scholarly journals Birth of lambs of a pre-determined sex after in vitro production of embryos using frozen–thawed sex-sorted and re-frozen–thawed ram spermatozoa

Reproduction ◽  
2004 ◽  
Vol 127 (5) ◽  
pp. 557-568 ◽  
Author(s):  
F K Hollinshead ◽  
G Evans ◽  
K M Evans ◽  
S L Catt ◽  
W M C Maxwell ◽  
...  
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Embryo Survival ◽  
Treatment Groups ◽  
Sperm Migration ◽  
And Control

The characteristics and functional capacity of ram spermatozoa frozen–thawed prior to and after flow cytometric sorting was assessed after incubation (37 °C; 6 h),in vitrofertilisation (IVF), and transfer of fresh and vitrifiedin vitroproduced embryos. Frozen-thawed spermatozoa from two rams were allocated to four treatment groups: (i) non-sorted (Control); (ii) sorted (FS); (iii) sorted then re-frozen (FSF) and (iv) re-frozen control (FCF). Frozen-thawed samples were separated into X- and Y-chromosome bearing spermatozoa using a high-speed sperm sorter after density gradient centrifugation (X: 88 ± 1.5% and Y: 87 ± 1.1% purity). After 6 h incubation (37 °C), the percentage of motile spermatozoa was higher (P< 0.001) for FS (84 ± 2.0%) compared with all other treatments (Control: 36 ± 3.3%, FSF: 28 ± 3.1%, FCF: 20 ± 2.0%). In a sperm migration test greater numbers of FS spermatozoa penetrated 5 mm into the artificial cervical mucus compared with spermatozoa from all other treatments (152 ± 39.4 vs 31 ± 9.2 spermatozoa respectively;P< 0.05). Fertilisation and cleavage rates were higher (P< 0.05) forin vitromatured oocytes inseminated with Control compared with FSF spermatozoa. However, the Day 7 blastocyst development rate was higher for oocytes inseminated with FSF (62.2%) than FS and Control spermatozoa (52.7 and 50.0%;P< 0.05). The number of ewes pregnant (Day 60), lambing and thein vivoembryo survival rate was greater (P< 0.01) after the transfer of fresh embryos rather than vitrified embryos derived from X- and Y-spermatozoa (67.6, 64.7 and 41.2% vs 29.6, 25.9 and 14.8% respectively). Twenty-six of the 30 (86.7%) lambs derived from sex-sorted spermatozoa were of the correct sex. These results demonstrate that frozen–thawed ram spermatozoa can be sex-sorted for immediate or future use after re-cryopreservation and, in conjunction with IVF and embryo transfer, can be used to efficiently produce offspring of pre-determined sex.

193 IMPROVED POST-THAW SURVIVAL OF BOVINE EMBRYOS PRODUCED IN SERUM-FREE IN VITRO PRODUCTION SYSTEM

2016 ◽  
Vol 28 (2) ◽  
pp. 227
Author(s):  
M. Nõmm ◽  
E. Mark ◽  
O. Sarv ◽  
S. Kõks ◽  
Ü. Jaakma
Keyword(s):  
Survival Rates ◽  
Slow Freezing ◽  
In Vitro Fertilisation ◽  
Ministry Of Education ◽  
In Vitro Production ◽  
Freezing And Thawing ◽  
Embryo Survival ◽  
Serum Free

