71 The Effect of Two Different In Vitro Culture Media and Mice Embryo Groupings on Hatchability After 24 Hours of Culture

2018 ◽  
Vol 30 (1) ◽  
pp. 174
Author(s):  
N. C. Negota ◽  
M. L. Mphaphathi ◽  
L. P. Nethenzheni ◽  
T. L. Rammutla ◽  
N. R. Serota ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Hatching Rate ◽  
Significant Difference ◽  
Autocrine Signalling ◽  
Blastocyst Hatching

Mammalian blastocysts must hatch out from the zona pellucida before implantation. In vitro embryo culture and grouping of mice blastocysts are conducive options of assisted reproductive technologies (ART) to speed up the hatching rate of mice embryos. The number of embryos per unit volume has the greatest impact on hatching rates due to autocrine signalling. The study aimed to determine the effect of two in vitro culture (IVC) media (TCM-199 and Ham’s F10) and embryo groupings (1, 2, 3, and 4 embryos per 50-µL droplet) after 24 h of culture on hatching rate. Breeds of C57BL/6 (n = 10) and BALB/c (n = 10) were raised until they reached maturity and bred naturally to produce the first filial generation. The photoperiod was 14 h of light followed by 10 h of darkness in the breeding house, and feed and water were provided ad libitum. Female mice were superovulated using eCG and hCG. The first filial generations from 2 breeds were used for the collection of 160 blastocysts and randomly allocated into 2 IVC media (80 embryos for TCM-199 and 80 embryos for Ham’s F10) and again subjected to 4 embryo groupings (1, 2, 3, and 4 embryos per droplet) treatments. Four replicates were done per treatment group. The general linear model of Minitab version 17 (Minitab Inc., State College, PA, USA) was used to analyse the data. The hatching rate of blastocyst stage was significantly higher for TCM-199 (56.9 ± 27.2) compared with Ham’s F10 (50.0 ± 35.1%). The comparison of all embryo groupings, 1 (20.0 ± 40.5), 2 (28.8 ± 29.7), 3 (59.1 ± 38.8), and 4 (43.8 ± 32.4%) per 50-µL droplet showed significant differences, irrespective of IVC medium and breed. In TCM-199, groupings of 1 (20.0 ± 41.0), 2 (30.0 ± 29.9), 3 (63.3 ± 40.3), and 4 (42.5 ± 33.5%) had a significant difference on blastocyst hatching percent. In Ham’s F10, groupings of 1 (20.0 ± 41.0), 2 (27.5 ± 30.2), 3 (55.0 ± 37.9), and 4 (45.0 ± 32.0%) were significantly different on blastocyst hatching rate. However, an increase in hatching rate was observed for the interaction of media and embryo groupings and especially when embryos were increased per droplet in all breeds. In conclusion, the use of TCM-199 and grouping of 3 embryos per 50-µL droplet during culture had the highest hatching rate compared with the use of Ham’s F10.

scholarly journals Comparative Study of Different Assisted Hatching Techniques and In Vitro Culture Media on Mice Embryo Hatching Rate

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Negota NC ◽  
◽  
Mphaphathi ML ◽  
Nethenzheni LP ◽  
Rammutla TL ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Abdominal Cavity ◽  
Blastocyst Stage ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Female Mice ◽  
Significant Difference ◽  
F1 Generation

