Successful recovery of preimplantation embryos by nonsurgical uterine flushing in the bonnet monkey

A major limitation to progress in primate embryology is the lack of an adequate supply of preimplantation embryos. We describe a method for recovering preimplantation-embryos in bonnet monkeys (Macaca radiata ) using a nonsurgical uterine flushing technique similar to the one previously employed in rhesus monkeys. Forty cyclic females were screened for cervical cannulation, and 10% of these had an impassable cervix. Eleven females suitable for cannulation were selected, and 27 menstrual cycles were monitored over a 5-mo period. Seventy-one percent of the cycles showed estrogen peaks, which were observed between Days 9 and 14 of the cycle. Following natural mating, uterine flushings were performed on Days 5 to 8 of pregnancy (Day 0 = the day following the estrogen peak). Of the 27 recovery attempts, 9 (33.3%) resulted in the recovery of ovulation products, including those of an unfertilized oocyte and empty zona (2 cases), retarded cleavage-stage (4 to 8-cell) embryos (4 cases), morula (1 case) and blastocysts (2 cases). These results show, for the first time, that the nonsurgical uterine flushing technique can be successfully performed to recover uterine-stage preimplantation embryos from bonnet monkeys.     

Non-surgical uterine flushing for the recovery of preimplantation embryos in rhesus monkeys: Lack of seasonal infertility

A non-surgical uterine flushing technique was employed to recover rhesus monkey preimplantation embryos during April–-September, a period thought to be associated with reduced fertility. A total of 22 females of proven fertility, maintained indoors under strict light and temperature control, were employed for the study in which 72 menstrual cycles were monitored. The average length of their menstrual cycle was 27.9 ± 3.8 days. The percentages of cycles that showed normal cyclic patterns of estrogen, LH, and sex-skin color were 84.7%, 91.7%, and 90.3%, respectively. Following natural mating, uterine flushing was performed on days 4–7 of pregnancy. Of 58 attempts, 27 (46.6%) resulted in the recovery of embryonic materials. Two recoveries produced unfertilized oocytes; 20 yielded embryos which were morphologically normal, and 7 yielded damaged and/or degenerate embryos. Luteal function in cycles involving uterine flushings was evaluated by examining ovaries by laparoscopy, ovulation being confirmed in 34 of 35 examinations. Serum progesterone was > 2 ng/ml in 39 of 45 cycles. The conception rate of 46.6% noted in this study was similar (P > 0.4) to those observed from other natural matings in our colony, either during the same period (49.3%; n = 67) or during October–-March (46.9%; n = 113). These results show that rhesus monkeys, maintained under appropriate environmental conditions, can experience normal fertile menstrual cycles throughout the summer months, and extend previous observations to demonstrate, for the first time, that preimplantation embryos can be recovered from this species year-round. © 1993 Wiley-Liss, Inc.     

Work in progress: A simple technique that may improve the rate of embryo recovery on uterine flushing in mares

Embryo recovery rates from uterine flushings of normal mares on Day 7 or later after ovulation currently range from 55% to 80%. In contrast, pregnancy rates at 14 d in experimental mares are often higher. There appears to be a discrepancy between pregnancy rates and recovery rates of embryos on uterine flushing, indicating that some embryos are not recovered from the uterus on flushing. Per rectum ultrasound examination of the uterus of mares during flushing suggested that in some mares, the infused fluid may accumulate in the uterine body and not extend to contact the entire uterus, even after massage of the filled uterus per rectum. To increase embryo recovery rates, the flusing technique was altered to allow 3 min contact time of the flush fluid with the uterus during each of three flushes. It was thought that during this time, if the embryo was not directly contacted by the infused fluid, mobility of the embryo might cause it to move into the fluid, and thus be collected. This technique was used in 20 flushes on 14 mares, from 7 to 11 d after ovulation. Embryos were recovered on 18 of the 20 flushes. A total of 21 embryos was recovered, for an embryo recovery rate of 105%. The recovery rate from mares with single ovulations was 13/15 (87%); the recovery rate from mares with multiple ovulations was 8/5 (160%). These rates appear to be higher than those obtained previously in our laboratory and those reported by other workers in the field. These results indicate that further investigation into the efficacy of this procedure is warranted.     

