Embryo survival and recipient pregnancy rates after transfer of fresh or vitrified, in vivo or in vitro produced ovine blastocysts.

The aim of this study was to assess the effect of production system and of cryopreservation of ovine embryos on their viability when transferred to recipients. The experimental design was an unbalanced 2 x 2 factorial design of two embryo production systems (in vivo versus in vitro) and two embryo preservation conditions prior to transfer (transferred fresh versus transferred after vitrification/warming). For the production of blastocysts in vivo, crossbred donor ewes (n=30) were synchronised using a 13-day intravaginal progestagen pessary. Ewes received 1500 IU equine chorionic gonadotropin (eCG) 2 days before pessary withdrawal, and were mated 2 days after pessary withdrawal and embryos were recovered surgically (6 days after mating). Blastocysts were produced in vitro (IVP) using standard techniques. Recipients (n=95) were synchronised using a progestagen pessary and received 500 IU eCG at pessary removal and were randomly assigned to receive (two per recipient) in vivo fresh (n=10), in vivo vitrified (n=10), in vitro fresh (n=35) or in vitro vitrified (n=40) blastocysts. Recipients were slaughtered at day 42 of gestation and foetuses recovered. Pregnancy and embryo survival rates were recorded and analysed using CATMOD procedures. Foetal weights and crown-rump lengths were recorded and analysed using generalised linear model (GLM) procedures. There were no statistically significant interactions between the effects of embryo production system and preservation status at transfer on pregnancy rate and embryo survival. The pregnancy rate following transfer of fresh IVP blastocysts was lower (P<0.07) than that of in vivo embryos (54.3% versus 90.0%, respectively). Vitrification resulted in a decrease in pregnancy rate, the effect being more pronounced in the case of IVP embryos (54.3-5.0%, P<0.001) compared with in vivo embryos (90.0-50.0%), although the absolute change was similar (49.3% versus 40.0%). Transfer of fresh IVP blastocysts resulted in a higher proportion of single (78.9% versus 33.3%) and lower proportion of twin (21.1% versus 66.7%) pregnancies than those produced in vivo. This was reflected in a significant difference in embryo survival rate (fresh: 32.8% versus 75.0%, P<0.01; vitrified: 2.5% versus 35.0%, P<0.001, for IVP and in vivo blastocysts, respectively). Similarly, all pregnancies resulting from the transfer of vitrified/warmed IVP blastocysts were single pregnancies, while 40% of those from vitrified/warmed in vivo blastocysts were twin pregnancies; this was reflected in an embryo survival rate of 35.0% versus 75.0%, respectively. There was a significant effect (P=0.0184) of litter size on foetal weight but not on foetal length (P=0.3304). Foetuses derived from the fresh transfer of IVP blastocysts were heavier (6.4+/-0.2g versus 5.8+/-0.2g, respectively, P<0.05) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.01) than those derived from fresh in vivo blastocysts. There was no difference in these parameters as a consequence of vitrification of IVP embryos. However, in vivo blastocysts subjected to vitrification resulted in heavier (6.6+/-0.3g versus 5.8+/-0.2g, respectively, P=0.055) and longer (5.2+/-0.1cm versus 4.8+/-0.1cm, respectively, P<0.05) foetuses than their counterparts transferred fresh.

Papadopoulos S ，Rizos D ，Duffy P ，Wade M ，Quinn K ，Boland MP ，Lonergan P ... -  《ANIMAL REPRODUCTION SCIENCE》

Vitrification of in vitro-produced and in vivo-recovered equine blastocysts in a clinical program.

There is a clinical demand for cryopreservation of both in vivo-recovered and in vitro-produced (IVP) equine embryos. We previously reported successful vitrification of expanded equine blastocysts in fine-diameter microloader pipette tips (MPTs) after blastocoel collapse, in a research setting. Here, we report the results of clinical application of the MPT vitrification technique for both in vivo-recovered and IVP blastocysts. In vivo-recovered blastocysts were obtained by referring veterinarians on Days 6 to 8 after ovulation, and shipped 1 to 10 hours to the laboratory before vitrification. IVP blastocysts (<300 μm in diameter) were produced by intracytoplasmic sperm injection and in vitro embryo culture. All vitrified-warmed embryos were shipped (0.5-12 hours) for transfer to recipient mares. In experiment 1, 47 IVP embryos from our clinical intracytoplasmic sperm injection program were vitrified using the MPT and transferred. The rates of initial pregnancy (59%) and foaling (45%) were equivalent to those for 52 IVP embryos from the same mare aspiration sessions and shipped for the same duration but transferred fresh (75% and 45%, respectively). The pregnancy and foaling rates for in vivo-recovered embryos were 76 and 71%, respectively for 17 small blastocysts (<300 μm in diameter), and 55 and 45%, respectively for 11 large blastocysts (303-608 μm in diameter, collapsed before vitrification; P > 0.1). In experiment 2, the MPT was cut lengthwise to form an open vitrification device, designated "Sujo". Research IVP blastocysts were vitrified at 1, 2, or 3 embryos per Sujo (n = 34 embryos), or singly on a commercial open device (Cryolock; n = 11). After warming, 97% and 91% of embryos, respectively, grew in culture. Similarly, culture of two in vivo-recovered large blastocysts after collapse and vitrification on Sujos both resulted in embryo growth. However, transfer of four in vivo-recovered expanded blastocysts after collapse, vitrification on Sujos, and warming resulted in only one foal. These data indicate that vitrification of equine IVP embryos and small in vivo-recovered embryos is efficient under clinical conditions. Collapse and vitrification of in vivo-recovered large blastocysts in MPT under our clinical conditions resulted in a 45% foaling rate. While numbers are low, use of an open vitrification system did not appear to improve results for these embryos.

