Artificial insemination and the assisted reproductive technologies in non-human primates

The experience with artificial insemination (AI) and the more invasive ARTs (assisted reproductive technologies) in the propagation of non-human primates (NHPs), although limited, has included representation from the Great Apes and both Old World and New World Macaques. The application of these technologies in NHPs is impacted by high cost, substantial technical requirements and the limited captive populations of available animals. A major incentive for their use would be to propagate endangered, underrepresented individuals or valuable founder animals. Detailed protocols and a substantial experience base for the ARTs are available for rhesus and cynomolgus macaques and form the basis of this review, including sperm recovery, processing and long-term storage at low temperatures, insemination techniques and timing. Controlled ovarian stimulation and subsequent oocyte recovery required for the invasive ARTs such as intracytoplasmic sperm injection (ICSI), is also described. Three recent AI reports in Old World Macaques are reviewed, along with examples of the use of the ARTs in the propagation of valuable founder animals, in the preservation of endangered macaques, and finally in the creation of neurodegenerative disease models for biomedical research purposes.     

Ultrasound-guided transfundal uterine sperm recovery from Macaca fascicularis

Previous studies from this center have indicated that the cynomolgus macaque (Macaca fascicularis) may serve as a model for human sperm interaction with the cervix and uterus. In some macaque species, transcervical aspiration of the uterine contents carries a significant risk of disturbing the cervical milieu due to the serpentine nature of the cervix. The only alternatives have been surgical procedures such as laparotomy or laparoscopy. In this paper, we report our experience with a new technique for ultrasound-guided sampling of spermatozoa in the macaque uterus. Twenty adult female cynomolgus macaques were monitored for menses (first day of menses = day l), and one mating per cycle was allowed on day 10, 11, or 12. In one group of ten animals, cervical mucus was sampled at 3 or 18 hr postcoitus (pc) and ultrasound-guided uterine aspiration was performed at 24 hours pc. In a second group of ten monkeys, uterine aspiration was at six hr pc and sperm numbers and motility were counted in the uterine fluid. Uterine fluid was obtained from fourteen of twenty monkeys. Pregnancy occurred in ten of the twenty experimental cycles. Ultrasound-guided uterine aspiration appears to be a reliable method for the evaluation of sperm transport in female macaques. The correlations between uterine sperm recovery and cervical mucus sperm populations arc discussed. The high conception rate in treatment cycles indicates that this procedure can be performed without apparent risk to pregnancy.     

New developments in low-dose insemination technology

New nonsurgical procedures for inseminating swine with a low number of spermatozoa have been developed and/or evaluated over the last few years. These procedures allow the deposition of the insemination dose into the uterine body (post-cervical insemination) or directly into the uterine horn (deep intrauterine insemination). With the use of the post-cervical insemination, a threefold reduction in the number of fresh sperm has been successfully used to achieve pregnancy. Using deep intrauterine insemination (DUI), up to a 20-fold reduction in the number of fresh spermatozoa or a sixfold reduction in the number of frozen/thawed spermatozoa can be achieved, with reproductive performance very similar to that obtained after standard AI. Complementing these nonsurgical insemination techniques, a new procedure for depositing spermatozoa into the oviduct by laparoscopy has been recently described. This laparoscopic technique has proven to be applicable to diluted and sex-sorted spermatozoa. The development of new insemination procedures will help achieve more efficient application of currently available sperm technologies. Using appropriate insemination procedures, it is now feasible to achieve high fertility rates with cooled, frozen-thawed, or sex-sorted semen.     