Over a few decades the bovine in vitro embryo production (IVP) systems have been improving rapidly. Still, the goal to produce the same quality embryos in vitro as in vivo has not yet been reached. The FCS is usually added to media during IVP to provide growth factors and energy sources. Currently, serum-free culture systems are often preferred due to the lower risk of contamination and prevention of the development of large offspring syndrome. The aim of this study was to establish whether complete elimination of FCS from the bovine IVP system has an effect on blastocyst rates, embryo quality, and embryo survival rates after slow freezing. We replaced our conventional in vitro maturation (IVM) medium [tissue culture medium-199, 10% (v/v) FCS, 10 µg mL–1 epidermal growth factor (EGF), 1500 U mL–1 serum gonadotropin and chorionic gonadotropin (PG600), Na-pyruvate 0.5 mM, gentamycin sulfate 50 µg mL–1 and l-glutamine 1 mM] with SOF (SOFaaci) supplemented with 0.4% fatty acid-free BSA fraction V, 10 µg mL–1 EGF, and 1500 U mL–1 PG600. Matured cumulus-oocyte complexes (COC) from both experimental groups (total of 1145 from serum-free IVP and 687 from our conventional IVP system) were used for in vitro fertilisation and culture. Blastocyst rates were similar in the serum-free and our usual IVP protocol, 18 and 22%, respectively. Seventy-seven Grade 1 (according to IETS) Day 7 blastocysts from the serum-free IVP system and 80 Grade 1 Day 7 blastocysts from our conventional IVP system were frozen in 1.5 M ethylene glycol and 0.1 M sucrose containing cryopreservation medium. The post-thaw survival rates after 24 h of culture and evaluated as percentages of re-expanded embryos were 63.6% for the serum-free IVP and 46.3% for the conventional IVP system (P < 0.05, Z Test for 2 population proportions). These results indicate that it is possible to have a completely serum-free bovine IVP system and based on the slow freezing and thawing results the quality of serum-free IVP embryos might be better than of the embryos matured in our conventional maturation media. However, more experiments and increased sample sizes are needed to confirm the results. This study was supported by Project 3.2.0701.12–0036 of Archimedes Foundation, AP 2.4 of CCRMB, and institutional research funding (IUT 08–01) of the Estonian Ministry of Education and Research.

scholarly journals Bugs and drugs: a systems biology approach to characterising the effect of moxidectin on the horse’s faecal microbiome

2020 ◽  
Vol 2 (1) ◽  
Author(s):  
S. P. Daniels ◽  
J. Leng ◽  
J. R. Swann ◽  
C. J. Proudman
Keyword(s):  
Gut Microbiota ◽  
Treated Group ◽  
Anthelmintic Treatment ◽  
Metabolic Profiles ◽  
Faecal Microbiota ◽  
Treatment Groups ◽  
Fermentation Kinetics ◽  
And Control

Abstract Background Anthelmintic treatment is a risk factor for intestinal disease in the horse, known as colic. However the mechanisms involved in the onset of disease post anthelmintic treatment are unknown. The interaction between anthelmintic drugs and the gut microbiota may be associated with this observed increase in risk of colic. Little is known about the interaction between gut microbiota and anthelmintics and how treatment may alter microbiome function. The objectives of this study were: To characterise (1) faecal microbiota, (2) feed fermentation kinetics in vitro and (3) metabolic profiles following moxidectin administration to horses with very low (0 epg) adult strongyle burdens. Hypothesis: Moxidectin will not alter (1) faecal microbiota, (2) feed fermentation in vitro, or, (3) host metabolome. Results Moxidectin increased the relative abundance of Deferribacter spp. and Spirochaetes spp. observed after 160 h in moxidectin treated horses. Reduced in vitro fibre fermentation was observed 16 h following moxidectin administration in vivo (P = 0.001), along with lower pH in the in vitro fermentations from the moxidectin treated group. Metabolic profiles from urine samples did not differ between the treatment groups. However metabolic profiles from in vitro fermentations differed between moxidectin and control groups 16 h after treatment (R2 = 0.69, Q2Y = 0.48), and within the moxidectin group between 16 h and 160 h post moxidectin treatment (R2 = 0.79, Q2Y = 0.77). Metabolic profiles from in vitro fermentations and fermentation kinetics both indicated altered carbohydrate metabolism following in vivo treatment with moxidectin. Conclusions These data suggest that in horses with low parasite burdens moxidectin had a small but measurable effect on both the community structure and the function of the gut microbiome.

scholarly journals 150 ENVIRONMENT OF THE EARLY EMBRYO AND ITS EFFECT ON DEVELOPMENT AND POSTNATAL LIFE

2005 ◽  
Vol 17 (2) ◽  
pp. 225
Author(s):  
A. Watkins ◽  
A. Wilkins ◽  
T. Papenbrock ◽  
C. Osmond ◽  
M. Hanson ◽  
...  
Keyword(s):  
Blood Pressure ◽  
Systolic Blood Pressure ◽  
Embryo Transfer ◽  
Litter Size ◽  
Early Embryo ◽  
Blastocyst Development ◽  
Treatment Groups ◽  
Elevated Systolic Blood Pressure