The study investigated the influence of Assisted Hatching (AH) techniques (mechanical, chemical, enzymatic thinning and laser) and two in vitro culture media (Ham’s F10 and TCM-199) on hatching rate of mice embryos (blastocyststage) following 24 hours of culture. The C57BL/6-black (n=30) and BALB/cwhite (n=30) mouse breeds were raised until maturity and naturally bred to produce a F1 generation. The light in the breeding house was controlled and the mice were fed ad libitum. Female mice (n=30) were injected (peritoneal) with 0.1 ml (5 IU) of eCG into the abdominal cavity with 1 ml syringe and 0.5x16 mm needle to stimulate follicular growth and 46-49 h later was injected with 0.1 ml (5 IU) of hCG to cause ovulation, maintain the corpus luteum and stimulate it to secrete progesterone for maintenance of pregnancy. After the injection, the male and female (1:1) could mate overnight. Female mice with vaginal plugs were observed and kept separately for blastocyst-stage embryos collection on day three following successful mating. They were euthanized, and abdomen sterilized with 70% ethanol. Using a sterile surgical scissor, a fine cut was made, holding the skin firmly above and below the incision, the skin was pulled apart using forceps. The embryos were flushed from the uterus using a 30-gauge needle with culture media. Following the AH techniques, embryos were cultured in TCM-199 or Ham’s F10 for 24 hours and zonal thickness of all hatched embryos were measured. Immediately after assisted hatching, the embryos were cultured into two different in vitro culture media. All embryos hatched were stained and the zonal thickness of embryos were measured. The number of blastomeres were counted and recorded 24 h later. Data collected were subjected to analysis of variance using PROC General Linear Model. The Tukey’s test was used to separate the means. A significant difference was observed between the thickness of Zona Pellucida (ZP) pre and post treatment after 24 hours of culture. However, there was no significance difference among blastocyst hatching rate and the blastomeres nuclei counted after staining. The thickness of the ZP decreased with individual AH techniques. The interaction between AH techniques and in vitro culture was found to be significantly different on blastocyst hatchability. However, laser AH technique had highest hatchability (56.3%) when embryos were cultured in TCM-199 followed by mechanical AH techniques (52.6%). The hatchability rate (33.3%) was recorded in the chemical AH technique group. The blastomeres nuclei counted under interaction of AH techniques and culture media was not differently affected, with the values ranging from 69 to 76%. In conclusion, the use of different AH techniques resulted in varying effect and increase outcomes towards the hatching rate.

160 COMPARATIVE STUDY OF IN VITRO CULTURE MEDIA AND ASSISTED HATCHING TECHNIQUES ON MOUSE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 188
Author(s):  
N. C. Negota ◽  
L. P. Nethenzheni ◽  
M. L. Mphaphathi ◽  
D. M. Barry ◽  
T. L. Nedambale
Keyword(s):  
In Vitro Culture ◽  
Chorionic Gonadotropin ◽  
Culture Medium ◽  
Culture Media ◽  
Assisted Hatching ◽  
Hatching Rate ◽  
Significant Difference ◽  
Equine Chorionic Gonadotropin ◽  
F1 Generation

The in vitro culture media and assisted hatching techniques remain challenging obstacles to be utilised widely. Mechanical, chemical, enzymatic thinning, and laser-assisted techniques have been used previously but information is still lacking on its application in livestock. The aim of this study was to compare the effect of 2 in vitro culture media (Hamster F10 and TMC-199) and 4 (mechanical, chemical, enzymatic, and laser) assisted hatching techniques on blastocyst formation and hatching rate using murine embryos as a model. The C57/b and Balb/c breeds were raised until they reached maturity and bred naturally to produce F1 generation. The light in the breeding house was controlled at 14 h light and 10 h dark. Feed and water were provided ad libitum for the mice. Superovulation of females were stimulated using equine chorionic gonadotropin and human chorionic gonadotropin. The F1 generation was used for the collection of the 400 blastocysts and randomly allocated into 4 assisted hatching techniques. Blastocysts were paired into a group of 10 and replicated 4 times for each assisted hatching technique. The general linear model of SAS version 9.4 (SAS Institute Inc., Cary, NC, USA) was used to analyse the data. Assisted hatching techniques of laser, mechanical, enzymatic, and chemical yielded 46.9 ± 37.1, 51.1 ± 40.2, 39.1 ± 35.8, and 33.3 ± 4.5%, respectively, under in vitro culture of Hamster F10. The TCM-199, laser, mechanical, enzymatic, and chemical assisted hatching techniques yielded 56.3 ± 43.3, 52.6 ± 35.5, 49.2 ± 37.5, and 33.9 ± 35.5%, respectively, with a significant difference. There was no significant difference observed in assisted hatching techniques and Hamster F10 culture medium. However, the hatching rate of embryos for all techniques was higher when in vitro cultured in TCM than cultured in Hamster F10. Hatching rate of blastocysts increased from chemical, enzymatic, mechanical, and laser with response to Hamster F10 and TCM; thus, laser is a suitable assisted hatching technique with TCM-199.

