Influence of oocyte donor on in vitro embryo production in buffalo

The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n = 40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A + B COC recorded in each 4-PS period had great repeatability (r = 0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A + B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r = 0.60; P < 0.001), SFL (r = 0.68; P < 0.001), COC (r = 0.48; P < 0.01) and Grade A + B COC (r = 0.40; P < 0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P < 0.05) number of TFL (8.3 ± 0.9 compared with 5.6 ± 0.7) and SFL (7.3 ± 0.3 compared with 3.8 ± 0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r = 0.80; P < 0.05) and COC (r = 0.76; P < 0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.     

Influence of CIDR treatment during superovulation on embryo production and hormonal patterns in cattle

One of the major sources of success in embryo transfer is timing of AI relative to the LH surge and ovulation. The aim of this study was to compare the embryo production following superovulation during a PGF2alpha (control cycle) or a CIDR-B synchronized cycle (CIDR-B cycle). CIDR-B (CIDR-B ND, Virbac, Carros, France) was inserted on Day 11 of a previously synchronized cycle and left for 5 days. A total dose of 350 microg FSH was administered (eight injections i.m. for 4 days; first on Day 13, decreasing doses) and PGFalpha analog (750 microg i.m.: Uniandine ND, Schering-Plough, Levallois-Perret, France) injected at the time of third FSH injection. Artificial inseminations were performed 12 and 24 h after standing estrus (Day 0). Embryos were collected on Day 7. Luteinizing hormone was measured by EIA (Reprokit Sanofi, Libourne, France) from blood samples collected every 3 h for 36 h, starting 24 h after PGF2alpha (control cycle) or 12 h after CIDR-B removal (CIDR-B cycle). The effects of treatment group and interval between the LH peak and AI (two classes, < 10 and > or = 10 h) on embryo production and quality were analyzed by ANOVA. No effect of treatment was observed on embryo production variables. The intervals between the end of treatment and onset of estrus and between end of treatment and LH surge were greater in heifers treated during a control than a CIDR-B cycle, respectively (45.5 +/- 1.4 versus 31.9 +/- 0.7; 42.0 +/- 1.6 versus 31.0 +/- 1.5; P < 0.05), but maximal LH and estradiol concentrations, at the preovulatory surge were similar in control and CIDR-B synchronized heifers. The numbers of viable and Grade I embryos were significantly increased (P < 0.01) when animals had an interval from LH peak to first AI > or = 10 h (7.2 +/- 0.9 and 3.5 +/- 0.6) when compared to shorter intervals (4.2 +/- 1.1 and 2.0 +/- 0.7) whereas total number of embryos was unchanged (11.8 +/- 1.4 versus 10.3 +/- 1.8). It is concluded that late occurrence of LH peaks in relation to estrous behavior is associated with a lower embryo quality when first AIs are performed systematically 12 h after standing estrus. Further studies are needed to know if results may be improved when making AI at a later time after standing estrus or if LH assays are useful to better monitor AI time.     

小鼠遗传背景对4n胚胎及2n-4n聚合胚制作效率的影响

不同品系小鼠的 2 细胞期胚胎 (二倍体 ,2n) ,经电融合后 ,获得发育的 4 细胞期四倍体胚胎 (4n)的能力上存在着差异。将不同品系小鼠的 2n、 4n胚胎分别配对作聚合 ,所获 2n 4n聚合胚的发育结果表明 :在着床前 ,2n 4n聚合胚的获得率因胚胎品系组合的不同而异 ;胚胎移植后 ,聚合胚在与 4n胚胎相同或相近品系的移植受体中 ,其着床率较高 ;在着床后胎儿及出生仔鼠的获得率上 ,采用遗传杂合性的 2n胚胎所组成的 2n 4n聚合组合较高。上述结果提示 :小鼠的遗传背景可影响到 4n胚胎及相应 2n 4n聚合胚的制作效率。以GFP标记跟踪2n 4n聚合胚 4n细胞着床后的发育命运 ,发现 :妊娠中后期的孕体中 ,4n细胞限制性地分布至胚外组织     

