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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Comparison of two different methods for the vitrification of hatched blastocysts from the dromedary camel (Camelus dromedarius).

The uteri of 32 donor camels were flushed non-surgically on Day 6, 7 or 8 after ovulation and a total of 184 embryos was recovered. Sixty Day 6 embryos and 61 Day 7 embryos were vitrified or frozen ultrarapidly using open pulled straws and a modified version of the Vajta protocol. These embryos were subjected to concentrations of either 10% and 20% or 20% and 40% ethanediol as the cryoprotectant before being loaded into open pulled straws (OPS) and plunged into liquid nitrogen. All embryos were subsequently thawed and rehydrated either directly into holding media or into holding media containing 0.2 M sucrose and were incubated for 5 or 10 min before being transferred to holding media before transfer to recipients. Although the survival rate of the embryos immediately after thawing was high (OPS 20%/40% ethanediol resulted in 97% and 100% survival for Day 6 and Day 7 embryos, respectively; OPS 10%/20% ethanediol resulted in 90% and 70% survival for Day 6 and Day 7 embryos, respectively), after 2 h in culture, survival rates had decreased to 46% and 53% for Day 6 and Day 7 embryos, respectively, using OPS 10%/20% and 53% and 63% for Day 6 and Day 7 embryos, respectively, using OPS 20%/40%; however, none of the embryos transferred resulted in a viable fetus. A further 63 embryos (Day 6: n = 31; Day 7: n = 16; Day 8: n = 16) were subsequently exposed to vitrification solution (20% glycerol + 20% ethylene glycol + 0.3 M sucrose + 0.375 M glucose + 3% polyethylene glycol) in three steps and after loading into 0.25 mL straws were plunged into liquid nitrogen. However, a much greater percentage of the Day 7 and Day 8 embryos (43.8% and 81.2% respectively) were fractured or torn after warming and none of the 12 intact embryos transferred resulted in a pregnancy. Better survival rates immediately after thawing and rehydration were obtained with the smaller Day 6 embryos (94%), which resulted in a total of eight fetuses from the 21 embryos transferred.

Hybridizing Old and New World camelids: Camelus dromedarius x Lama guanicoe.

Thirty female dromedary camels were inseminated on a total of 50 occasions with 2-4 ml of fresh guanaco semen diluted with an equal volume of commercially available camel semen extender. Similarly, nine female guanacos were inseminated on 34 occasions with 4-6 ml of fresh, diluted camel semen. Only two of the dromedary females conceived; one aborted a female foetus on day 260 of gestation and the other gave birth to a stillborn female calf on day 365. Six conceptions occurred in the female guanacos. Two of these conceptuses, diagnosed by ultrasound, were resorbed between days 25 and 40 of gestation, one female foetus was aborted on day 291, another female foetus was aborted on day 302, and one female calf was stillborn on day 365 of gestation. The sixth foetus, a male, was born prematurely but alive after a 328-day gestation. It had a phenotypic appearance intermediate between that of a camel and a guanaco and its hybrid parentage was confirmed by the DNA fingerprinting of eight llama microsatellites. To our knowledge, this is the first viable hybrid ever to be produced between Old World and New World camelids, which have been reproductively isolated from one another for at least 11 million years. The preponderance of female hybrids is in accordance with Haldane's law. Histological examination of their ovaries revealed a failure of meiosis, with only an occasional abnormal oocyte surrounded by follicle cells. Although the diploid chromosone number of camels and guanacos is the same (2n = 74), sufficient genetic change has taken place to make the pairing of homologous chromosomes no longer possible.

Implantation and early placentation in the one-humped camel (Camelus dromedarius).

The ovaries and uteri were removed from four pregnant camels on days 14, 25, 35 and 56 after ovulation. The day 14 and 25 uteri were perfuse-fixed with 3 per cent glutaraldehyde: 3 per cent paraformaldehyde whereas the day 35 and 56 specimens were opened dorsally for photography before biopsies of allantochorion attached to endometrium were taken and fixed in either 3 per cent glutaraldehyde: 3 per cent paraformaldehyde or Bouin's fluid. Samples of each uterus were then processed and sectioned for light and transmission electron microscopy. By day 14 the majority of the trophoblast had become closely apposed to the luminal epithelium of the endometrium to form the start of an epitheliochorial placenta with microvillar interdigitation initiated in some places. By day 25 a well-developed microvillar junction had formed between the fetal and maternal tissues. The fetus was situated in the middle of the left uterine horn in the day 35 and 56 specimens and, histologically, large multinucleate giant trophoblast cells had developed at frequent but irregular intervals along the, otherwise single-cell, trophoblast. These giant cells were often situated over the mouth of an endometrial gland but their actual function in pregnancy is not yet known.

Patterns of hormone secretion throughout pregnancy in the one-humped camel (Camelus dromedarius).

