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 ... -  《-》

Survival of vitrified in vitro-produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure.

Vitrification is an alternative to slow-rate freezing for cryopreserving bovine embryos. However, this technology requires simplification if it is to be used under field conditions. The main objective of this work was to develop a new system for the direct transfer of vitrified embryos to be used under farm conditions. For this, three objectives were set: (1) to compare the effect of vitrification, using the cryologic vitrification method (CVM), and slow-rate freezing on bovine embryo development and quality; (2) to develop a one-step warming procedure for bovine in vitro-produced (IVP) vitrified (by CVM) embryos; and (3) to assess the effects on embryo survival of a new method for the direct transfer of vitrified IVP bovine blastocysts. In vitro-produced blastocysts were initially either vitrified by CVM or subjected to slow freezing to compare embryo survival and quality (experiment 1). No differences were detected between these cryopreservation techniques in terms of the survival and quality variables at 24 hours or in terms of the proteins expressed. However, at 48 hours the vitrified embryos showed higher hatching rates, greater total cell numbers, and lower apoptotic indices (P < 0.05). In experiment 2, CVM-vitrified IVP blastocysts were warmed by the conventional two-step or one-step warming procedure by incubating them at 41 °C in 0.25 M sucrose for 10 minutes, 0.15 M sucrose for 10 minutes, or 0.25 M sucrose for 5 minutes. In addition, embryo transfer (ET) was performed using vitrified embryos warmed by the one-step procedure in 0.25 M sucrose solution for 5 minutes. As a control group, IVP fresh embryos were transferred to recipient females. No differences were observed in embryo survival or total cell number between any of the warming procedures. Moreover, no significant differences for pregnancy at 60 days were found between the ET groups. In experiment 3, expanded IVP blastocysts were then either vitrified using a conventional or a modified fiber plug designed to allow direct ET after in-straw cryoprotectant (CP) dilution. They were warmed using the one-step process (0.25 M sucrose, 5 minutes) in a 0.25 mL French straw. Embryo recovery associated with the modified fibreplug system was less reliable than with the conventional system. However, no differences were seen between the systems in terms of in vitro embryo survival among those finally recovered. Finally, IVP blastocysts were vitrified using conventional fibreplugs to maintain a high embryo recovery rate, and then warmed using the one-step warming in-straw CP dilution procedure, but using an adapter with a wider opening coupled to the French straw and a heated metal chamber to protect and keep the straw at 41 °C (experiment 4). No differences were seen in embryo survival rates between the two groups. The CVM combined with this new one-step warming in-straw CP dilution procedure could be used for direct ET under field conditions.

Caamaño JN ，Gómez E ，Trigal B ，Muñoz M ，Carrocera S ，Martín D ，Díez C ... -  《-》

Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in-straw warming of in vitro-produced bovine embryos.

The cold-adapted bacterium Pseudomonas sp. ID1 produces the extracellular exopolysaccharide ID1 (EPS ID1) with cryoprotective activity. This study was designed to optimize the vitrification/in-straw warming protocol of in vitro-produced (IVP) blastocysts by adding EPS ID1 to the vitrification media. Day 7-expanded blastocysts were vitrified/warmed using the VitTrans device after the addition of 0 or 100 μg/mL EPS ID1 to the vitrification media. Blastocysts vitrified by the Cryotop method and fresh non-vitrified blastocysts served as controls. Outcomes were assessed in the warmed embryos in terms of survival rates and mRNA relative abundances of BAX, BCL2, GPX1, and CDX2 genes. No differences in survival rates were observed at 3 h post-warming between vitrification treatments. At 24 h post-warming, the addition of EPS prior to vitrification with the VitTrans device produced similar survival rates to Cryotop-vitrified embryos and similar hatching rates to fresh non-vitrified or Cryotop-vitrified embryos. No differences emerged in BCL2 gene expression. Lower BAX (p < .05) and higher GPX1 (p < .05) and CDX2 (p < .1) gene expression were observed in expanded and/or hatched blastocysts derived from VitTrans-EPS-vitrified embryos when compared to those from the non-supplemented group. In conclusion, addition of EPS not only promoted blastocyst survival and hatching after VitTrans vitrification/warming but also modified the expression of genes associated with better embryo quality.

Martínez-Rodero I ，Salas-Huetos A ，Ordóñez-León A ，Hidalgo CO ，Yeste M ，Mercadé E ，Mogas T ... -  《-》

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》

New device for the vitrification and in-straw warming of in vitro produced bovine embryos.

Two experiments were designed to test the use of a new device designed to vitrify and in-straw warm in vitro produced (IVP) embryos, which can potentially be used for their direct transfer to recipient females in field conditions. In experiment 1, IVP embryos from both prepubertal and adult animals were vitrified on cryotops and warmed in steps (1, 0.5 and 0M sucrose; protocol W3) or directly in 0.5M (protocol W1/0.5) or 0M sucrose (protocol W1/0). Similar survival rates were recorded 24h after warming for calf embryos irrespective of the warming procedure (W3: 79.2%, W1/0.5: 62.5%, W1/0: 66.7%). For cow embryos, survival rates at 24h post-warming were significantly higher when embryos were warmed using the W3 (85.7%) or W1/0.5 (89.1%) protocols compared to the W1/0 protocol (70.5%). In experiment 2, IVP embryos were vitrified on the new designed device followed by their in-straw cryoprotectant (0.5M sucrose) dilution/warming and different warming temperatures (45, 50, 60 and 70°C) were tested. When warming solution passed through the new vitrification/warming device at 45°C, 61.5% of blastocysts were fully re-expanded or hatched at 24h post-warming, being not significantly different to the control (65%). Other warming temperatures triggered significantly lower survival rates at 24h post-warming. No significant differences were detected in total cell numbers and blastocyst apoptosis indices in response to vitrification followed by warming at 45°C respect to the control. Our findings indicate that the new device allows vitrification and in-straw warming of IVP bovine embryos, being a useful option for their direct transfer in field conditions.

Morató R ，Mogas T 《-》