Choi YH ，Hinrichs K 《-》

Survival and viability of vitrified in vitro and in vivo produced ovine blastocysts.

Dattena M ，Ptak G ，Loi P ，Cappai P ... -  《THERIOGENOLOGY》

Vitrification and in-straw warming do not affect pregnancy rates of biopsied bovine embryos.

In the cattle industry, in vivo or in vitro embryo production combined with genotyping and cryopreservation technologies allows the selection and conservation of embryos carrying genes for desirable traits. This study aimed to assess the efficiency of a vitrification method suitable for in-straw warming of biopsied in vivo derived (IVD) bovine embryos. Three experiments were carried out using two methodologies: the Cryotop®, the gold standard vitrification and 3-step warming methodology, or the VitTrans, a vitrification/in-straw 1-step warming method that enables direct embryo transfer to the uterus. In experiment 1, intact and biopsied in vitro produced (IVP) day 7 expanded blastocysts were vitrified using the Cryotop® and warmed in 1- or 3-steps. No differences in survival rates were recorded at 24 h after warming for intact or biopsied IVP blastocysts irrespective of the warming procedure. In experiment 2, the effect of the time from trophectoderm (TE) biopsy to vitrification/in-straw warming on post-warming survival rate was assessed. No significant differences in survival were observed when blastocysts were vitrified/in-straw warmed immediately after biopsy or after 3 h in culture when compared to intact blastocysts. In experiment 3, IVD embryos were vitrified 3 h after biopsy using the Cryotop® or the VitTrans method and pregnancy rates were assessed at day 60 after transfer. Fresh, biopsied embryos served as control. Similar pregnancy rates were observed when IVD biopsied embryos were transferred fresh or vitrified/warmed by the Cryotop® or VitTrans method. No significant effect of the embryo quality or developmental stage was detected on the percentage of pregnant recipients when IVD biopsied embryos were transferred fresh or after vitrification. While fresh female IVD embryos produced significantly higher pregnancy rates than male embryos, there were no differences in pregnancy rates when male or female vitrified/warmed embryos were transferred. About 81% from the biopsies analyzed successfully determined the embryo sex, confirming that DNA was there, and it was efficiently amplified. To conclude, our findings indicate that both vitrification methodologies produced similar post-warming outcomes for both intact and biopsied IVP embryos. Besides, vitrification/in-straw warming of biopsied IVD bovine embryos did not affect the viability to originate pregnancy, being a useful option for their direct transfer in field conditions.

González-Rodríguez N ，Martínez-Rodero I ，Scherzer J ，Jung S ，Reichenbach M ，Zablotski Y ，Otzdorff C ，Zerbe H ，Mogas T ... -  《-》

Effect of warming method on embryo quality in a simplified equine embryo vitrification system.

Equine embryo vitrification is still not a well-established technique in equine practice. Notably, little work has been done on the effect of the warming system on viability of vitrified embryos. Our goal was to evaluate the effect of warming without cryoprotectants on in vitro - produced (IVP) embryo viability in culture, quality assessment parameters, and pregnancy after transfer. Equine IVP blastocysts were vitrified using commercial embryo vitrification media and a semi-closed vitrification device. In Exp. 1, we evaluated two warming temperatures (room temperature, RT, ∼22 °C; and 38 °C) for each of three warming systems: commercial warming solution (Kit); commercial embryo holding medium (EHM) with decreasing concentrations of sucrose (EHM + SS); or EHM alone without added sucrose. Embryos (n = 9 to 14 per treatment) were cultured in vitro for 24 h, stained with DAPI, TUNEL, and fluorophore-labelled phalloidin, and evaluated for nucleus number, mitotic rate, apoptotic rate, and actin filament distribution. In Exp. 2, to survey embryo viability in vivo, vitrified IVP blastocysts were shipped to an embryo transfer facility, then warmed immediately before transfer to recipient mares, using the warming treatments associated with the nominally best (Kit-RT, Kit-38, EHM-RT) and poorest (EHM + SS-38) assessed embryo quality in Exp. 1 (n = 7 to 8 per treatment). Subsequently, IVP blastocysts produced as part of our clinical program were vitrified and shipped, then warmed in embryo holding medium at an embryo transfer facility before transfer to recipient mares; fresh IVP embryos were shipped and transferred as controls. In Exp. 1, embryos increased significantly in diameter after culture (P < 0.01), with no difference among treatments. There was no difference (P > 0.05) in the number of viable nuclei, apoptotic rate, or microfilament distribution among treatments, or between vitrified-warmed and Control embryos. The mitotic rate was higher (P = 0.021) for Kit-RT (3.6%) when compared with the other treatment groups (1.5-2.0%). In Exp. 2, there was no difference (P > 0.05) in initial pregnancy (71.4-87.5%) or heartbeat (57.1%-85.7%) rates among warming treatments. In the clinical trial, there was no difference (P > 0.05) between vitrified-warmed and Control embryos in initial pregnancy (90.9% and 66.6%, respectively) or heartbeat (81.8% and 66.6%, respectively) rates. These results indicate that a semi-closed vitrification system using commercially-available media, and incorporating warming in the field in a single step using commercial embryo holding medium without cryoprotectants, can provide high pregnancy rates with IVP equine embryos.

Canesin HS ，Ortiz I ，Rocha Filho AN ，Salgado RM ，Brom-de-Luna JG ，Hinrichs K ... -  《-》