85 DOMESTIC CAT KITTENS BORN AFTER TRANSFER OF CRYOPRESERVED EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERM

2011 ◽  
Vol 23 (1) ◽  
pp. 148 ◽  
Author(s):  
C. E. Pope ◽  
E. G. Crichton ◽  
M. C. Gómez ◽  
C. Dumas ◽  
B. L. Dresser
Keyword(s):  
X Chromosome ◽  
Domestic Cat ◽  
Assisted Reproductive Techniques ◽  
Developmental Competence ◽  
Embryo Survival ◽  
Male Birth ◽  
Reproductive Techniques ◽  
Cryopreserved Embryos

For successful application of sperm sorting technology to a particular species, the various assisted reproductive techniques required for in vivo or in vitro embryo production must be established. Previously, we have demonstrated the in vivo developmental competence of cat embryos derived by IVF 1) with presumptive X-chromosome bearing sperm and 2) after controlled-rate cryopreservation. Here, our intent was to determine in vivo viability by transfer to recipients of cryopreserved cat embryos produced by IVF with flow-sorted sperm. Semen collected from a single male using an artificial vagina was extended in electrolyte-free solution and shipped overnight (4°C) to the sorting facility. After sorting immediately upon arrival, sperm were concentrated by centrifugation, suspended in TEST yolk buffer, and return-shipped to the IVF laboratory at 4°C. At 48 h after semen collection, in vivo matured oocytes (n = 64) recovered from 2 gonadotropin-treated donors were inseminated in vitro with presumptive X-chromosome-bearing sperm. At 5 h post-insemination, oocytes were rinsed and placed in modified Tyrodes+BSA and NEAA (IVC-1) at 38°C in a humidified atmosphere of 5% O2, 5% CO2, and 90% N2. At 27 h post-insemination (Day 1), 43 two-cell embryos (67% cleavage rate) were equilibrated (3 steps) in 1.4 M propylene glycol, 0.125 M sucrose, and 10% dextran 70, cooled at a slow controlled rate (0.3°C min–1) from –6°C to –30°C, and plunged into liquid nitrogen. After 26 to 43 months in cryostorage, 4 straws, each containing 8 or 9 embryos, were thawed in air (22°C) and cryoprotectant was removed from the embryos by a 5-step rinse. Then, embryos were cultured for 17 to 20 h in IVC-1, as described earlier. On Day 2, 35 embryos were transferred (8 or 9 per recipient) by laparoscopy to the oviduct of four Day 1 gonadotropin-treated recipients at 24 h after laparoscopic oocyte retrieval (16 to 25 oocytes/donor). Most embryos (83%) were at the four- to eight-cell stages at transfer. Three recipients established pregnancies and delivered, without assistance, litters of 1, 2, and 3 live kittens (27% embryo survival) between Day 64 and Day 67 of gestation. Four kittens were female, and two were male. Birth weights ranged from 73 to 133g (mean = 117 g). Overall, of 18 kittens [12 from fresh (2009 Theriogenology, 71, 864) +6 from frozen embryos] born after transfer of embryos produced by IVF with sex-sorted sperm, 16 (89%) have been female. The variation in sex ratios between kittens born from fresh v. cryopreserved embryos probably is a reflection of the sort purity of the different sperm samples used for IVF. The ability to produce kittens after transfer of cryopreserved embryos derived by in vitro insemination with sex-sorted sperm represents an expansion in the assortment of assisted reproductive techniques that may be useful for selective propagation of biomedically relevant and endangered felids.