131 Effect of Oviductal Fluid During In Vitro Culture on Bovine Embryo Development and Quality

2018 ◽  
Vol 30 (1) ◽  
pp. 205 ◽  
Author(s):  
R. Emmerstorfer ◽  
K. Radefeld ◽  
V. Havlicek ◽  
U. Besenfelder ◽  
H. Yu ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Specific Effect ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Significant Difference

The aim of this work was to establish an in vitro culture approach using bovine oviducal fluid (OF) to improve embryo quality and to provide an in vitro system to study oviduct function. Bovine oviducts ipsilateral to ovulation were collected at the slaughterhouse, 1 to 4 days after ovulation. The OF was collected by flushing the oviducts with 1 mL of Charles Rosenkrans 1 medium (CR1). Samples from 21 oviducts were pooled and proteins were concentrated using centrifugal filter devices. Aliquots of 3 different protein concentrations, determined by Bradford assay, were prepared and stored at –20°C. Abattoir-retrieved cumulus–oocyte complexes were used for standard in vitro maturation (IVM) and IVF (Day 0). On Day 1, presumptive zygotes (n = 1498) were randomly allocated to 4 different culture groups and cultured up to Day 9. The presumptive zygotes of the control group (n = 364) were cultured in CR1 with 5% oestrous cow serum (OCS) supplemented with 1 mg mL−1 hyaluronan. In the experimental groups, OCS was replaced by OF, resulting in 3 groups with final protein concentrations of 0.1 mg mL−1 (n = 380), 0.5 mg mL−1 (n = 380) or 1 mg mL−1 (n = 374). Cleavage rate was recorded on Day 2 and blastocyst yield on Days 7, 8, and 9 after fertilization. On Day 7, blastocysts were removed and either stained (Hoechst 33342) for cell number or subjected to a slow freezing protocol using 1.5 M ethylene glycol. After thawing, the re-expansion and hatching rate of blastocysts were determined at 24, 48 and 72 h. Eight replicates were carried out and data were analysed by ANOVA. Cleavage rate increased with increasing protein concentration (0.1 mg mL−1: 80.9 ± 4.2%; P > 0.05; 0.5 mg mL−1: 83.4 ± 2.5%; P < 0.1) and was significantly higher in the 1 mg mL−1 group (84.5 ± 4.4%; P < 0.05) compared with the control group (79.7 ± 3.4%). The cumulative blastocyst rate on Day 9 was significantly lower (P < 0.05) in all experimental groups (0.1 mg mL−1: 15.8 ± 8.9%; 0.5 mg mL−1: 18.7 ± 12.0%; 1 mg mL−1: 17.0 ± 11.2%) compared with the control group (34.1 ± 5.4%). The total number of cells was not affected by OF (P > 0.05). There was no significant difference (P > 0.05) in the post-thaw re-expansion rate between the experimental groups (0.1 mg mL−1: n = 26 thawed blastocysts; 0.5 mg mL−1: n = 27; 1 mg mL−1: n = 23) and the control group (n = 58). The post-thaw hatching rate was significantly higher at 24 and 72 h, respectively, in the 0.5 mg mL−1 group (44.4% and 74.1%; P < 0.05) and the 1 mg mL−1 group (47.8%; P < 0.05; and 82.6%; P < 0.01) compared with the control group (18.9% and 44.8%). The replacement of serum with OF during in vitro culture of bovine embryos had a stage specific effect, resulting in higher cleavage rates but lower blastocyst rates. To address this issue, OF will be collected at different stages and applied in the matching in vitro culture phases in future studies. Interestingly, the post-thaw hatching rate was up to twice as high in the experimental groups, indicating better quality of those embryos developing to blastocyst stage.

scholarly journals Reproductive fluids, used for the in vitro production of pig embryos, result in healthy offspring and avoid aberrant placental expression of PEG3 and LUM.