We have investigated the impact of mouse early embryo in vitro culture environment on (a) short-term blastocyst development and (b) long-term postnatal growth and physiology after embryo transfer. In vitro-developed blastocysts, cultured from the 2-cell stage, had reduced inner cell mass (ICM) and trophectoderm (TE) cell numbers when compared to in vivo-derived blastocysts at 96 h post-hCG (n = 13–39, P < 0.05). Despite the retardation in blastocyst development, the ICM:TE ratio was equivalent in both treatment groups. Using embryo transfer techniques, we compared the postnatal development of embryos cultured in vitro from the 2-cell to the blastocyst stage (termed “in vitro” mice) with offspring generated from blastocysts developed in vivo, but which also underwent embryo transfer (termed “in vivo” mice). These two treatment groups were in turn compared with mice derived from naturally mated mothers, which had their mean litter size at birth adjusted to a size comparable with that of the in vitro and in vivo mice (a mean of 6 animals) and which had not been transferred. All data were analyzed using a multilevel random effects regression model which took into account between-mother and within-mother variation in litter size for parameters measured from individual animals. No significant differences in birth weight were observed between in vitro and in vivo offspring. However, in vitro offspring were significantly lighter than in vivo offspring in a gender-dependent manner at 2 weeks of age (males, P = 0.009) and at 6 and 11 weeks of age (females, P = 0.037 and 0.035, respectively). In addition, at 4 weeks of age, the in vivo males became significantly lighter when compared to the naturally mated males (P = 0.034). At 8 weeks of age, the in vivo females had a significantly elevated systolic blood pressure when compared to the in vitro females (P = 0.003); however, at 21 weeks of age, both in vitro males and females had a significantly elevated blood pressure when compared to in vivo offspring (P < 0.003). At 8, 15, and 21 weeks of age, offspring derived from transferred embryos developed with significantly elevated systolic blood pressure when compared to non-embryo transfer offspring (P < 0.05). No significant differences in serum angiotensin-converting enzyme activity (a potent regulator of systolic blood pressure) was observed between the treatment groups. Significantly altered liver:body weight ratios were observed between the in vitro and in vivo males, and between the in vitro and the naturally mated (6) females (P < 0.038). All of the above data are independent of litter size. These data support the hypothesis that early embryo environment can influence postnatal growth and cardiovascular physiology. This research was funded by an MRC research grant to TPF, and by a DOHaD studentship.

Effects of different sources and levels of tannins on live performance and antioxidant response of Ossimi lambs

2020 ◽  
Vol 158 (4) ◽  
pp. 339-348
Author(s):  
Tamer M. M. Hassan ◽  
Omar A. Ahmed-Farid ◽  
Fathy A. I. Abdel-Fattah
Keyword(s):  
Control Diet ◽  
Protein Digestibility ◽  
Antioxidant Response ◽  
Live Performance ◽  
Treatment Groups ◽  
Stress Markers ◽  
Mango Leaves ◽  
And Control

AbstractPomegranate peels (PP) and mango leaves (ML) were analysed for nutrients and tannin contents. In an in vitro test, ten diets were prepared; six contained 2, 4 and 6% of PP or ML, three diets supplemented with mixed levels of PP and ML (1 + 1%, 2 + 2% and 3 + 3%) and control diet free of them. Gas was measured after 3, 6, 12, 24, 48 and 72 h of incubation. Methane and rumen parameters were estimated. In an in vivo experiment, 40 Ossimi lambs were divided into four groups; the first was control, other groups (T1, T2 and T3) fed diets containing 6% PP, 6% ML or mix levels (3% PP + 3%ML), respectively, for 2 months. Results showed that PP and ML were rich in tannins. In the in vitro test, a maximum reduction in gas, methane and NH3-N was in 6% PP, 6% ML and mixed levels (3% PP + 3% ML). In the in vivo experiment, there were no differences in growth and digestibility of DM and nutrients between treatment groups. Only a lowered DM intake and protein digestibility in lambs fed 6% PP. Gas and methane emission was decreased significantly in lambs fed 6% PP, compared to other groups. TVFAs and NH3-N were decreased for treatment groups. Also, all treatments did not show any pathological changes in liver function or on oxidative stress markers. In conclusion, PP and ML can be used in sheep diets at inclusion levels of 6% and mixture without detrimental effects on general health of Ossimi lambs.

scholarly journals 233EXPRESSION OF INSULIN-LIKE GROWTH FACTOR (IGF) MRNA IN BOVINE CONCEPTUSES FROM EMBRYOS PRODUCED IN VIVO OR IN VITRO