Baseline and stress levels of cortisol in conscious and unrestrained Japanese macaques (Macaca fuscata)

A vest and tethering system was applied to Japanese macaques (Macaca fuscata) to collect blood samples under less stressful conditions. The level of plasma cortisol was assayed to assess the amount of stress. The pattern of plasma cortisol fluctuation during 24 hours was consistent under non-stress conditions. The plasma cortisol level was increased significantly by encounter and venipuncture. The increase was especially noteworthy when the experimental macaques encountered a large adult male, reflecting the strong stress on macaques. As venipuncture is a common medical procedure, and the encounter between macaques who have never met before occurs frequently, this vest and tethering system is useful for the assessment of the amount of stress in the daily care of macaques in a laboratory.     

Randomized Clinical Trial Comparing Ultrasound-Guided Procedure with the Seldinger’s Technique for Placement of Implantable Venous Ports

The aim of this randomized controlled study was to compare ultrasound-guided procedure with the Seldinger’s technique for placement of implantable venous ports. A total of 214 patients were randomized to receive TIAP placement by either ultrasound-guided procedure or the Seldinger’s technique. Complications and pain perception were compared between these two groups. No severe perioperative or periinterventional complication occurred. Significantly (P < 0.05) lower pain perception was observed in the ultrasound-guided group. Seldinger’s technique group showed higher rate in incidence of early and late complications including catheter dislocation, catheter occlusion, venous thrombosis, fever of unknown origin, skin necrosis, and sepsis. In conclusion, both techniques, the TIAP implantation via ultrasound-guided jugular vein puncture and via Seldinger’s technique subclavian vein puncture, are feasible and safe. Regarding intrainterventional pain perception and implantation-related complications, the jugular vein puncture under ultrasound guidance seems to be advantageous.     

In vitro fertilization in the pigtailed macaque (Macaca nemestrina)

The objective of this study was to investigate the possibility of collecting oocytes and semen from pigtailed macaques (Macaca nemestrina) and to establish a protocol for the production of viable embryos that would be suitable for transfer into surrogate females. A total of 82 oocytes were collected from a total of four females (on 2 d with two females each). Semen was collected from the same male on both occasions with respective ejaculate volumes of 0.55 and 0.1 mL containing 2 x 10(9) and 6.6 x 10(8)sperm/mL. Following insemination and after 48 h in culture, 42 (51.2%) of the oocytes had cleaved. Of these, 21 were selected based on developmental stage and their morphology and cryopreserved. The remainder was kept in culture for an additional 5 d, at which time three had reached the expanded blastocyst stage. A total of five transfers were performed with frozen-thawed embryos; two of these resulted in pregnancies and the birth of infants. The results of this study demonstrated that oocytes can be retrieved from pigtailed macaques and that such oocytes can be inseminated and cultured in vitro to the blastocyst stage and give rise to viable offspring after transfer into surrogate females.     

Transcervical artificial insemination of Australian Merino ewes with frozen-thawed semen

We compared conventional methods for laparoscopic and cervical artificial insemination (AI) to a transcervical AI procedure (Guelph System for Transcervical AI; GST-AI) for use with frozen semen in Merino ewes. The GST-AI procedure was performed by an experienced operator in Experiment 1 (771 ewes) and by 2 inexperienced operators in Experiment 2 (555 ewes). In Experiment 1, intrauterine insemination by GST-AI was achieved in 76% of the ewes. The pregnancy rate at Day 70 for ewes inseminated by laparoscopy (48%, 120 251 ) was higher (P<0.01) than for ewes inseminated by either intrauterine GST-AI (32%, 64 201 ) or cervical AI (9%, 24 256 ). The overall (intrauterine and intracervical) pregnancy rate for GST-AI was 26% (68 264 ) and was unaffected by depth of insemination within the cervix. Pregnancy rates were unaffected by ram or day of insemination. In Experiment 2, the operators achieved intrauterine inseminations by GST-AI in 43% (78 182 ) of the ewes, with a significant operator effect (P<0.01) on depth of cervical penetration. The pregnancy rate to intrauterine GST-AI (40%, 31 78 ) did not differ from that to laparoscopic insemination. The total pregnancy rate for GST-AI in Experiment 2 (19%, 34 182 ) was lower (P<0.05) than that for laparoscopic AI (39%, 72 187 ) but superior (P<0.05) to that for cervical AI (1%, 1 186 ). The GST-AI pregnancy rates were affected by depth of AI (P<0.01) and by operator (P<0.05). It is concluded that GST-AI is superior to cervical AI, and may have application in Merinos if cervical penetration rates can be improved.     