Nonsurgical uterine stage preimplantation embryo collection from the common marmoset

A nonsurgical technique for the recovery of uterine stage preimplantation embryos was developed for the common marmoset (Callithrix jacchus). In 54 flush attempts, using 19 different animals, 54 morphologically normal embryos, seven unfertilized oocytes or degenerate embryos, and five empty zonae pellucidae were recovered, giving a recovery rate of 1.0 embryo per flush or 1.2 ovulation products per flush. Because the ovarian cycles of common marmosets can be synchronized with prostaglandin PGF2α, multiple marmosets can be flushed in a short period, providing age-matched embryos for controlled experiments.     

Collection of tubal stage bovine embryos by means of endoscopy. A technique report

Here we describe the development and optimization of endoscopy-mediated transvaginal access for collecting ova and embryos from the bovine oviduct. The novel technique was developed in three experimental setups: In Experiment 1 embryos were collected unilaterally from nonstimulated heifers. We flushed the oviducts of superovulated heifers unilaterally (Experiment 2) and bilaterally (Experiment 3). In Experiment 1 the oviducts of 18 heifers were successfully cannulated, which resulted in the collection of twelve 1-cell to 8-cell embryos and one empty zona. Unilateral flushing of 13 animals (Experiment 2) resulted in 84 ova with 6.3 +/- 3.2 observed ovulation sites. Bilateral flushing of 25 animals (Experiment 3) resulted in 293 ova plus 10 empty zonae from 11.8 +/- 5.4 ovulation sites. Given our experience from these studies we optimized the technical equipment by improving the flushing metal catheter (Experiment 4). The novel catheter hermetically sealed the lumen of the ampulla at the moment, the medium was flushed through the oviduct. This resulted in a visible flow of medium via oviducts toward the embryo filter connected to an embryo flushing catheter that was fixed in the uterine horns. Our endoscopy-guided method is minimally invasive and facilitates the flushing of tubal stage embryos.     

Effect of repeated flushing and a prostaglandin analogue on the estrous cycle of pony mares

An experiment was conducted to test the effect of repeated transcervical (non-surgical) uterine flushing and a prostaglandin analogue (PG) on the estrous cycle of pony mares. Uteri in group A were trancervically flushed for embryos 7 to 9 days post ovulation. In addition, group B mares were given 5 ml of PG by intramuscular injection on the day of flushing. Group C served as controls and were not flushed or given PG but were allowed to cycle normally. All mares (except controls) were bred A.I. every other day during estrus. There was no effect on embryo recovery rate from repeated flushing or PG administration. The number of days in estrus was greater for groups A and B than for group C (P<0.05). Length of diestrus was longer for group C than for the other two groups. The total estrous cycle length was similar for all three groups (P>0.05).     

Non-surgical recovery of bovine embryos

A simple one-way cannula technique for non-surgical recovery of bovine embryos has been described. The collector was very reliable, stable, and easy to use. It could be guided through cervix and without damage to the genital tract into both uterine horns on all attempts. Ninety-six per cent (96%) of the flushing medium was recovered. Bleeding occurred rarely. The post-flushing reproductive performance was satisfactory. An average of 4.1 eggs per dairy cow were recovered yielding a recovery rate of 46%. The method was performed under farm conditions with and without the use of a chute or stanchion. Several general practitioners have used the method with success after a brief training period. Good manipulative skills were a prerequisite for good results.     

Bovine embryo recovery by filtration of non-surgical flushings

A simple filtration system has been developed for the rapid collection of bovine embryos from large fluid volumes such as non-surgical uterine flushings. The technique utilizes a nylon plankton net sieve of 56 mum pore size and was evaluated on the non-surgical flushings of 18 superovulated cows. Approximately 500 ml of flushings from each uterine horn was collected in sedimentation flasks and two aliquots of 20 ml removed from the bottom of the flask after standing for 20 min, and searched for embryos. The remainder of the flushings was passed through the sieve and the sieve examined for embryos. Seven days (Day 7) after insemination, 53.3% (40 75 ) of embryos were found on the sieve or 47.2% of all normal embryos. On Day 12,28% (7 25 ) of eggs were found on the sieve, all of which were unfertilized or degenerate. All embryos were located within 10 min of starting filtration.     