2004 ◽  
Vol 16 (2) ◽  
pp. 237
Author(s):  
C.E. Farin ◽  
J.E. Alexander ◽  
K.F. Rodriguez ◽  
P.W. Farin
Keyword(s):  
Mrna Expression ◽  
Agricultural Research ◽  
Pcr Analysis ◽  
Blastocyst Development ◽  
Globin Mrna ◽  
Treatment Groups ◽  
The North ◽  
Gapdh Mrna

The objective of this study was to determine the effect of in vitro embryo production (IVP) on expression of mRNAs for IGF-1, IGF-2, IGF-1 receptor (IGF-1R), IGF-2R and GAPDH in bovine conceptuses at Day 17 of gestation. In vivo embryos (In vivo) were recovered from superovulated Holstein cows. For IVP, Holstein oocytes were matured, fertilized and then cultured in M199 with 10% serum (IVPS) or 1% BSA (IVPSR) to 72hpi. All embryos were then transferred to M199 with 10% serum and cultured to 168hpi. The same Holstein sire was used to produce all embryos. Single grade 1 blastocysts were transferred into recipient heifers. On day 10 after transfer, conceptuses were recovered, measured and stored at −80°C prior to extraction of whole cell (wc) RNA and genomic (g) DNA. wcRNA was assessed for quality based on A260/A280 ratio and visualization of 18S and 28S ribosomal RNA. Only complete (intact) conceptuses with at least 10μg high-quality wcRNA recovered after extraction were included in the analysis (In vivo, n=8; IVPS n=8; IVPSR n=7). Conceptus sex was determined by PCR analysis of gDNA. Semi-quantitative PCR assays were used to assess relative expression of all mRNAs. For each conceptus, 2μg wcRNA were mixed with 3pg rabbit globin mRNA, DNase-treated and reverse-transcribed using random hexamers. Conceptus cDNA samples (140ng each for IGF-1, IGF-2 mRNAs;; 100ng each for IGF-1R, IGF-2R, GAPDH and globin mRNAs) were assayed in duplicate within single assays. Relative mRNA expression was calculated as the ratio of band intensities representing the mRNA of interest to globin mRNA. All data were analyzed for effects of treatment (In vivo, IVPS, IVPSR or In vivo, IVP), sex, stage of blastocyst development at transfer (early, mid, expanded) and the interactions of treatment sex and treatment stage. Conceptus length (LSM±SEMmm) did not differ with treatment (In vivo: 182±55; IVPS: 257±47; IVPSR: 310±72). Expression of GAPDH mRNA was greater (P=0.02) in female conceptuses (1.31±0.11) than in males (0.96±0.07) and did not differ (P=0.14) between treatment groups (In vivo: 1.30±0.11, IVPS: 1.00±0.09, IVPSR: 1.10±0.14). However, when the IVP groups were combined and compared to In vivo controls, expression of GAPDH mRNA was both greater (P=0.002) in females (1.38±0.10) than in males (0.94±0.06) and differed (P=0.03) with treatment (In vivo: 1.30±0.10, IVP: 1.01±0.07). Expression of IGF-1 and IGF-2 mRNA was not detected in the majority of conceptuses. Expression of IGF-1R mRNA was greater (P=0.02) in female (1.19±0.10) than in male conceptuses (0.87±0.07) and differed (P=0.02) with stage of blastocyst development at transfer (early: 0.79±0.07; mid: 1.23±0.13; expanded: 1.07±0.11). There was no effect of treatment on expression of IGF-1R mRNA. Expression of IGF-2R mRNA differed (P=0.03) with treatment (In vivo: 0.85±0.10; IVPS: 0.76±0.09; IVPSR: 0.33±0.13). In conclusion, expression of both imprinted and non-imprinted genes is altered in conceptuses resulting from embryos produced in vitro. Supported by the North Carolina Agricultural Research Service.

scholarly journals 332IN VIVO DEVELOPMENTAL CAPACITY OF IN VITRO-PRODUCED EMBRYOS DERIVED FROM SEX-SORTED AND RE-CRYOPRESERVED FROZEN-THAWED RAM SPERM

2004 ◽  
Vol 16 (2) ◽  
pp. 286 ◽  
Author(s):  
J.K. O'Brien ◽  
F.K. Hollinshead ◽  
G. Evans ◽  
W.M.C. Maxwell
Keyword(s):  
Potential Application ◽  
High Speed ◽  
Fisher Exact Test ◽  
Embryo Survival ◽  
Food Dye ◽  
Frozen Semen ◽  
Developmental Capacity ◽  
Ram Sperm