66 DEVELOPMENT OF IN VITRO-PRODUCED CAT EMBRYOS AFTER VITRIFICATION AND NONSURGICAL EMBRYO TRANSFER: PRELIMINARY RESULTS

2009 ◽  
Vol 21 (1) ◽  
pp. 133
Author(s):  
E. Iacono ◽  
B. Merlo ◽  
M. Regazzini ◽  
D. Zambelli
Keyword(s):  
Ethylene Glycol ◽  
Embryo Transfer ◽  
Domestic Cat ◽  
Embryo Survival ◽  
Abdominal Ultrasonography ◽  
Preliminary Results ◽  
Pregnancy Status ◽  
Viable Embryo

There are no refereed reports on vitrification of domestic cat embryos derived from in vitro-matured oocytes and transferred using a nonsurgical embryo transfer technique. The aim of this study was to verify the effects of vitrification on the in vitro and in vivo developmental ability of in vitro-produced (IVP) cat blastocysts. Oocytes recovered from minced ovaries were matured, fertilized, and cultured in vitro as previously reported (Merlo B et al. 2005 Theriogenology 63, 2032–2039). On Day 7 of in vitro culture (IVC), blastocysts were selected and vitrified in straws (Cristal ET 0.25 mL, 133 mm, IMV-Technologies, Paillette Crista, France). For vitrification (modified from Campos-Chillòn LF et al. 2006 Theriogenology 65, 1200–1214), the embryos were transferred in 1 mL of V1 [ethylene glycol 3.5 m in HEPES synthetic oviductal fluid (HSOF)] for 3 min, and then in 10 μL of V2 (ethylene glycol 7 m, galactose 0.5 m, Ficoll 70 18% in HSOF) for 20 s. Finally, the embryos were loaded in straws preloaded with 190 μL of dilution solution (galactose 0.5 m in HSOF). Straws were heat sealed and immediately plunged into liquid nitrogen. Vitrified embryos were warmed in air for 10 s, and then in a waterbath at 37°C for 30 s. For developmental ability and in vitro evaluation, 27 embryos were warmed and immediately examined: 25 re-expanded, 2 did not re-expand, and 1 had damaged zona pellucida. Re-expanded embryos were cultured in SOF plus amino acids, 16 mg mL–1 BSA, and 5% fetal bovine serum at 38.5°C in 5% O2, 5% CO2, 90% N2. After 24 h of IVC, only 4 blastocysts were expanded, and after 48 h, embryos were clearly degenerated or shrunk. in vivo developmental ability was tested by nonsurgical embryo transfer of 8 vitrified-warmed embryos and 6 IVP fresh embryos into 2 natural estrus queens, injected with 200 IU of hCG i.m. (Day 0) for induction of ovulation. Ovulation was confirmed by plasmatic progesterone assay on Day 5. Nonsurgical embryo transfer was made on Day 8 using the catheter proposed by Zambelli et al. 2001 for transcervical insemination in the cat. The catheter was connected to a 1-mL syringe and loaded with the embryos. Then, it was inserted in the vagina and transrectally guided into the uterus, where the embryos were deposited. To assess pregnancy status, abdominal ultrasonography was done on recipients on Day 13, 25, and 40. On Day 13, an embryonic vesicle was observed in both queens, although a smaller diameter than expected was detected in the recipient of the vitrified embryos. On Day 25, a viable embryo was detected only in the recipient of fresh IVP embryos. On Day 40, the gestational chamber was still present but no sign of a viable embryo was detected. Further studies are in progress to improve the nominal incidence of pregnancy and frequency of embryo survival after vitrification. Nevertheless, the preliminary results obtained using an AI catheter for nonsurgical embryo transfer are encouraging, and the improvement of the technique could make it reliable in the cat. Supported by Animal Stem Cells Laboratory, Regione Emilia Romagna, PRRIITT Project Number M-404AIWTSV.

scholarly journals Developmental Competence of Domestic Cat Vitrified Oocytes in 3D Enriched Culture Conditions

Animals ◽  
2019 ◽  
Vol 9 (6) ◽  
pp. 329 ◽  
Author(s):  
Martina Colombo ◽  
Maria Giorgia Morselli ◽  
Mariana Riboli Tavares ◽  
Maricy Apparicio ◽  
Gaia Cecilia Luvoni
Keyword(s):  
Developmental Stages ◽  
Culture Conditions ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Vitro Fertilization ◽  
In Vitro Embryo Production ◽  
Physical And Chemical