Ultrasonographic and hormonal monitoring of pregnancy in the saddle back tamarin,Saguinus fuscicollis

Ultrasonography was used in six saddle back tamarin females (Saguinus fuscicollis) to diagnose pregnancy, monitor the patterns of uterine growth and embryonic/foetal development and examine the incidence loss of single embryos/foetuses. Pregnancy was reliably diagnosed 17 days after conception, 10 days earlier than by plasma progesterone measurement. The patterns of uterine and embryonic/foetal growth paralleled those reported for the common marmoset, including a delay in embryonic development. The results support the hypothesis of retardation of organogenesis in most callitrichid species. Individual embryos could be reliably identified from day 50 of pregnancy; a loss of single embryos/foetuses after this stage did not occur. All pregnancies were carried to term, resulting in five times twins and one singleton. The smaller litter size compared to the common marmoset may be due to loss of single embryos at earlier stages of pregnancy or to a lower ovulation rate.     

Improvement in recovery of embryos/ova using a shallow uterine horn flushing technique in superovulated Holstein heifers

The aim of this study was two-fold: (1). to compare recovery of embryos/ova from superovulated Holstein heifers by flushing the uterine horns through insertion of the catheter very close to the tip of the horn (deep) or just after the uterine bifurcation (shallow) and (2). to evaluate the hormonal and superovulatory response to estradiol benzoate (EB) treatment prior to superovulation. Ten Holstein heifers (12-16 months) underwent two superovulatory treatments in a cross-over design. Heifers were treated with decreasing doses of FSH from Days 8 to 12.5 of a synchronized estrous cycle. At 4 days prior to superovulation, half of the heifers received EB (5mg, i.m.) or served as Controls, followed by the alternative treatment in the subsequent superovulation. At embryo recovery, one uterine horn was flushed with deep ( approximately 7 cm caudal to the tip of the horn) and the other with shallow ( approximately 5 cm cranial to the beginning of the uterine bifurcation) flushing techniques. Embryos/ova were recovered, counted, and scored. Number of ovulations was estimated by ultrasound. Pretreatment with EB reduced circulating FSH and regressed the first wave dominant follicle with no change in number of large follicles, number of ovulations, number of embryos/ova recovered, or number of transferable embryos. The shallow flushing technique was superior to the deep technique for number of embryos/ova recovered per horn (5.4+/-1.1 versus 3.9+/-0.8) or percentage of embryos/ova recovered per CL (63.9+/-8.6% versus 37.4+/-6.5%). Thus, flushing the entire uterine horn increased recovery of embryos/ova.     

Lack of association of Mycobacterium avium subsp. paratuberculosis with oocytes and embryos from moderate shedders of the pathogen

Paratuberculosis is a chronic and progressive disease of the intestine in ruminants caused by Mycobacterium avium subsp. paratuberculosis (Map). The bacterium is transmitted to young animals, becomes manifest in adulthood and leads to economic losses. The aim of this study is to investigate if cows shedding Map possess oocytes and embryos that are carriers of the bacterium. New genetical material can enter the dairy farm using embryo transfer but the question as to whether this technique is safe with respect to transmission of paratuberculosis has yet to be addressed. We selected and bought 16 cows, all proven to be moderate shedders of the bacterium in the faeces immediately prior to the experiment but none were clinically sick. One sample of uterine content was collected from each animal by flushing the uterus on the day of heat and five samples of homogenised uterine tissue were collected on the eighth day of the same cycle by biopsy. In addition, 217 cumulus-oocyte complexes (COCs), ranging from 3 to 35 COCs per animal, were collected using ultrasound guided transvaginal puncture of the ovarian follicles (OPU). On the seventh day of the subsequent cycle 31 embryos were obtained using the classic technique of super ovulation induction, artificial insemination (AI), followed by flushing of the uterus. These embryos have been washed and trypsinised. Fourteen of the 16 cows were treated again for super ovulation in the subsequent cycle and 19 foetuses were collected by opening of the uterus after euthanasia on Days 35-49 of the cycle. All samples were cultured for presence of Map and checked every 2 months during 1 year for bacterial growth. None of the samples showed growth of Map after 12 months of culture. Pathological examination of the cows revealed different degrees of severity of pathological alterations of the intestinal tract and mesenteric lymph nodes. However, the results suggest that neither in vivo embryo's nor oocytes are carriers of the bacteria and do not form an extra risk at transfer. However, due to the limited size of the experiment (sample size of 16 cows), a certain margin for error remains.     