A STUDY OF THE DIFFUSIBLE FACTOR RELEASED BY THE JELLY OF THE EGG OF THE TOAD, BUFO ARENARUM

Some properties of an essential factor for fertilization released from the oocyte strings of the toad, Bufo arenarum were studied. All the experiments were carried out with a partially purified preparation of this diffusible factor, and the general procedure consisted in assaying under different conditions its capacity to recover the fertilizability of oocytes previously deprived of this active substance. Using this procedure, a test was devised for estimating the diffusible factor activity.
As regards the mechanism of action of this fertilization factor the data reported here are not consistent with the previous assumption that the factor plays its role in fertilization through a simple pH rise of the inseminating medium.
Treatment of the gametes with the diffusible factor before insemination proved to be ineffective to promote fertilization; the factor must be present at the very moment of insemination.     

Intraperitoneal insemination in repeat-breeder cows: a preliminary report

Intraperitoneal insemination of 62 repeat-breeder dairy cows resulted in 9 pregnancies. The pregnancy rate was not different for intraperitoneal or uterine inseminations. In nonpregnant cows, reproductive variables such as the percentage of cows returning to estrus, the average interval from insemination to the next estrus, and the pregnancy rate following subsequent uterine insemination were not influenced by intraperitoneal insemination. Our results suggest that intraperitoneal insemination could be an alternative procedure to the usual deposition of semen into the uterus in repeat breeder cows.     

Refinement of a macaque transplantation model: application of a subcutaneous port as a means for long-term enteral drug administration and nutritional supplementation

A new application of a device enabling the long-term enteral administration of drugs or nutritional supplementation was developed for implementing in research entailing the use of macaques (Macaca fascicularis). After implanting a subcutaneous port, a surgically-placed gastrostomy (SPG) was completed to afford access to the gastric lumen and enable the administration of substances. In this study, the device was left in place for a period ranging between two and 12 months in macaques (n= 16). In five cases, the SPG was used successfully for 8-12 months, until the experimental endpoint was reached. In six cases, the SPG had to be removed earlier due to local infection at the implant site, which promptly regressed after the SPG was removed and antibiotic treatment was administered. One SPG-implanted macaque was euthanized for reasons unrelated to the SPG or the xenotransplantation procedure. In four cases, the SPG was implanted without any complications but has yet to be used to administer substances to the animals. From an ethical standpoint, the SPG device described here minimizes the forced handling of macaques otherwise needed for the oral administration of viscous or unpalatable substances by gavage. The device thus represents an effective refinement that fully complies with the tenet of the '3 Rs' that should be considered by primate centres exposing non-human primates to the long-term daily administration of substances by oral gavage.     

Traçabilité en assistance médicale à la procréation

An ART traceability procedure provides a written record of the controls performed to ensure precise allocation of gametes or embryos during insemination or IVF techniques. This procedure requires analysis of the various steps associated with a risk of error and optimization of safety systems. The author provides a basis for further thought on this subject.     

Low dose insemination of mares using non-sorted and sex-sorted sperm.