Effect of timing of prostaglandin PGF 2 alpha injection subsequent to embryo collection on the resumption of normal follicular development following superovulatory treatment in cattle

Nonlactating Holstein and Jersey cows (n = 24) were superovulated and ovarian follicular development was monitored by transrectal ultrasound during the period after embryo recovery. Luteolysis was induced by two injections of prostaglandin F(2)alpha (PGF; 25 mg Lutalyse; 12-h interval) at specific times after superovulatory induced estrus (Treatment 1, Day 9; Treatment 2, Day 12; Treatment 3, Day 17; Treatment 4, Day 25; superovulatory estrus = Day 0 of Cycle 1). Follicular development was monitored during Cycle 1 before and after PGF injection and continued through the ensuing estrous cycle (Cycle 2). Superovulation led to more than one embryo collected in 14 cows (mean = 8.71 embryos: positive superovulatory response [PSR] cows), while 10 cows were not successfully superovulated (mean = 0.1 embryo; negative superovulatory response [NSR] cows). These cows differed in terms of number of unovulated follicles detected at embryo collection (4.21 vs 17.2, PSR vs NSR) and plasma progesterone during the superovulatory estrous cycle (32.3 ng/ml PSR vs 8.6 ng/ml NSR). Follicular development during Cycle 1 started sooner in NSR than in PSR cows (day by class by response P<0.03) and was initiated on Days 11 to 12 in NSR cows and on Days 19 to 20 in PSR cows. Interval to estrus after PGF averaged 6.3 d. Cows having short intervals to estrus had follicles at the time of PGF injection. Treatment influenced the length of Cycle 1, but it did not affect the interval to estrus after PGF, the length of Cycle 2, or follicular development during Cycle 2. The results indicate that 1) the timing of PGF injection after embryo collection does not influence subsequent follicular populations, 2) elongated estrous cycles and intervals to estrus after PGF in superovulated cattle are a function of decreased follicular activity, and 3) the presence of numerous corpora lutea and not the superovulatory treatment, per se, seem to attenuate follicular growth.     

Ultrasound-guided non-surgical embryo collection in the common marmoset

Experimental primate embryology has been hampered by limited access to embryos. In addition to surgical techniques, the less stressful non-surgical technique of uterine flushing has been developed but has had only limitedly used in recovering pre-implantation embryos from marmoset monkeys. In this study, we introduce the use of ultrasonography during marmoset non-surgical uterine flushing to make the cannulation easier, to further reduce stress, and to ensure thorough uterine flushing. We were able to cannulate in 99% of the transcervical cannulation attempts, repeat the flushing up to 17 times with the same animal, and recover up to 90% of the ovulation products. We also found that 8-cell or earlier stage embryos could be frequently obtained by non-surgical uterine flushing at 4 or 5 days after ovulation. The easiness and effectiveness of this novel ultrasound-guided technique will enable more research groups to study marmoset embryology and facilitate progress in this field.     

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.     

Automation of somatic embryo production

Automation could enhance the use of somatic embryogenesis for micropropagation in two ways: as effective tools for research on somatic embryogenesis, and for improving the efficiency of embryo production by reducing labor costs. Processes expected to be automated for somatic embryo production are: (1) evaluation of embryogenic cultures, (2) embryo development, (3) harvesting, (4) post-harvesting (pre-delivery) processes for enhancing conversion and preparing for delivery. In this review, the techniques related to the automation of each process are introduced and discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of the male on embryo quality

Early pregnancy failure or loss (EPL) represents a major source of wastage and inefficiency in livestock production systems. Although successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, potential adverse male affects on embryo quality and development are probably often underestimated. Of adverse male effects which have been identified, those associated with sperm and semen "quality" have been best characterized. In turn, although many factors can adversely impact semen quality, the mechanisms involved are relatively few. This presents opportunities for identifying biological markers for spermatogenic damage, as well as protective measures.     

A method for transcervical embryo collection in the pig

Five cyclic primiparous sows were used to test a surgical procedure for in vivo transcervical collection of pig embryos. The procedure consisted of shortening the uterine horns. After surgery, all sows returned to estrus and embryos were recovered following artificial insemination. Transcervical uterine flushing was carried out in four sows. On average 3.6 +/- 1.5 (mean +/- SD) embryos were recovered from the five sows. The results indicate that it is possible to recover embryos transcervically from sows with a resectioned uterus.     