Cryoinjuries severely affect the competence of vitrified oocytes (VOs) to develop into embryos after warming. The use of culture conditions that provide physical and chemical support and resemble the in vivo microenvironment in which oocytes develop, such as 3D scaffolds and coculture systems, might be useful to improve VOs outcomes. In this study, an enriched culture system of 3D barium alginate microcapsules was employed for the in vitro embryo production of domestic cat VOs. Cryotop vitrified-warmed oocytes were in vitro matured for 24 h in the 3D system with or without fresh cumulus-oocyte complexes (COCs) in coculture, whereas a control group of VOs was cultured in traditional 2D microdrops of medium. After in vitro fertilization, presumptive embryos were cultured in 3D or 2D systems according to the maturation conditions. Vitrified oocytes were able to mature and develop into embryos in 3D microcapsules (17.42 ± 11.83%) as well as in 2D microdrops (14.96 ± 8.80%), but the coculture with companion COCs in 3D resulted in similar proportions of VOs embryo development (18.39 ± 16.67%; p = 1.00), although COCs presence allowed for blastocyst formation (0.95 ± 2.52%). In conclusion, embryos until late developmental stages were obtained from cat VOs, and 3D microcapsules were comparable to 2D microdrops, but improvements in post-warming conditions are still needed.

267 BIRTH OF DOMESTIC CAT KITTENS OF PREDETERMINED SEX AFTER TRANSFER OF EMBRYOS PRODUCED BY IN VITRO FERTILIZATION OF OOCYTES WITH FLOW-SORTED SPERMATOZOA

2008 ◽  
Vol 20 (1) ◽  
pp. 213 ◽  
Author(s):  
C. E. Pope ◽  
E. B. Crichton ◽  
M. C. Gmez ◽  
C. Dumas ◽  
B. Dresser
Keyword(s):  
X Chromosome ◽  
Egg Yolk ◽  
Sex Selection ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
Single Male ◽  
Vitro Fertilization ◽  
Food Dye

In cats, sex selection by fertilization of oocytes with sperm sorted into X- andY-chromosome-bearing populations has credible biomedical, commercial, and conservation connotations. Our objectives were (1) to evaluate the efficiency with which embryos could be produced by IVF of in vivo- and in vitro-matured oocytes with cooled sex-sorted sperm after overnight shipment to the sorting facility and overnight return delivery to an IVF laboratory, and (2) to determine if live kittens of predetermined sex (female) could be produced after transfer of embryos derived by IVF of in vivo-matured oocytes with X-chromosome-bearing sperm to recipient females. Semen samples (n = 5) collected from a single male using an artificial vagina were extended in electrolyte-free solution (glucose–BSA) and shipped overnight in an Equitainer (4�C) to the sorting facility. Upon arrival, sperm were stained (9 µm Hoechst 33 342; 75 � 106 sperm mL–1), adjusted to 50 � 106 sperm mL–1 with 4% egg yolk TALP containing 0.002% food dye, and sorted on an SX MoFlo� flow cytometer (Dako, Fort Collins, CO, USA). Purities from resort analysis averaged 94% (X) and 83% (Y). After sorting, sperm were concentrated by centrifugation, suspended in TEST-yolk buffer (Irvine Scientific, Santa Ana, CA, USA), and return-shipped to the IVF lab where they were received 48 h after collection. In vivo-matured oocytes recovered from gonadotropin-treated donors were inseminated in vitro with X-chromosome-bearing sperm. In vitro-matured oocytes obtained from ovaries donated by local clinics were inseminated in vitro with control (cooled/shipped/non-sorted) or X- orY-chromosome-bearing sperm. At 5 h or 18 h post-insemination, in vivo- and in vitro-matured oocytes, respectively, were rinsed and placed in IVC-1 medium (Pope et al. 2006 Theriogenology 66, 59–71). Embryos produced from in vitro-matured oocytes were allowed to develop until Day 8 in a three-step culture system (Pope et al. 2006). Cleavage frequency of in vitro-matured oocytes after insemination with X-, Y-, and control sperm was 33% (40/120), 35% (52/150), and 42% (48/115), and blastocyst development was 50% (11/22), 55% (23/42), and 53% (21/40), respectively (P > 0.05). Incidence of cleavage after insemination of in vivo-matured oocytes with X-sperm was 62% (54/87). On Day 2, 45 embryos (9–16 per recipient) produced by in vitro insemination of in vivo-matured oocytes with X-sperm were transferred by laparoscopy to the oviducts of four Day 1 gonadotropin-treated recipients. Three recipients established pregnancies and delivered litters of one, four, and seven female kittens between Day 62 and Day 66 of gestation. We have demonstrated that sperm-sorting technology can be applied and used effectively in domestic cats and, potentially, should be relevant to the selective breeding of endangered cats.