Mares are generally inseminated with 500 million progressively motile fresh sperm and approximately 1 billion total sperms that have been cooled or frozen. Development of techniques for low dose insemination would allow one to increase the number of mares that could be bred, utilize stallions with poor semen quality, extend the use of frozen semen, breed mares with sexed semen and perhaps reduce the incidence of post-breeding endometritis. Three low dose insemination techniques that have been reported include: surgical oviductal insemination, deep uterine insemination and hysteroscopic insemination.Insemination techniques: McCue et al. [J. Reprod. Fert. 56 (Suppl.) (2000) 499] reported a 21% pregnancy rate for mares inseminated with 50,000 sperms into the fimbria of the oviduct.Two methods have been reported for deep uterine insemination. In the study of Buchanan et al. [Theriogenology 53 (2000) 1333], a flexible catheter was inserted into the uterine horn ipsilateral to the corpus luteum. The position of the catheter was verified by ultrasound. Insemination of 25 million or 5 million spermatozoa resulted in pregnancy rates of 53 and 35%, respectively. Rigby et al. [Proceedings of 3rd International Symposium on Stallion Reproduction (2001) 49] reported a pregnancy rate of 50% with deep uterine insemination. In their experiment, the flexible catheter was guided into position by rectal manipulation.More studies have reported the results of using hysteroscopic insemination. With this technique, a low number of spermatozoa are placed into or on the uterotubal junction. Manning et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 84] reported a 22% pregnancy rate when 1 million spermatozoa were inserted into the oviduct via the uterotubal junction. Vazquez et al. [Proc. Ann. Mtg. Soc. Theriogenol. (1998) 82] reported a 33% pregnancy rate when 3.8 million spermatozoa were placed on the uterotubal junction. Recently, Morris et al. [J. Reprod. Fert. 188 (2000) 95] utilized the hysteroscopic insemination technique to deposit various numbers of spermatozoa on the uterotubal junction. They reported pregnancy rates of 29, 64, 75 and 60% when 0.5, 1, 5 and 10 million spermatozoa, respectively, were placed on the uterotubal junction.Insemination of sex-sorted spermatozoa: One of the major reasons for low dose insemination is insemination of X- or Y-chromosome-bearing sperm. Through the use of flow cytometry, spermatozoa can be accurately separated into X- or Y-bearing chromosomes. Unfortunately, only 15 million sperms can be sorted per hour. At that rate, it would take several days to sort an insemination dose containing 800 million to 1 billion spermatozoa. Thus, low dose insemination is essential for utilization of sexed sperm. Lindsey [Hysteroscopic insemination with low numbers of fresh and cryopreserved flow-sorted stallion spermatozoa, M.S. Thesis, Colorado State University, Fort Collins, CO, USA, 2000] utilized either deep uterine insemination or hysteroscopic insemination to compare pregnancy rates of mares inseminated with sorted, fresh stallion sperm to those inseminated with non-sorted, fresh stallion sperm. Hysteroscopic insemination resulted in more pregnancies than ultrasound-guided deep uterine insemination. Pregnancy rate was similar for mares bred with either non-sorted or sex-sorted spermatozoa.In a subsequent study, Lindsey et al. [Proceedings of 5th International Symposium on Equine Embryo Transfer (2000) 13] determined if insemination of flow-sorted spermatozoa adversely affected pregnancy rates and whether freezing sex-sorted spermatozoa would result in pregnancies. Mares were assigned to one of four groups: group 1 was inseminated with 5 million non-sorted sperms using hysteroscopic insemination; group 2 was inseminated with 5 million sex-sorted sperms using hysteroscopic insemination; group 3 was inseminated with non-sorted, frozen-thawed sperm; and group 4 was inseminated with sex-sorted frozen sperm. Pregnancy rates were similar for mares inseminated with non-sorted fresh sperm, sex-sorted fresh sperm and non-sorted frozen sperm (40, 37.5 and 37.5%, respectively). Pregnancy rates were reduced dramatically for those inseminated with sex-sorted, frozen-thawed sperm (2 out of 15, 13%). These studies demonstrated that hysteroscopic insemination is a practical and useful technique for obtaining pregnancies with low numbers of fresh spermatozoa or low numbers of frozen-thawed spermatozoa. Further studies are needed to determine if this technique can be used to obtain pregnancies from stallions with poor semen quality. In addition, further studies are needed to develop techniques of freezing sex-sorted spermatozoa.     