2020 ◽  
Author(s):  
Evelynne Paris-Oller ◽  
Sergio Navarro-Serna ◽  
Cristina Soriano-Úbeda ◽  
Jordana Sena Lopes ◽  
Carmen Matas ◽  
...  
Keyword(s):  
Reproductive Performance ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
In Vitro Production ◽  
Aberrant Expression ◽  
Low Efficiency ◽  
The Impact

Abstract Background: In vitro embryo production (IVP) and embryo transfer (ET) are two very common assisted reproductive technologies (ART) in human and cattle. However, in pig, the combination of either procedures, or even their use separately, is still considered suboptimal due to the low efficiency of IVP plus the difficulty of performing ET in the long and contorted uterus of the sow. In addition, the potential impact of these two ART on the health of the offspring is unknown. We investigated here if the use of a modified IVP system, with natural reproductive fluids (RF) as supplements to the culture media, combined with a minimally invasive surgery to perform ET, affects the output of the own IVP system as well as the reproductive performance of the mother and placental molecular traits.Results: The blastocyst rates obtained by both in vitro systems, conventional (C-IVP) and modified (RF-IVP), were similar. Pregnancy and farrowing rates were also similar. However, when compared to in vivo control (artificial insemination, AI), litter sizes of both IVP groups were lower, while placental efficiency was higher in AI than in RF-IVP. Gene expression studies revealed aberrant expression levels for PEG3 and LUM in placental tissue for C-IVP group when compared to AI, but not for RF-IVP group.Conclusions: The use of reproductive fluids as additives for the culture media in pig IVP does not improve reproductive performance of recipient mothers but could mitigate the impact of artificial procedures in the offspring.

474 Effect of four assisted hatching techniques and two in vitro culture media on embryo hatching rate

2017 ◽  
Vol 95 (suppl_4) ◽  
pp. 231-231
Author(s):  
N. C. Negota ◽  
L. P. Nethenzheni ◽  
N. R. Serota
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Assisted Hatching ◽  
Hatching Rate

246 IN VITRO PRODUCTION OF SPRINGBOK (ANTIDORCAS MARSUPIALIS) EMBRYOS

2007 ◽  
Vol 19 (1) ◽  
pp. 239 ◽  
Author(s):  
R. Krisher ◽  
A. Auer ◽  
K. Clark ◽  
K. Emsweller ◽  
S. Rogers ◽  
...  
Keyword(s):  
South Africa ◽  
Oocyte Maturation ◽  
Assisted Reproductive Technologies ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Genetic Management ◽  
Blastocyst Development ◽  
Antidorcas Marsupialis