The ability to sort and re-freeze frozen-thawed sperm would significantly increase the potential application of sperm sexing technology to species management. Frozen-thawed, sorted, re-frozen and then thawed ram sperm appear fully functional in vitro with blastocyst production greater than that for frozen-thawed, non-sorted sperm (Hollinshead FK et al. 2003 Theriogenology 59, 209 abst). The aim of this study was to evaluate the in vivo capacity of in vitro-produced embryos derived from frozen-thawed sperm after sorting and a second cryopreservation/thawing step. Frozen semen from 2 rams (n=3 ejaculates per ram) was used throughout. Post-thaw sperm treatments comprised (i) non-sorted (Control); (ii) sorted (Froz-Sort) and (iii) sorted, then re-frozen (Froz-Sort-Froz). X and Y sperm were separated using a high-speed sorter (SX MoFlo®, DakoCytomation, Fort Collins, CO, USA) after incubation with Hoechst 33342 and food dye to eliminate nonviable sperm. Reanalysis revealed high levels (mean±SEM) of purity for X- and Y-enriched samples for all treatments (89±1.2%). At Day 6 post-insemination, 2 embryos (blastocyst stage or greater) were transferred per recipient. Data were analyzed by chi-square and Fisher Exact Test. In vivo embryo survival was similar across sperm treatments (28/64, 43.8% overall) and 20 of 23 (87.0%) sexed lambs were of the predicted sex (Table 1). These results demonstrate high in vivo developmental capacity of in vitro-produced sexed embryos derived from frozen-thawed ram sperm after sorting and a second cryopreservation/thawing step, and increase the potential application of sperm sexing technology. Research support by XY, Inc., Australian Research Council and Zoological Parks Board of NSW. Table 1 In vivo survival of transferred in vitro produced embryos derived from frozen-thawed non-sorted (Control), frozen-thawed and sorted (Froz-Sort) and frozen-thawed, sorted, then frozen-thawed (Froz-Sort-Froz) ram sperm.

scholarly journals In vitro production of bovine embryos using flow-cytometrically sexed sperm

2006 ◽  
Vol 49 (2) ◽  
pp. 133-140 ◽  
Author(s):  
L. Kątska-Książkiewicz ◽  
B. Ryńska ◽  
M. Bochenek ◽  
J. Opiela ◽  
J. Jurkiewicz
Keyword(s):  
Bovine Embryos ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Bovine Spermatozoa ◽  
Embryo Cleavage ◽  
And Control ◽  
Vitro Production ◽  
Sperm Freezing

Abstract. The investigation aimed to compare the effect of fresh and frozen-thawed X and Y fractions of flow-cytometrically sorted bovine spermatozoa on in vitro fertilization of bovine in vitro matured oocytes and subsequent blastocyst development. Sperm cells sorted in MoFloSX cytometer were used either for IVF or frozen and stored in liquid nitrogen. Immature oocytes recovered from ovaries of slaughtered animals and matured in vitro in TCM-199 containing 20% estrus cow serum and additional granulosa cells were fertilized in vitro with fresh or frozen-thawed fractions of sorted sperm. Simultaneously, control, fresh or frozen/thawed sperm was used for IVF. A total number of 2712 IVM oocytes were fertilized with sorted and control sperm of 6 bulls. Embryo cleavage rates were significantly affected by bull (P<0.0001), sperm sexing (P<0.0001) and sperm freezing (P<0.01). Blastocysts development was affected by sperm freezing (P<0.04) and sperm sexing (P<0.01). The significant differences were shown between unsorted and sorted sperm, however no differences in embryo cleavage rates and blastocysts rates were observed between X- and Y-sperm fractions, both fresh and frozen/ thawed. There were significant differences in cleavage rates among fresh, control sperm (52.7%), X fraction (26.8%) and Y fraction (24.7%). Similar differences in cleavage rates were shown for frozen/thawed control sperm (52.8%), X fraction (33.9%) and Y fraction (26.2%). The female blastocysts were frozen for further transfer, while the hatched male blastocysts were analysed by PCR revealing 76.2% accuracy. The results suggest that there were significant differences in cleavage rates and blastocyst rates due to sperm sorting in comparison to unsorted sperm and no differences between effectiveness of X and Y fractions of spermatozoa.