Ovarian stimulation of marmoset monkeys (Callithrix jacchus) using recombinant human follicle stimulating hormone

To reduce the number of animals required for controlled studies of marmoset oocytes and early embryos, a superovulation protocol was developed for the common marmoset. Females were given up to 50 i.u./day recombinant human follicle stimulating hormone (FSH)--(r-hFSH) for 6 days. Ovaries were visualized by a modified laparoscopic technique and follicular aspiration was performed using a needle and suction apparatus inserted directly through an otoscope speculum. The number of follicles + ovulation points (+/- S.E.) was 2.9 (+/- 0.2) in controls and 14.1 (+/- 1.6; P < or = 0.001) in the 50 i.u. r-hFSH per day animals. Oocytes, typically at the germinal vesicle stage at collection, extruded a first polar body within 26 hours. In vitro fertilization was performed and embryos developed to the hatched blastocyst stage (34%). With many high quality oocytes and the ability to synchronize cycles, the marmoset is a valuable primate model for examining nuclear reprograming and early embryonic events.     

Pregnancy resulting from cattle oocytes matured and fertilized in vitro

Follicular oocytes (n = 81) collected from cattle at a local slaughterhouse were matured and fertilized in vitro. Of 27 ova 19 (70%) were penetrated by spermatozoa and 40/54 (74%) inseminated ova transferred surgically to the oviducts of a synchronized heifer were recovered by non-surgical flushing of the uterine horns 6 days later. Of the 40 ova 15 (38%) were at the morula, early blastocyst or diminutive morula stages. Culture in vitro sustained further development of all embryos and 9 were expanding or expanded blastocysts. One pregnancy resulted from non-surgical transfer of 2 blastocysts. The results demonstrate that immature oocytes from cattle can be matured and fertilized in vitro, subsequently develop to the blastocyst stage, and develop into a normal pregnancy after non-surgical transfer.     

Endoscopic embryo collection and embryo transfer into the oviduct and the uterus of pigs

We describe the first complete embryo transfer program, including flushing of embryos from the oviducts via the uterine horns, transfer of embryos into the Fallopian tubes or the uterine horns and recording of the number of piglets born live. The described procedure is minimally invasive and allows the use of pigs simultaneously for embryo collection and production of normal pregnancies. A 30 degrees forward oblique endoscope provided optimal visualization of the reproductive organs and free access to the organs for embryo flushing and transfer. In contrast to surgical and nonsurgical methods, endoscopy allows to pre-examine the genital tract for reproductive abnormalities and successful ovulation. A total of 95 prepuberal gilts or cyclic sows were used in this trial. Embryos or oocytes were collected from hormonally treated pigs via endoscopy(n = 17) on Day 3 and via laparotomy or post mortem after slaughter (control group, n = 38) on Day 3 and 6 after insemination. One (unilateral collection, n = 7) or both oviducts (bilateral collection, n = 10) were flushed endoscopically. We recovered 114 (average 16/pig) and 279 (average 28/pig) oocytes or embryos with fertilization rates of 89% and 72%, respectively. In the control group 834 oocytes or embryos were collected at Day 3 and 6 after insemination (fertilization rate 64%, total 534 embryos, 33 at 2-, 367 at 4-, 2 at 8-cell stage, 24 morulae and 108 blastocysts). Of 836 embryos recovered by endoscopy, surgery or slaughter 528 Day 3 embryos at 2- to 4-cell stage were transferred into (one) oviducts (n = 27 pigs, about 20/pig) resulting in 9 pregnant pigs diagnosed at Day 28 by sonography. Of the 9, 8 carried a total of 49 piglets to term. A total of 195 Day 6 embryos were transferred into uterine horns (n = 12 pigs, about 16/pig), resulting in 5 pregnant pigs carrying a total of 38 offspring to term. The use of endoscopy in assisted reproduction of pigs has the advantages of allowing easy access to the ovary, oviduct and uterus, clear view of the organ manipulation without exposure and exteriorization of viscera during surgery.     