The objective of this experiment was to develop in vitro embryo production (IVP) technologies in springbok (Antidorcas marsupialis), a southern African antelope. Springbok, a fairly common species on game farms in parts of South Africa, may be used as a model species for gamete rescue and IVP techniques to be applied to the conservation of other threatened antelope species. Springbok belong to the family bovidae, subfamily antilopinae, tribe antilopini, which comprises about twenty species in genera Gazella, Antilope, Procapra, Antidorcas, Litocranius, and Ammodorcas. In this tribe alone, there are 4 species or subspecies that are critically endangered, 3 that are endangered, and 10 that are considered vulnerable, demonstrating the need for antelope conservation efforts. In addition, our studies contributed to the South African biological resource bank, so that banked springbok semen and embryos might be used in the future for managed genetic contribution to isolated captive or wild populations via assisted reproductive technologies. Oocytes were recovered (3 replicates) from ovaries obtained at supervised culls for management purposes in South Africa, and cultured in defined Gmat or undefined TCM-199 with FCS maturation medium for 28-30 h (Brad et al. 2004 Reprod. Fertil. Dev. 16, 223). Oocytes were fertilized with frozen-thawed springbok epididymal spermatozoa in modified SOF fertilization medium with caffeine (Herrick et al. 2004 Biol. Reprod. 71, 948–958). Eighteen hours after insemination, a randomly selected subset of the zygotes were fixed to determine fertilization success. The remaining zygotes were cultured in G1/G2 media. On Day 7 of culture, embryos were analyzed for development to the morula or blastocyst stage. A total of 259 selected oocytes were collected from 50 females (5.2 selected oocytes/female on average). There was no difference in the percentage of oocytes normally fertilized (2 pronuclei, PN) between oocytes matured in Gmat (n= 43; 12%) and those matured in TCM-199 (n= 42; 10%). There were significantly (P &lt; 0.05) more oocytes penetrated (e2 PN) when matured in TCM (50%) compared to Gmat (23%). There were no differences in embryonic cleavage or morula/blastocyst development (of total oocytes inseminated) between treatments (Gmat,n= 89, 54%, 9.0%; TCM-199, n= 85, 68%, 9.4%, respectively). In both treatments, the average blastocyst grade was 2.125 using the standard bovine grading system (Curtis, Cattle Embryo Transfer Procedure, 1991). In conclusion, in vitro oocyte maturation, fertilization, and embryo culture to the blastocyst stage is possible in springbok. Importantly, blastocysts can be produced in vitro under semi-defined conditions, demonstrating that oocyte maturation without serum does support developmental competence. This is important for the potential international movement of IVP embryos to be used for genetic management in the conservation of antelope species.

163 EVALUATION OF VIABILITY OF BULL SEMEN COLLECTED BY ELECTRO-EJACULATION USING COMMERCIAL SEMEN EXTENDER AND 2 CULTURE MEDIA AT CONTROLLED ROOM TEMPERATURE

2017 ◽  
Vol 29 (1) ◽  
pp. 190
Author(s):  
A. M. Raseona ◽  
O. A. Ajao ◽  
L. D. Nethengwe ◽  
L. R. Madzhie ◽  
T. L. Nedambale ◽  
...  
Keyword(s):  
Sperm Motility ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Sperm Viability ◽  
Progressive Motility ◽  
Bull Semen ◽  
Significant Difference ◽  
Semen Extender ◽  
Motility Rate

Preservation of semen is an important process to ensure that semen quality is sufficient for assisted reproductive technologies. The aim of this study was to evaluate the viability of bull semen collected by electro-ejaculation using commercial semen extender and 2 modified culture media stored at controlled RT (24°C) for 72 h. Two Nguni bulls were used for semen collection; after collection, the semen was evaluated macroscopically for volume, pH, and colour, and microscopically for sperm motility, viability, and morphology. Uncontaminated semen samples with progressive motility >70% and morphological defects <20% were pooled after collection before being aliquoted into 3 extenders, namely Triladyl, modified Ham’s F10, and TCM-199 culture media, at a dilution ratio of 1:4 and then stored at controlled RT (24°C). Sperm motility rate was analysed using the computer-aided sperm analyser after 0, 24, 48, and 72 h of storage. Sperm morphology and viability was performed after staining the sperm cells with spermac and nigrosine-eosin stain, respectively. The study was replicated 4 times and data were analysed using ANOVA. Triladyl had a higher sperm viability rate (41.3%) and total motility rate (96.3%) for 72 h (P < 0.01) compared with the 2 modified culture media, Ham’s F10 (26.5 and 86.8%) and TCM-199 (25.0 and 86.7%), respectively. However, Ham’s F10 had higher progressive motility rate (37.8%) as compared with the other extenders, TCM-199 (31.7%) and Triladyl (23.4). There was no significant difference (P > 0.05), in viability rate between Ham’s F10 (26.5%) and TCM-199 (25.0%). No significant difference (P > 0.05) in straight line velocity was observed for the three extenders. Furthermore, no significant difference was observed in total sperm abnormalities, except for reacted acrosomes and absent tails (P > 0.05), between the 2 Nguni bulls. Nguni semen can be preserved in Triladyl or modified Ham’s F10 and TCM-199 culture media stored at 24°C and stay viable for 72 h. Triladyl proved to be the best suitable extender of the 3 extenders, showing higher sperm viability and total motility rate as compared with Ham’s F10 and TCM-199 modified culture media.