scholarly journals 38BIRTH OF AFRICAN WILD CAT CLONED KITTENS

2004 ◽  
Vol 16 (2) ◽  
pp. 141 ◽  
Author(s):  
M.C. Gomez ◽  
C.E. Pope ◽  
A.M. Giraldo ◽  
L. Lyons ◽  
R.F. Harris ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Nuclear Transfer ◽  
Pcr Amplification ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Felis Silvestris ◽  
Cell Nuclei ◽  
Oocyte Aspiration

The African wild cat (AWC, Felis silvestris lybica; 2n=38) is one of the smallest wildcats, and it’s future is threatened by hybridization with domestic cats (Felis silvestris catus; 2n=38). Nuclear transfer (NT) is a potentially valuable tool for retaining genetic variability, and could assist in the continuation of species with few remaining individuals. Inter-species nuclear transfer into domestic cat (DSH) supports development of somatic cell nuclei from AWC (Gomez et al., 2003, Biol Reprod 69, 1032–1041). Therefore, the purpose of the present study was to evaluate the in vivo developmental competence of nuclear transfer embryos derived by fusion of African wildcat fibroblasts with domestic cat cytoplasts, after transfer into domestic cat recipients. In vivo- and in vitro-matured domestic cat oocytes were mechanically enucleated in modified Tyrodes salt solution supplemented with 20μgmL−1 of cytochalasin B (CCB) and 2mgmL−1 of sucrose, and reconstructed with AWC fibroblast cells derived from an adult male; cultured and passaged 1 to 3 times before serum-starved with DMEM +0.5% FBS and cultured for 5 additional days before use. Fusion took place in fusion medium (0.3M mannitol and 0.1mMMg+2), and membrane fusion was induced by applying a 3s AC pre-pulse of 20V, 1MHz; followed by two 30μs DC pulses of 240V/mm at intervals of 0.5s. Fused couplets were activated 2–3h after fusion by placing the couplets between two electrodes in a fusion chamber containing 3mL of fusion medium and exposing them to two 60μs DC pulses of 120V/mm. Then, couplets were incubated in 30μL drops of Tyrodes solution containing 1% MEM nonessential amino acids, 3mgmL−1 BSA (IVC-1 medium), and supplemented with 10μgmL−1 cycloheximide and 5μgmL−1 CCB at 38°C in 5% CO2 for 4h. After activation, cloned embryos were cultured in 500μL of IVC-1 medium until the day of the transfer. Derived AWC NT embryos were transferred into the oviducts (Day 1) or uteri (Days 5, 6, 7) of 36 gonadotrophin-treated DSH recipients on Day 1 after ovulation or on Days 5, 6, or 7 after oocyte aspiration, respectively. Pregnancy was assessed by ultrasonography on Days 21 to 23. One domestic cat was still pregnant and ongoing on Day 60. Kittens were delivered by Cesarean section in each of the seven pregnant recipients on days 61 to 67 of gestation. The kittens weighed an average of 86.2g (50.0 to 103g) and died within 36h after delivery. The post-mortem pathology reports revealed that most of them had an immature respiratory system. The clonal status of the kittens was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. Results from these assays confirmed that the AWC kittens had originated from the AWC donor somatic cell line and were not related to the DSH recipient cats. In summary, these results indicate that AWC cloned kittens can be produced by ET of embryos derived from AWC cells into DSH cytoplasts. Research was funded partially by the John & Shirley Davies Foundation. Table 1

scholarly journals hCG Priming Before Ovary Collection Increasing The Oocyte Quality In The Domestic Cat