Stimulation of immunoreactive inhibin production by preimplantation embryos during early pregnancy in the marmoset monkey (Callithrix jacchus).

The role of the embryo in promoting increased plasma concentrations of immunoreactive inhibin after conception in the marmoset monkey was determined by flushing embryos from the uterus between days 5 and 9 after ovulation (implantation commences on days 11-12). Blood samples were taken from each animal (three times a week) after ovulation until the end of the luteal phase. Plasma inhibin concentrations were measured using a radioimmunoassay based on antisera against a synthetic fragment of the alpha-subunit of human inhibin. When embryos were flushed on days 5 and 6 (n = 6) after ovulation inhibin concentrations did not exceed 250 ng ml-1 for the duration of the luteal phase. In contrast when embryos were flushed on days 7 (n = 4), 8 (n = 4) and 9 (n = 3) maximum concentrations of inhibin always exceeded 250 ng ml-1, reaching > 400 ng ml-1 when embryos were flushed on days 8 and 9. Inhibin concentrations remained high for the duration of the luteal phase, which varied in length between 20 and 32 days. Significantly (P < 0.01) higher mean plasma concentrations of immunoreactive inhibin were first recorded on days 7-8 after ovulation in animals that had embryos flushed on days 7, 8 and 9 compared with concentrations in animals that had embryos flushed on days 5 and 6. Inhibin could not be detected in the medium of embryos cultured for up to 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early implantation stages in the marmoset monkey (Callithrix jacchus)

The morphology of the initial stages of implantation in the marmoset monkey (Callithrix jacchus) was studied by obtaining embryos and associated endometrium at timed intervals after ovulation. Estrus cycles were detected by measuring daily levels of plasma progesterone. Following a short follicular phase, circulating levels of progesterone above 20 ng/ml were taken as representing day 1 after ovulation. On this basis, single, twin, and triplet embryos were recovered from six perfused-fixed females on days 13, 16, 19, 23, and 29 after ovulation and prepared in resin for light microscopy. Early implantation stages, 13 and 16 days after ovulation, were characterized by the intrusion of syncytial trophoblast between epithelial cells of the endometrium with minimal cellular damage. Some hyperplasia of epithelium at the margin of the implantation site was evident. The consolidation of the initial attachment was achieved by an increase in syncytial trophoblast underlying the inner cell mass of the embryo which rapidly surrounded and breached maternal capillaries. Although initially separate, the chorions of twin or triplet embryos started to fuse by day 19 after ovulation. This process was complete by day 29 such that embryos shared a common uterine exocoelom surrounded by continuous trophoblast. It was concluded that implantation in the marmoset monkey commenced on days 11-12.5 after ovulation and involved an intrusive mechanism. Although trophoblast penetration of endometrium was superficial, maternal capillaries were tapped at an early stage of implantation. The fusion of chorions of twins and triplets first occurred around day 19 after ovulation.     

New method to deliver exogenous material into developing planarian embryos

The ability to report or modify the embryological processes in living embryos is pivotal for developmental biology research. Planarian embryology has experienced renewed interest as the genetic pathways that drive adult regeneration were found to be involved in the development of embryos. The major drawback to the study of planarian embryology is the absence of methods that give access to the embryos and enable their manipulation. Herein, we report on a novel method for delivering external material into developing embryos using nanosecond laser pulses. When focused on the eggshell surface under optimal parameters, laser pulses ablate the protective case and open a pathway throughout which foreign material can be delivered. In this study, we used egg capsules from Schmidtea polychroa (Schmidt, 1861) to microinject 1 microm fluorescein isothiocyanate fluorescent beads into the live embryos. We obtained viability values ranging from 15% in early egg capsules to 100% in late developmental stages. Moreover, we measured the delivery effectiveness as the number of hatchlings containing fluorescent beads per microinjected egg capsule, reaching 100% in early stages and almost 40% in late stages. This is the first time that planarian embryos have been modified without compromising normal development. We consider that this technique will be of extreme value to future work on planarian developmental biology and regeneration, enabling the application of modern functional tools to the study of this Lophotrochozoan.     