Embryo Culture Conditions and the Epigenome

2018 ◽  
Vol 36 (03/04) ◽  
pp. 211-220 ◽  
Author(s):  
Sneha Mani ◽  
Monica Mainigi
Keyword(s):  
Embryo Culture ◽  
Assisted Reproductive Technologies ◽  
Culture Media ◽  
Reproductive Technologies ◽  
Common Mechanism ◽  
Success Rates ◽  
Blastocyst Development ◽  
Increased Risk ◽  
Modifiable Factors

AbstractAssisted reproductive technologies (ARTs) lead to an increased risk for pregnancy complications, congenital abnormalities, and specific imprinting disorders. Epigenetic dysfunction is thought to be one common mechanism which may be affecting these outcomes. The timing of multiple ART interventions overlaps with developmental time periods that are particularly vulnerable to epigenetic change. In vitro embryo culture is known to impact blastocyst development, in vitro fertilization (IVF) success rates, as well as neonatal outcomes. Embryo culture, in contrast to other procedures involved in ART, is obligatory, and has the highest potential for causing alterations in epigenetic reprograming. In this review, we summarize progress that has been made in exploring the effects of embryo culture, culture media, and oxygen tension on epigenetic regulation in the developing embryo. In humans, it is difficult to isolate the role of embryo culture on epigenetic perturbations. Therefore, additional well-controlled animal studies isolating individual exposures are necessary to minimize the epigenetic effects of modifiable factors utilized during ART. Findings from these studies will likely not only improve IVF success rates but also reduce the risk of adverse perinatal outcomes.

Expression of mRNA encoding leukaemia inhibitory factor (LIF) and its receptor (LIFRβ) in buffalo preimplantation embryos produced in vitro: markers of successful embryo implantation

Zygote ◽  
2012 ◽  
Vol 21 (2) ◽  
pp. 203-213 ◽  
Author(s):  
S. Eswari ◽  
G. Sai Kumar ◽  
G. Taru Sharma
Keyword(s):  
Culture Media ◽  
Total Cell ◽  
Inhibitory Factor ◽  
Preimplantation Embryos ◽  
Leukaemia Inhibitory Factor ◽  
Hatching Rate ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Significant Difference

SummaryThe objective of this study was to evaluate the effect of supplementation of recombinant leukaemia inhibitory factor (LIF) in culture media on blastocyst development, total cell number and blastocyst hatching rates and the reverse transcription-polymerase chain reaction analysis of preimplantation buffalo embryos to determine whether they contain the LIF-encoding mRNA and its beta receptor (LIFRβ) genes in different stages of preimplantation buffalo embryos. Cumulus–oocyte complexes retrieved from slaughterhouse buffalo ovaries were matured in vitro and fertilized using frozen buffalo semen. After 18 h of co-incubation with sperm, the presumptive zygotes were cultured in modified synthetic oviductal fluid without (control) or with rhLIF (100 ng/ml). There was no significant difference in the overall cleavage rate up to morula stage however the development of blastocysts, hatching rate and total cell numbers were significantly higher in the LIF-treated group than control. Transcripts for LIFRβ were detected from immature, in vitro-matured oocytes and in the embryos up to blastocyst stage, while transcripts for the LIF were detected from 8–16-cell stage up to blastocyst, which indicated that embryo-derived LIF can act in an autocrine manner on differentiation process and blastocyst formation. This study indicated that the addition of LIF to the embryo culture medium improved development of blastocysts, functional (hatching) and morphological (number of cells) quality of the blastocysts produced in vitro. The stage-specific expression pattern of LIF and LIFRβ mRNA transcripts in buffalo embryos indicated that LIF might play an important role in the preimplantation development and subsequent implantation of buffalo embryos.