226 CRYOPRESERVED EJACULATED AND EPIDIDYMAL SPERM COLLECTED FROM THE SAME HOLSTEIN BULLS USED FOR IN VITRO FERTILIZATION

2013 ◽  
Vol 25 (1) ◽  
pp. 261 ◽  
Author(s):  
M. A. Stout ◽  
J. R. Saenz ◽  
J. F. Chenevert ◽  
G. T. Gentry ◽  
K. B. Bondioli ◽  
...  
Keyword(s):  
Embryo Development ◽  
Epididymal Sperm ◽  
In Vitro Production ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Mean Values ◽  
Treatment Groups ◽  
Reproductive Techniques ◽  
Holstein Bulls

Exposure to seminal plasma may modify the ability of sperm to survive cryopreservation, undergo capacitation, and fertilize oocytes. The present work was designed to compare embryo development after IVF of oocytes with ejaculated and epididymal bovine sperm from bulls previously tested and showing similar responses to freezing. However, we also found that this ejaculated and epididymal sperm differed in their in vitro culture dynamics (capacitation, viability, and auto-acrosome reaction) and ability to fertilize oocytes in vitro. Ejaculated and epididymal sperm were collected from the same fertile mature Holstein bulls (n = 4) by artificial vagina and post-castration retrograde caudal epididymal flush, respectively. Collection of epididymal sperm was conducted 2 weeks after the last collection of ejaculated sperm. After collection, ejaculated and epididymal sperm were cryopreserved and stored in LN until use. Before IVF, a viable sperm population was isolated by centrifugation through a Percoll density gradient. Ejaculated and epididymal sperm were then added to fertilization drops at a final concentration of 1 × 106 mL–1, and IVF was conducted with and without the capacitation agent heparin. Oocytes were washed and randomly assigned to one of four treatment groups (ejaculated ± heparin or epididymal ± heparin). Embryo development was determined at 72 and 186 h after IVF. Differences in the mean values among treatment groups were analysed by one-way ANOVA, followed by the Holm-Sidak pairwise multiple comparisons. Embryo cleavage after IVF using ejaculated sperm without heparin (45.2%) was significantly lower (P < 0.05) than in all other groups. Cleavage rates of ejaculated sperm with heparin (56.9%) and epididymal sperm with (58.1%) and without (57.5%) heparin were found to be similar. No difference was noted between ejaculated and epididymal sperm in blastocyst development, although the inclusion of heparin did significantly (P < 0.01) increase blastocyst development in both ejaculated (9.3 compared with 23.6%) and epididymal sperm (2.5 compared with 23.3%). In conclusion, cryopreserved ejaculated and epididymal sperm collected from the same bulls can be successfully used for the in vitro production of bovine blastocysts without changing the existing protocols. This may increase the efficiency when using epididymal sperm in assisted reproductive techniques.

49 SUCCESSFUL CRYOPRESERVATION USING LOW ETHYLENE GLYCOL CONCENTRATION FOR IN VITRO-PRODUCED BOVINE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 132
Author(s):  
M. Takayama ◽  
S. Sato ◽  
Y. Nishimura ◽  
K. Imai ◽  
O. Dochi
Keyword(s):  
Ethylene Glycol ◽  
Calf Serum ◽  
Bovine Embryos ◽  
Embryo Survival ◽  
Lower Survival Rate ◽  
Treatment Groups ◽  
Chi Squared ◽  
Hatching Rates