Disarming canine teeth of nonhuman primates using the submucosal vital root retention technique.

Removing or reducing the size of canine teeth of baboons and macaques has become an accepted practice to minimize the potential for injury to laboratory animal care personnel. A submucosal vital root retention procedure was adapted from the technique of root banking human teeth. In this technique, the crown of a tooth is amputated below the level of the alveolar bone crest, and the exposed pulp covered by a mucoperiosteal gingival flap. Our aim was to disarm the canine teeth of baboons and macaques with a single surgical procedure that would preserve a vital tooth root buried in alveolar bone under normal mucosa. Our long-term objective was to develop a technique that would not require further clinical management during the life of the animal. This paper presents the surgical techniques used.     

Circumventing antivector immunity by using adenovirus-infected blood cells for repeated application of adenovirus-vectored vaccines: proof of concept in rhesus macaques

Adenovirus has been extensively exploited as a vector platform for delivering vaccines. However, preexisting antiadenovirus immunity is the major stumbling block for application of adenovirus-vectored vaccines. In this study, we found that freshly isolated peripheral blood mononuclear cells (PBMCs), mostly CD14(+) cells, from adenovirus serotype 5 (Ad5)-seropositive primates (humans and rhesus macaques) can be efficiently infected with Ad5 in vitro. On the basis of this observation, a novel strategy based on adenoviral vector-infected PBMC (AVIP) immunization was explored to circumvent antivector immunity. Autologous infusion of Ad5-SIVgag-infected PBMCs elicited a strong Gag-specific cellular immune response but induced weaker Ad5-neutralizing antibody (NAb) in Ad5-seronegative macaques than in macaques intramuscularly injected with Ad5-SIVgag. Moreover, Ad5-seropositive macaques receiving multiple AVIP immunizations with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines elicited escalated Env-, Gag-, and Pol-specific immune responses after each immunization that were significantly greater than those in macaques intramuscularly injected with these Ad5-SIV vaccines. After challenged intravenously with a highly pathogenic SIVmac239 virus, macaques receiving AVIP immunization demonstrated a significant reduction in viral load at both the peak time and set-point period compared with macaques without Ad5-SIV vaccines. Our study warranted further research and development of the AVIP immunization as a platform for repeated applications of adenovirus-vectored vaccines.     

超声引导与传统PICC 置管术在老年患者中应用的对比性研究

目的:比较超声引导下PICC置管术与传统PICC置管术在老年患者中的应用情况,评价其效果。方法:回顾性分析2009年1月至2010年12月期间解放军总医院南楼老年患者PICC置管情况,记录超声引导及传统PICC置管两种方法的成功率与并发症情况。结果:超声引导组共512例次,传统PICC组共384例次,超声引导组均选择肘上静脉置管,传统PICC置管组均选择肘下静脉;超声引导组一次置管成功率及总成功率分别为96.7%,99.6%,传统PICC组分别为78.6%,86.2(P<0.01);超声引导组相关并发症发生率5.9%,对照组为12.0%(P<0.01)。结论:采用超声引导下PICC穿刺置管术显著提高了置管成功率,降低了PICC相关并发症发生率,与传统PICC组比较优势明显,值得临床推广。     

Lambing rates and litter sizes following intrauterine or cervical insemination of frozen/thawed semen with or without oxytocin administration