2021 ◽  
pp. 123-126
Author(s):  
Karisma Mardatillah ◽  
Rini Widyastuti ◽  
Diah Nugrahani Pristihadi ◽  
Wahyudin ◽  
Sigit Prastowo ◽  
...  
Keyword(s):  
Oocyte Quality ◽  
Domestic Cat ◽  
Developmental Competence ◽  
Control Group ◽  
Oocyte Competence ◽  
Slicing Method ◽  
Determining Factor

Oocyte competence is a determining factor that influences the embryo development. Embryos produced in vitro have a reduced developmental competence than embryos produced in vivo. Therefore, human Chorionic Gonadotropin (hCG) injection was carried out to improve the quality of the oocytes. The objective of this study was to evaluate the effect of ovarian stimulation with hCG before ovary collection on oocyte quality in the domestic cat. Oocyte donors were either 1) treated with a single dose of 200 IU hCG four days before ovary collection (hCG group), or, 2) no treatment before ovary collection (control group). The oocytes were collected by the slicing method. Immature cumulus oophorus complexes (COCs) from both groups were pooled and matured in vitro for 24-26 hours. Then mature oocytes were fertilized with epididymal sperm and cultured in vitro for seven days. The results study showed that the number of the dominant follicle (DF) and the number of COCs in the hCG group was higher than the control group in right and left ovaries (p<0.05). The morulae and blastocyst rates from cleavage embryos were 88% and 75%, respectively. These results demonstrate that hCG priming of oocytes donors before ovary collection improve oocyte quality.

scholarly journals 36 IMPROVING THE APPLICATION OF NUCLEAR TRANSFER FOR PRODUCING NON-DOMESTIC FELIDS

2005 ◽  
Vol 17 (2) ◽  
pp. 168 ◽  
Author(s):  
M.C. Gomez ◽  
C.E. Pope ◽  
L. Lyons ◽  
A. Cole ◽  
M. Lopez ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Cell Lines ◽  
Nuclear Transfer ◽  
Implantation Rate ◽  
Domestic Cat ◽  
Developmental Competence ◽  
In Vitro Production ◽  
Fetal Survival

One of the most remarkable aspects of somatic cell nuclear transfer (NT) is the possibility of avoiding extinction when there are few remaining animals of a specific felid population. Previously, we produced live male African Wildcat (AWC; Felis lybica) cloned kittens using inter-species nuclear transfer (Gomez et al. 2004 Cloning and Stem Cells 6, 217–228). The production of females is a primary objective of most breeding programs. Therefore, the purpose of the present study was to determine (1) if we could produce live female AWC cloned kittens at a proportion similar to that previously demonstrated with males, and (2) if our inter-species NT technique used to produce AWC is applicable to in vitro production of another non-domestic felid species. Specifically, we evaluated the in vivo developmental competence of NT embryos derived by fusion of Black footed cat (BFC, Felis nigripes) and AWC fibroblasts with domestic cat (DSH, Felis catus) cytoplasts, after transfer into domestic cat recipients. Fibroblast cell lines were established from skin biopsies of BFC (6-year-old), and AWC (12-year-old) adult females. After at least three passages, cells were serum-starved for 5 days and injected into the perivitelline space of enucleated domestic cat oocytes. Fusion of cell-cytoplast couplets was induced by applying a 3-s AC pre-pulse of 20 V, 1 MHz, followed by two 30-μs DC pulses of 240 V/mm. Fused couplets were activated 2 to 3 h after fusion by exposure to two 60 μsec DC pulses of 120 V/mm, followed by 4 h incubation with 10 μg/mL cycloheximide and 5 μg/mL cytochalasin B. Reconstructed BFC (n = 16) and AWC (n = 536) NT Day 1 embryos were transferred by laparoscopy into the oviducts of 1 and 12 gonadotrophin-treated DSH recipients, respectively, on Day 1 after induced ovulation. Pregnancy was assessed by ultrasonography on Day 22. One cat (100%) receiving BFC NT embryos and 5 (41.6%) cats receiving AWC NT embryos became pregnant. Twenty-three AWC cloned embryos implanted and 11 kittens were born. Three BFC NT embryos implanted and the pregnancy is currently ongoing. AWC cloned kittens were phenotypically and genetically identical to their somatic cell donor. Their clonal identity was assessed by multiplex PCR amplification of 20 microsatellite markers, including seven markers that are known to be on the X chromosome. In summary, these results indicate that female AWC cloned kittens can be produced and BFC pregnancy can be established in domestic cat recipients. The embryo implantation rate and viability of AWC female cloned embryos was higher than that observed after the transfer of AWC male cloned embryos. The difference may be due to improvements in the NT procedure, rather than to differences in the sex of the cell lines. Table 1. Implantation rate and fetal survival to term of AWC and BFC NT embryos in pregnant domestic cat recipients