In vitro-produced (IVP) bovine embryos tend to have a lower survival rate after cryopreservation than in vivo embryos do. Therefore, the freezing medium (FM) and concentration of cryoprotectant are very important factors. This study was to investigate the effect of 1.2 M ethylene glycol (EG) with 0.1 M sucrose (SUC) on survival of IVP embryos after freezing. The COC were matured in 25 mM HEPES-buffered TCM199 (TCM199) supplemented with 5% calf serum (CS) and 0.02 AU mL−1 FSH. Oocytes (20 to 25) were cultured in 100-μL droplets of maturation medium for 20 h. After 6 h of gamete co-culture (5 × 106 sperm/mL), the presumptive zygotes were cultured in CR1aa medium supplemented with 5% CS for 9 days (fertilization = Day 0). Only the expanded blastocysts from Days 7 to 9 were used in this experiment and separated into 3 treatment groups. The first and second groups were frozen in Dulbecco’s phosphate-buffered saline (D-PBS) supplemented with 20% CS, 0.1 M SUC, and 1.2 or 1.5 M EG (groups 1.2 or 1.5 M EG), respectively. The third group was D-PBS supplemented with 20% fetal calf serum (FCS), 0.25 M SUC, and 1.4 M glycerol (group GLY). In each group, embryos were equilibrated with their FM for 10 min and loaded into 0.25-mL straws individually. These straws were placed into the cooling chamber of a programmable freezer precooled to −7°C. After 2 min, the straws were seeded and then held for a further 13 min at −7°C. Then, the straws were cooled to −30°C at −0.3°C/min before being plunged into liquid nitrogen. The cryopreserved embryos were thawed by allowing the straws to stand in air for 7 s and then warming them in a 30°C water bath for 20 s. The thawed embryos were washed twice using 38°C D-PBS supplemented with 20% FCS. Subsequently, they were immersed in the same medium, held at 38°C for 10 min, and then each embryo was cultured in 20-μL droplets of TCM199 supplemented with 20% FCS and 0.1 mM β-mercaptoethanol for 72 h. The rates of embryos developing to the re-expanded and hatching blastocyst stages were determined 72 h after thawing. All data were analysed by the chi-squared test with Yates’ correction. The re-expanded and hatching rates of frozen-thawed embryos after 72 h in culture were not significantly different between 1.2 M EG (n = 39: 71.8% and 69.2%), 1.5 M EG (n = 38: 76.3% and 63.2%), and 1.4 M GLY (n = 37: 75.7% and 64.9%) groups (P > 0.05). Survival and hatching rates according to embryo quality were also not significantly different between 1.2 M EG (good n = 18: 88.9% and 88.9%; fair n = 21: 57.1% and 52.4%), 1.5 M EG (good n = 19: 89.5% and 84.2%; fair n = 19: 63.2% and 42.1%), and 1.4 M GLY (good n = 18: 77.8% and 66.7%; fair n = 19: 73.7% and 63.2%) (P > 0.05). In conclusion, cryoprotectant type and concentration did not affect embryo survival or development after cryopreservation in this study. Therefore, the ethylene glycol concentration used for the cryopreservation of IVP embryos can be reduced.

Effect of embryo source and recipient progesterone environment on embryo development in cattle

2007 ◽  
Vol 19 (7) ◽  
pp. 861 ◽  
Author(s):  
P. Lonergan ◽  
A. Woods ◽  
T. Fair ◽  
F. Carter ◽  
D. Rizos ◽  
...  
Keyword(s):  
Artificial Insemination ◽  
Recovery Rate ◽  
In Vitro Maturation ◽  
Strong Support ◽  
Embryo Survival ◽  
Embryo Recovery ◽  
The Mean ◽  
And Control

The aim of the present study was to examine the effect of embryo source (in vivo v. in vitro) and the progesterone environment into which it was transferred on Day 7 on embryo survival and size on Day 13. Day 7 blastocysts were produced either in vivo using superovulation, artificial insemination and non-surgical embryo recovery or in vitro using in vitro maturation, fertilisation and culture. In order to produce animals with divergent progesterone concentrations, following synchronisation recipients were either superovulated (High progesterone; n = 10) or not (Control progesterone; n = 10). Ten blastocysts, produced either in vivo or in vitro, were transferred to each recipient on Day 7. Both groups were killed on Day 13. The mean progesterone concentration from Day 7 to Day 13 (the period when the embryos were in the uterus) in the High and Control progesterone recipients was 36.32 ± 1.28 and 10.30 ± 0.51 ng mL–1, respectively. Of the in vivo embryos transferred, the overall recovery rate at Day 13 was 64%, which was higher (P < 0.001) than that of 20% for the in vitro embryos transferred. The mean area of embryos recovered from High progesterone recipients was 3.86 ± 0.45 mm2 (n = 28) compared with 1.66 ± 0.38 mm2 (n = 24) for embryos recovered from Control progesterone recipients (P < 0.001). Similarly, the origin of the embryo used for transfer affected embryo size on Day 13. In summary, the recovery rate of blastocysts was higher for in vivo- than in vitro-derived embryos. Blastocyst size was approximately 2.3-fold greater in recipients with high compared with normal progesterone. The present study lends strong support to the hypothesis that an earlier rise in progesterone after conception stimulates blastocyst growth and the development of competent embryos.

Sign in / Sign up

Export Citation Format

Share Document