Intrauterine insemination by laparoscopy is required to achieve acceptable lambing rates in ewes when using frozen semen but the procedure has evoked welfare concerns. Oxytocin has been used to dilate the cervix as a means of accessing the uterus during conventional cervical insemination, but its effect on fertility is not well documented. Three hundred crossbred ewes were synchronised in estrus and randomly allocated to one of three insemination procedures using frozen/thawed semen containing 400 x 10(6)/ml progressively motile sperm: single cervical (0.2 ml), multiple cervical (4 x 0.05 ml) or laparoscopic (0.05 ml per uterine horn). The effects of each insemination procedure on lambing rate (percentage of treated ewes lambing) and litter size (lambs per ewe lambing) were tested with and without oxytocin (10 IU given i.m.) prior to fixed-time insemination. Oxytocin did not permit complete cervical penetration in any ewes and neither lambing rate nor litter size was influenced by the number of inseminations. Lambing percentages were 69 and 42 (P < 0.01) for the laparoscopic and cervical insemination methods, respectively, and oxytocin reduced these to 58 (NS) and 10 (P < 0.001) percent, respectively. Corresponding litter sizes for ewes not receiving oxytocin were 1.91 and 1.51 and for those receiving oxytocin, 1.83 and 1.41 (laparoscopic versus cervical, P < 0.02). Thus, in the absence of complete cervical penetration at insemination, 10 IU oxytocin decreased the number of ewes lambing but had no effect on their litter size.     

Insemination results with slow-cooled stallion semen stored for 70 or 80 hours

An insemination trial was conducted to evaluate the fertility of extended slow-cooled stallion spermatozoa stored for 70 h or 80 h at 5 to 7 degrees C before insemination. Then, 1 or 2 of the first sperm-rich fractions were collected with an open-ended vagina from 4 stallions. Semen from each stallion was diluted within 2 to 3 min after collection with a modified Kenney skim milk extender (6). The proportion of raw semen in the insemination doses was 24+/-6%. One insemination dose (25 to 50 ml) consisted of approximately 2 billion total spermatozoa. In the trial, palpation per rectum and ultrasonography of 34 mares (40 cycles) were performed every 12 h. The pregnancy rate per cycle (30-d) with semen stored for 70 h before insemination was 77% (17 cycles) and, with semen stored for 80 h, 57% (23 cycles). The difference was not statistically significant. The combined pregnancy rate per cycle was 65%. These results indicate that stallion semen can retain its fertilizing capacity for up to 80 h when collected and diluted using this procedure and when the inseminations are done less than 12 h after ovulation.     

Incorporación de la ecografía y la punción de tiroides a la actividad de endocrinología en una consulta de alta resolución

IntroductionFine-needle aspiration biopsy (FNAB) is considered the reference diagnostic procedure for thyroid nodules.Materials and methodsRoutine performance of thyroid ultrasound and ultrasound-guided FNAB by endocrinologists allows a more efficient approach in the setting of a high-resolution practice, thus reducing costs and the time elapsed until diagnosis.ResultsWe present our initial results of this procedure 2 years after its introduction, with a total of 286 biopsies performed. After a progressive learning curve over time and according to the endocrinologists’ previous experience, 72.72% samples were considered satisfactory for diagnosis. Greater difficulty was observed in obtaining optimal cytological specimens in smaller nodules. In conclusion, we have successfully incorporated thyroid ultrasound and ultrasound-guided FNAB into routine endocrine practice.ConclusionRoutine performance of thyroid ultrasound in endocrine practice will considerably aid the management of nodular thyroid disease.     

Psychiatric Aspects of Artificial Insemination (Donor)

Artificial insemination (donor) [A.I.D.] in humans is a medical procedure that has been carried out for roughly 50 years. Its legal status has not yet been established; its moral implications are still hotly contested, and its psychological and psychiatric implications are only now coming under scientific scrutiny.The use of this procedure in couples who are psychologically unsuited for it can have unfortunate consequences. The obstetrician should seek the assistance of a dynamically oriented psychiatrist in screening couples who ask for artificial insemination (donor).Parenthood, in line with psychoanalytic ego psychology, is seen as a phase of ego development. The potential for mothering and fathering children is a later stage in growth than the capacity to conceive and sire them. It is the psychiatrist''s role to assess the couple''s motivation for A.I.D. in the light of the extent to which they have achieved this degree of ego development.