X-chromosome reactivation: a concise review

2021 ◽  
Author(s):  
Alessandra Spaziano ◽  
Dr Irene Cantone
Keyword(s):  
X Chromosome ◽  
X Chromosome Inactivation ◽  
Epigenetic Modifications ◽  
X Chromosomes ◽  
Cell Divisions ◽  
Concise Review ◽  
Inactive X Chromosome ◽  
Silencing Pathways

Mammalian females (XX) silence transcription on one of the two X chromosomes to compensate the expression dosage with males (XY). This process — named X-chromosome inactivation — entails a variety of epigenetic modifications that act synergistically to maintain silencing and make it heritable through cell divisions. Genes along the inactive X chromosome are, indeed, refractory to reactivation. Nonetheless, X-chromosome reactivation can occur alongside with epigenome reprogramming or by perturbing multiple silencing pathways. Here we review the events associated with X-chromosome reactivation during in vivo and in vitro reprogramming and highlight recent efforts in inducing Xi reactivation by molecular perturbations. This provides us with a first understanding of the mechanisms underlying X-chromosome reactivation, which could be tackled for therapeutic purposes.

Effects of bovine oviduct epithelial cells, fetal calf serum and bovine serum albumin on gene expression in single bovine embryos produced in the synthetic oviduct fluid culture system

2005 ◽  
Vol 17 (8) ◽  
pp. 751 ◽  
Author(s):  
Mona E. Pedersen ◽  
Øzen Banu Øzdas ◽  
Wenche Farstad ◽  
Aage Tverdal ◽  
Ingrid Olsaker
Keyword(s):  
Gene Expression ◽  
Fetal Calf Serum ◽  
Calf Serum ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Glut 1 ◽  
Significant Difference ◽  
Oviduct Fluid

In this study the synthetic oviduct fluid (SOF) system with bovine oviduct epithelial cell (BOEC) co-culture is compared with an SOF system with common protein supplements. One thousand six hundred bovine embryos were cultured in SOF media supplemented with BOEC, fetal calf serum (FCS) and bovine serum albumin (BSA). Eight different culture groups were assigned according to the different supplementation factors. Developmental competence and the expression levels of five genes, namely glucose transporter-1 (Glut-1), heat shock protein 70 (HSP), connexin43 (Cx43), β-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), analysed as mRNA by using reverse transcription–polymerase chain reaction, were measured on bovine embryos cultured for 9 days. Gene expression of these in vitro-produced embryos was compared with the gene expression of in vivo-produced embryos. There was no significant difference found in embryo developmental competence between the Day 9 embryos in BOEC co-culture, FCS and BSA supplements in SOF media. However, differences in gene expression were observed. With respect to gene expression in in vivo and in vitro embryos, BOEC co-culture affected the same genes as did supplementation with FCS and BSA. HSP was the only gene that differed significantly between in vitro and in vivo embryos. When the different in vitro groups were compared, a significant difference between the BOEC co-culture and the FCS supplementation groups due to Glut-1 expression was observed.

scholarly journals 183PREGNANCY RATE FOLLOWING TRANSFER OF IN VITRO- AND IN VIVO-PRODUCED BOVINE EMBRYOS TO LH-TREATED RECIPIENTS