Nonsurgical embryo collection from sows with surgically shortened uteri

To allow for the nonsurgical collection of swine embryos, the uteri of sows (n=7) were surgically shortened. A section of each uterine horn was resected to facilitate a transcervical flushing procedure. All sows with a shortened uterus exhibited natural estrus at least once after the operation. Four to six days after insemination, embryos were collected with a two-way Foley catheter. Embryos were collected (n=55, 6.3+/-6.0: x +/-SD ) from every treated sow. Although treated sows often did not exhibit estrus beyond 1 to 9 natural estruses, those sows (n=27) with persistent corpora lutea (CL) over a 4 to 5 wk period were given prostaglandin F(2alpha) (PGF(2alpha)) and they returned to estrus in 5.2+/-1.1 d: x +/-SD .     

Live birth of a bear cub following nonsurgical embryo collection

In the near future, 6 of 8 bear species will face extinction mainly because of loss of their natural habitat. This loss of habitat will ultimately require some of these bears to be maintained in zoos and wildlife preserves in the hope of conserving genetic diversity. If the giant panda is representative of other bear species, reproductive performance will be inhibited in such an environment. In this study, we used the nonendangered American black bear (Ursus americanus) as the model for developing appropriate embryo transfer procedures. The donor bear mated numerous times between late May and early June. In late July we anesthetized her and used a series of telescoping sheaths to gain access to the uterus Then we passed a catheter through the largest sheath, inflated the balloon, and, using a 20-mL syringe, repeatedly infused into and then aspirated from the uterus PBS + BSA. We emptied the syringe into Petri dishes and observed 2 embryos. We rinsed the embryos, placed them in human tubal fluid + HSA + HEPES and then held them at 35 degrees C for 5 h. The recipient mated during mid-June; in late July we anesthetized her and, with the aid of laparoscopy, transferred an embryo into the cranial portion of the uterine horn ipsilateral to the ovary containing a CL. The recipient delivered 2 cubs in January. Necropsy results indicated that the neonates lived for 6 to 8 wk before succumbing to flooding in the den. The DNA from hair samples belonging to the neonates indicated that the male cub belonged to the donor, the female cub to the recipient. The delayed implantation mechanism in bears probably allowed for the successful development of the embryo in the presence of a substantial asynchrony between the donor and the recipient (13 d). We conclude that embryo transfer is possible in the American black bear and can lead to the birth of live cubs.     

Repeated surgical embryo recovery and embryo production in rabbits

The purpose of this study was to assess the embryo production after repeated surgical recovery of embryos in Gigante de Espa?a does. A total of 195 ovulatory treatments and embryo recoveries were performed from 1995 to 1999. Ovulation was induced by an intramuscular injection of 20 microg GnRH immediately after mating. Each doe was induced to ovulate up to four consecutive times at intervals of at least 50 days. Embryos were surgically collected from oviducts 68-69 h post-coitus. An average of 8.6 corpora lutea and 6.4 recovered embryos (90% of them classified as viable) were recorded from the 195 treatments. The process seemed to be less efficient in the fourth treatment, with a drop of more than two recovered and viable morulae with reference to the third (P<0.05 for both parameters) or the second recovery (P<0.1 and P<0.05, respectively). More than 20 recovered embryos and 18 viable embryos per donor doe were recorded considering the three first ovulatory treatments performed in 33% of the does (30/90). Results indicate that the methodology used in the present study could be an efficient way to maximize in vivo embryo production from rabbits.     

Effect of age of donor cows on embryo production

The effect of age on the total, transferable and percent of transferable embryos was examined in 144 embryo collections from 56 Texas Longhorn cows aged from 3 to 22 years. At 10 years of age, the percent of embryos collected that were transferable declined because of a decline in the viability of the embryos. The total embryos recovered per collection remained the same for all ages, which suggested that the pool of follicles present in the ovary was sufficient to respond to superovulation in a similar manner for all ages studied.