Pregnancies following transfer of equine embryos cryopreserved by vitrification

The objective of this study was to investigate the in vitro and in vivo developmental abilities of equine embryos cryopreserved by vitrification. Twenty-eight embryos were recovered from Native pony and Thoroughbred mares at Days 5 to 7 by nonsurgical uterine flushing (detection of ovulation=Day 0). The vitrification solution contained 40% ethylene glycol, 18% Ficoll, and 0.3 M sucrose in PBS. The embryos were placed for 1 to 2 min in vitrification solution (Group 1) or following exposure to 20% ethylene glycol in PBS for 10 to 20 min (Groups 2 and 3). Single embryos were loaded in 0.25-ml straws, cooled for 1 min in liquid nitrogen vapor and immersed in liquid nitrogen. Straws were warmed in water (20 degrees C, 20 sec), and the contents were expelled with 0.5 M sucrose in PBS. Then the sucrose was diluted in 1-step (Groups 1 and 2) or 4-steps (Group 3). Embryos (n=21) were cultured for 120 h in TCM199 supplemented with 10% fetal bovine serum at 37 degrees C in 5% CO(2) in air and evaluated morphologically. Development to the hatching or hatched blastocyst stage was obtained in 0 7 , 4 7 and 4 7 embryos in Groups 1, 2 and 3, respectively. An additional 7 embryos were vitrified-warmed according to the treatment of Group 2 (4 embryos) and Group 3 (3 embryos). Five embryos were selected after in vitro culture for 4 h and were transferred nonsurgically into the uterine horn of Day-4 recipient mares. Transfer of 2 embryos (both Day-6 blastocysts: Group-2 treatment) resulted in pregnancies with a viable fetus at Day-60 of the gestation period.     

Interstrain inseminations and embryo transfers between the SLA miniature pig and standard crossbred pig

The purpose of this study was to determine fertilization and karyotypes of embryos after interstrain insemination and survival of embryos after reciprocal transfers between the National Institutes of Health SLA miniature pig and standard crossbred pig. Recovery of viable embryos indicated fertilization rates were not different in the two strains. Karyotypes of cells from embryos of both strains had the same chromosome number. The wide variation (within animal) in developmental stages of embryos recovered from the SLA minipig suggests the possibility of a prolonged ovulation interval, or a super imposed recruitment of a second set of follicles ovulating a few hours later. Cystic endometrial hyperplasia in the SLA minipig reduced the number of embryos recovered due to mechanical blockage of the uterine horns, thus preventing adequate flushing. SLA minipig recipients with no morphological evidence of cystic endometrial hyperplasia have a similar pregnancy rate to the standard pig. Cystic endometrial hyperplasia may contribute to reduced reproductive efficiency of the SLA minipig as a result of a detrimental effect on early embryo development and/or implantation.     

A non-surgical uterine lavage technique in large cats intended for treatment of uterine infection-induced infertility

This paper presents the successful use of a non-surgical, transcervical uterine lavage technique for the treatment of uterine infection-induced infertility in three female large cats. We developed a non-surgical uterine lavage technique, which allowed repeated flushing of the uterine lumen and installation of therapeutic antibiotics. The entire procedure was performed under general anaesthesia (duration of anesthesia ranged from 40 to 70 min). It was successfully applied in a Sumatran tiger (Panthera tigris sumatrae), a Corbett tiger (Panthera tigris corbetti) and an Amur leopard (Panthera pardus orientalis). The tigers were treated only once, whereas the leopard received four uterine treatments, due to re-infection after mating. Decisions to conduct uterine treatments were based on detection of uterine fluid during previous transrectal ultrasound examinations. The catheter was guided into the vagina, with the aid of an endoscope, passing the urethra, and then into the uterus, with the aid of transrectal ultrasonography. Both uterine horns were separately flushed with approximately 300 mL of cell medium M199, followed by an antibiotic infusion. Upon ultrasonographic re-examination, the topical uterine treatments resulted in an apparent decline in the inflammatory and/or degenerative processes. The Corbett tiger had the most severe uterine alterations, in addition to an aseptic pyometra. As a result, she was treated 1 month prior to ovariohysterectomy (in order to reduce the surgical risk). The Sumatran tiger was artificially inseminated twice after hormone-induced estrus, and the Amur leopard expressed a spontaneous estrus and re-initiated mating behaviour.