2004 ◽  
Vol 16 (2) ◽  
pp. 213 ◽  
Author(s):  
J. Small ◽  
M. Colazo ◽  
D. Ambrose ◽  
R. Mapletoft ◽  
J. Reeb ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Animal Health ◽  
Beef Cows ◽  
Water Bath ◽  
Bovine Embryos ◽  
Chi Square ◽  
Embryo Survival ◽  
Frozen Embryos

The objective was to evaluate the effect of pLH treatment on pregnancy rates in recipients receiving in vivo- or in vitro-produced bovine embryos. Heifers (n=37) and lactating (n=28) and non-lactating (n=150) beef cows were treated at random stages of the cycle with 100μg GnRH i.m. (Cystorelin, Merial Canada Inc., Victoriaville, Quebec, Canada) on Day −9, 500μg cloprostenol i.m. (PGF; Estrumate, Schering Plough Animal Health, Pointe-Claire, Quebec, Canada) on Day —2 and GnRH on Day 0 (66h post-PGF; without estrus detection). Cattle were placed at random, by class, into three groups: no further treatment (Control; n=71), or 12.5mg pLH (Lutropin-V, Bioniche Animal Health, Belleville, Ontario, Canada) on Day 5 (n=72) or on Day 7 (n=72) after the second GnRH. On Day 7, cattle with a CL &gt;10mm in diameter (determined ultrasonically) received in vivo-produced, fresh (Simmental) or frozen (Holstein), or in vitro-produced frozen (Holstein) embryos (embryo type balanced among groups). Embryos were cryopreserved in 10% ethylene glycol; in vivo-produced frozen embryos were thawed 5 to 10s in air, 15s in a water-bath at 30°C and then “direct-transferred” nonsurgically. In vitro-produced frozen embryos (donated by IND Lifetech Inc., Delta, British Columbia, Canada) were thawed in a water-bath at 27°C for 10s and placed in ViGro Holding Plus medium (AB Technology, Pullman, WA, USA) at room temperature, evaluated and then transferred nonsurgically. Pregnancy was determined by ultrasonography on Day 35. Data were analyzed with CATMOD, chi-square and GLM procedures (SAS Institute, Cary, NC, USA.). Twenty cattle (9.3%) did not receive embryos; five heifers had cervical problems, and five heifers and 10 cows did not have a CL &gt;10mm. Overall, 7.1% of the recipients had two CL on the day of embryo transfer. There was no effect (P&gt;0.05) of treatment, embryo type (or interaction) or class of recipient on pregnancy rate (overall, 44.1%, 86/195; Table 1). Similarly, mean (±SD) CL diameter and luteal area did not differ (P&gt;0.05) among groups or between pregnant and open recipients (overall, 22.0±3.4mm and 352.0±108.7mm, respectively). However, recipients with a CL diameter ≥18mm tended (P&lt;0.1) to have a higher pregnancy rate (45.8 vs 25.0%). In a subset of 40 recipients examined ultrasonically on Day 12, 50% of those treated on Day 5 and 70% of those treated with pLH on Day 7 had two CL. In summary, overall pregnancy rate in GnRH-synchronized recipients receiving in vitro- or in vivo-produced embryos by nonsurgical transfer was 44.1%. Embryo survival to Day 35 was not affected by type of embryo or treatment with pLH 5 or 7 days after ovulation. Table 1 Pregnancy rate in recipients on Day 35 based on pLH treatment and embryo-type

scholarly journals Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos

2020 ◽  
Author(s):  
Vera A van der Weijden ◽  
Meret Schmidhauser ◽  
Mayuko Kurome ◽  
Johannes Knubben ◽  
Veronika L Flöter ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Early Stage ◽  
Developmental Competence ◽  
Molecular Signaling ◽  
Developmental Potential ◽  
Stage Of Development ◽  
Molecular Signaling Pathways ◽  
Canonical Pathways

Abstract Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation, tRNA charging, and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, signaling of Rho family GTPases, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.

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