Peripheral serum samples were collected from 8 pregnant dromedary camels and hormone secretion patterns were examined at specific time intervals. Mean serum progesterone concentrations began to rise 3-4 days after ovulation and remained reasonably constant at 3-5 ng mL-1 for the first 90-100 days of gestation. Concentrations then showed a definite fall, but thereafter remained constant again at 2-4 ng mL-1 throughout the rest of pregnancy. In contrast, serum oestrogen concentrations showed pronounced fluctuations during the first 100 days of gestation. Mean oestradiol-17 beta concentrations increased at around Day 50 to about 100 pg mL-1 and then remained relatively constant from Day 90 to Day 300. Mean oestrone sulfate concentrations, however, showed two definite peaks in early gestation, each reaching about 10 ng mL-1, with the first peak occurring around Day 25 and the second peak around Day 75. Oestrogen production then remained fairly constant until around Day 300, after which concentrations of both oestrone sulfate and free oestradiol-17 beta rose steeply over the next 80 days to reach mean peak values of 46 ng ML-1 and 518.7 pg mL-1, respectively, at the time of parturition. Concentrations of 13,14 dihydro-15-keto prostaglandin F2 alpha (PGFM) remained low and reasonably steady at 100-200 pg mL-1 during the first 320 days of pregnancy; thereafter, PGFM concentrations rose steeply over the next 50 days, before an explosive further increase to a peak of 1900 +/- 141 pg mL-1 mean +/- sem on the day of calving. These results suggest that, as in the cow, a major change in steroid synthetic capability and/or enzyme content of the placenta may occur at around 80% (Day 300) of gestation in the pregnant camel.

Developmental competence in vitro and in vivo of cryopreserved, hatched blastocysts from the dromedary camel (Camelus dromedarius).

The present paper describes experiments designed to investigate methods for cryopreserving embryos from dromedary camels. Because preliminary studies had shown ethanediol to be the best cryoprotectant to use for camel embryos, the current experiments were performed to determine the minimum exposure time to 1.5 m ethanediol required to achieve cryoprotection. The uteri of 30 donor camels were flushed non-surgically 8 days after mating. Embryos were recovered and 158 were assigned to one of three groups, which were exposed to 1.5 m ethanediol for either 10 min (n = 67), 5 min (n = 51) or 1 min (n = 40). Embryos were subsequently thawed and rehydrated by expelling either directly into holding medium (HM; HEPES-buffered Tyrode's medium containing sodium lactate and 3 mg mL(-1) bovine serum albumin, 10% fetal calf serum, 100 IU mL(-1) penicillin G, 100 microg mL(-1) streptomycin and 25 microg mL(-1) amphotercin B) or initially into HM containing 0.2 m sucrose for 5 or 10 min. The survival rate of all embryos immediately post-thawing, as judged by the morphological appearance of the embryos, was high (91%), but was greatly reduced after 2 h culture (59%). Ninety-two embryos were transferred to recipient camels resulting in 18 viable fetuses (1 min ethanediol exposure, n = 1/15; 5 min ethanediol exposure, n = 3/34; 10 min ethanediol exposure, n = 14/43). Of the embryos rehydrated directly in HM, six of 65 resulted in viable fetuses and those rehydrated initially in 0.2 m sucrose for 5 or 10 min resulted in nine of 47 and three of 46 fetuses respectively. From these experiments, we conclude that camel embryos can be cryopreserved using ethanediol as a cryoprotectant when the embryos are cooled slowly (to 33 degrees C) before being plunged into liquid nitrogen for storage.

Assisted reproductive techniques for hybridization of camelids.

The camelid family comprises the Old World camelids (or dromedary and Bactrian camels) and the New World camelids (namely the llamas, alpacas, guanacos and vicunas). Although the species within each group can hybridize among themselves to produce fertile offspring, it is only recently that a hybrid between New and Old World camelids has been reported. To create this hybrid, semen was collected from male camels by artificial vagina (AV) and inseminated into female guanacos (n = 9) and llamas (n = 3) at the appropriate stage of their follicular wave cycle. Similarly, guanaco and llama semen was collected, also by AV, and inseminated into female camels (n = 42). Although several conceptions occurred, only one hybrid (camel sire x guanaco dam) continued to term and was born alive after 328 days of gestation, and another is pregnant at the time of writing (camel sire x llama dam). Further studies are presently being carried out using extraspecific embryo transfer to try and improve the success rate of live offspring being born. Female guanacos (n = 4) are treated with hormones to stimulate their ovaries to produce several follicles before being inseminated with camel semen. Of the 12 camel recipients that have to date received hybrid embryos (camel sire x guanaco dam), 10 conceived, but 9 of these subsequently aborted between 30 and 365 days and only one recipient was still pregnant at the time of writing.

Reproductive aspects and storage of semen in camelidae.

The characteristics of male and female reproductive tracts and reproductive physiology in camelids are described. An account is given on methods of collection, characteristics and storage of semen, and fertility after artificial insemination (AI) with fresh, liquid-stored and frozen-thawed lamoid and camel semen.