94 EFFECTS OF 6- OR 12-HOUR CULTURE IN A Micro Q STRAW INCUBATOR ON DEVELOPMENT OF IN VITRO-PRODUCED BOVINE EMBRYOS

2016 ◽  
Vol 28 (2) ◽  
pp. 177
Author(s):  
C. R. Looney ◽  
J. H. Pryor ◽  
M. Snyder ◽  
A. Ilercil ◽  
C. R. Long
Keyword(s):  
Embryo Transfer ◽  
Uv Light ◽  
Tween 20 ◽  
Treatment Groups ◽  
Cell Counts ◽  
Stage 4 ◽  
Embryo Stage ◽  
Transfer Services ◽  
In Transit

Transporting in vitro-produced (IVP) embryos can be challenging when an embryo transfer destination is more than 6 h away or electricity is not available on site to unload embryos for transfer. The objective of this study was to determine if development rates would be compromised for Day 6.5 IVP embryos when placed in warmed Vigro holding medium (Vetoquinol, Pullman, WA, USA) loaded and plugged into 1/4 cc straws (Professional Embryo Transfer Services, Canton, TX, USA) for a period of either 6 or 12 h in a 38.5°C Micro Q straw block incubator (Micro Q iQ1T 64). Bovine oocytes were shipped and matured in transit from a commercial abattoir (DeSoto Biosciences, Seymour, TN, USA), fertilized (IVF = Day 0) with frozen-thawed semen, and cultured in Bovine Evolve (Zenith Biotech, Guilford, CT, USA) supplemented with 4 mg mL–1 BSA (Probumin, EMD Millipore, Norcross, GA, USA) under oil in a 5% CO2, 5%O2, 90% N2 humidified incubator (Pryor et al. 2011 Theriogenology 75, 24–33). Cleavage rates of 87.7% (664/757) from three replicates produced 273 (36.0%) viable embryos on Day 6.5 post-IVF, which were evenly distributed by IETS stage (4–7) and grade (1 and 2) into three treatment groups (0 = control, 6 or 12 h straw incubation) before in vitro culture for an additional 24 h. For each replicate, the average embryo stage was calculated by multiplying the number of embryos in each treatment by their IETS stage and dividing by total embryos per group. The change in stage for each treatment was calculated by subtracting the initial average stage from the final average stage on Day 8. Grade 1 and 2 embryos at stage 6–8 were counted and used to calculate total viable rates. Day 8 (post-IVF) embryos were fixed in cold methanol, washed in PBS/0.1%Tween 20, mounted in 10 μg mL–1 Hoechst 33342/glycerol and viewed under UV light to count nuclei. Percentage data were transformed using arcsine square root function before analysis, and means were compared using a one-way ANOVA and Tukey’s HSD. Although viability decreased with increasing time in straw incubation, there were no statistical differences between control, 6 and 12 h treatments for total viable rates (90.8, 80.3, and 70.8%, respectively). Average embryo stage on Day 8 for control, 6 and 12 h (7.0 ± 0.66, 6.6 ± 0.24, and 6.2 ± 0.30 s.e.m., respectively) was not different, but tended to be higher in control (P = 0.08). The change in stage, however, was different between control and 12 h (1.46 ± 0.33 and 0.66 ± 0.24, respectively; P < 0.05). Likewise, cell numbers were greater in control and 6 h embryos compared with 12 h straw incubation (149.8 ± 9.14, 138.7 ± 7.94, and 101.8 ± 5.29; n = 47, 50, and 46, respectively P < 0.01). In conclusion, 6.5 day IVP embryos held in warm Vigro holding medium for 12 h in 1/4 cc straws fail to develop at the same rate and incurred lower cell counts than either control or 6 h treatments. Further research to evaluate pregnancy rates following transfer and utilising different incubation or media and/or temperature is warranted to further evaluate the utility of in straw incubation for extended periods of time.

86 IMPROVED BOVINE EMBRYO PRODUCTION USING NOVEL IN VITRO CULTURE SYSTEMS

2016 ◽  
Vol 28 (2) ◽  
pp. 172
Author(s):  
J. H. Pryor ◽  
J. F. Hasler ◽  
L. Strøbech ◽  
B. Avery ◽  
N. Hashem ◽  
...  
Keyword(s):  
Production Systems ◽  
Uv Light ◽  
Cumulus Cells ◽  
Tween 20 ◽  
Bovine Embryo ◽  
In Vitro Production ◽  
Embryo Viability ