) and a new follicle wave (n=3) emerged 3-6 days later. At the initiation of superovulation, the diameters of the largest follicle in GnRH and progestogen groups were 7.4+/-0.59 and 20.5+/-2.26 mm, respectively but after superovulation and mating there was no significant differences in the number of unovulated follicles or CLs between groups. In conclusion, two GnRH injections, 14 days apart, may be used to synchronize follicle wave emergence in Bactrian camel.

Studies on liquefaction and storage of ejaculated dromedary camel (Camelus dromedarius) semen.

The purpose of this study was to evaluate seminal liquefaction and quality of ejaculated camel semen during storage in different extenders at room (23 degrees C) and refrigeration (4 degrees C) temperature. Semen was collected using an artificial vagina and diluted immediately (1:1), using a split-sample technique, in five extenders [(1) Tris-tes egg yolk, (2) Tris-lactose egg yolk, (3) citrate egg yolk, (4) sucrose egg yolk and (5) Tris-fructose egg yolk], while one fraction was kept without an extender to act as control. The semen was transported to the lab at 37 degrees C, in a portable incubator within half an hour, and thereafter liquefaction of semen was monitored every 15 min. After complete liquefaction of the semen it was evaluated for sperm concentration and morphology and then was extended to a final ratio of 1:3. Aliquots of each semen sample were then stored at refrigeration and room temperature. The average volume of an ejaculate was 4.3+/-0.4 mL and it had a very viscous consistency. The average concentration of spermatozoa was 230.4+/-10.7 x 10(6)mL(-1) and the proportion of spermatozoa with protoplasmic droplets averaged 1.02+/-0.2, while 2.7+/-0.6 and 9.7+/-2.9% had mid-piece and tail abnormalities, respectively. All extended semen samples liquefied within 1.5h at 37 degrees C, however, there was slow liquefaction in the sample without an added extender (control). Best liquefaction was observed in Tris-lactose extender followed by Tris-fructose and citrate egg yolk diluents whereas in the other two extenders there was head-to-head agglutination of the spermatozoa. There was no difference in the initial motility of the spermatozoa in extenders 1-5 after its liquefaction, however, after 24 and 48 h of storage a higher proportion of spermatozoa were motile in extenders 1, 2 and 4 (P<0.05) at both the temperatures. There was a gradual decline in viability of the spermatozoa in all extenders at both the temperatures, although, a high portion of the spermatozoa had intact acrosomes throughout the storage period. It may be concluded that dromedary semen, when added to an extender (1:1) immediately after collection, liquefies within 60-90 min at 37 degrees C. It maintains a high proportion of motile and viable spermatozoa that can survive storage up to 48 h in Tris-lactose egg yolk, Tris-tes egg yolk and sucrose egg yolk diluents. However, best liquefaction and progressive sperm motility is achieved in Tris-lactose egg yolk extender.

Comparison of pregnancy rates in dromedary camels (Camelus dromedarius) after deep intra-uterine versus cervical insemination.

The ovarian follicular wave patterns of sixty adult female camels were monitored by serial trans-rectal ultrasound examinations and when the dominant follicle reached 1.3-1.8 cm in diameter they received a single intravenous injection of 20 microg buserelin, to induce ovulation, and were inseminated with a known number of spermatozoa 24 h later. Ejaculates were collected from the male camels and diluted 1:1 in Green Buffer with 20% egg yolk (v:v) added. Sperm concentration and motility were assessed and a dose of 40, 80 or 150 x 10(6) motile spermatozoa were deposited either just through the cervix into the uterine body or at the tip of the horn ipsilateral with the ovary containing the dominant follicle. Insemination of 150, 80 and 40 x 10(6) spermatozoa into the uterine body resulted in conception rates of 53, 7 and 0%, respectively, whereas insemination at the tip of the uterine horn resulted in conception rates of 43, 40 and 7%, respectively.