scholarly journals 146COMPARISON OF EMBRYO CULTURE MEDIA FOR BLASTOCYST DEVELOPMENT AFTER INTRACYTOPLASMIC SPERM INJECTION OF IN VITRO-MATURED EQUINE OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 195
Author(s):  
Y.H. Choi ◽  
D.D. Varner ◽  
K. Hinrichs
Keyword(s):  
In Vitro Culture ◽  
Embryo Culture ◽  
Intracytoplasmic Sperm Injection ◽  
Culture Media ◽  
Polar Body ◽  
Developmental Competence ◽  
Blastocyst Development ◽  
Vitro Fertilization ◽  
Significant Difference

Research on in vitro culture of equine embryos has been scant, due to failure of equine in vitro fertilization to be repeatably successful. We have recently obtained high fertilization rates of equine oocytes via intracytoplasmic sperm injection (ICSI) using a piezo drill (Choi et al., 2002 Reproduction 123, 455–465). Culture of presumptive zygotes in G1.2/2.2 medium resulted in 63% cleavage and an average of 15 cells at 4d, but only 2 to 9% blastocyst development at 7 days (Choi et al., 2003 Theriogenology 59, 1219–1229). In the present study, we evaluated the effect of two different culture media, G1.3/G2.3 v. DMEM/F-12, with or without FBS, on blastocyst development after ICSI. Oocytes were collected from slaughterhouse-derived ovaries by follicular scraping and were matured in vitro for 24h in M199 with 10% FBS and 5μUmL−1 FSH. After culture, oocytes having a polar body (198/305; 65%) were fertilized by ICSI with frozen-thawed equine sperm using a piezo drill. Presumptive zygotes were cultured in 1 of 4 media: G1.3/G2.3 (which includes 0.8% BSA) with or without 10% FBS, or in DMEM/F-12 with 0.5% BSA, with or without 10% FBS. Culture was performed in microdroplets at 5μL/zygote under oil at 38.2°C in an atmosphere of 5% CO2, 5% O2 and 90% N2 for 7.5 days. In G1.3/2.3 treatments, G1.3 media were completely refreshed at 48h, zygotes were transferred to G2.3 (with or without FBS as per the first stage) at 96h, and were completely refreshed with the same media at 144h. In DMEM/F-12 treatments, media were completely refreshed every other day. Three to 5 replicates were performed in each treatment, and data were analyzed by chi-square test. There were no significant differences in cleavage rates (59–64%) among treatments. The rate of development to blastocyst, per oocyte injected, in G1.3/G2.3/BSA (1/49, 2%) was significantly lower (P&lt;0.05) than that for the other three treatments: G1.3/2.3/BSA/FBS (9/49, 18%), DMEM/F-12/BSA (9/50, 18%), or DMEM/F-12/BSA/FBS (10/50, 20%). There was no significant difference in blastocyst development among the latter three treatments. These findings indicate that G1.3/2.3 media with BSA only do not adequately support growth of equine embryos. Development of up to 20% of injected oocytes to the blastocyst stage in G media supplemented with FBS, in DMEM/F-12/BSA or in DMEM/F-12/BSA/FBS represents the highest in vitro equine blastocyst rate in medium alone (i.e. without co-culture) yet reported. The success of DMEM/F-12 as an embryo culture medium may provide a relatively simple basis for equine in vitro culture programs. To determine whether this medium was able to support further developmental competence, we cultured equine embryos resulting from nuclear transfer of in vitro-matured oocytes in DMEM/F-12+10% FBS (without BSA). We transferred 4 resulting blastocysts to recipient mares by transcervical transfer; one pregnancy is ongoing at 230d gestation at the time of this writing. This work was supported by the Link Equine Research Endowment Fund, Texas A&amp;M University.

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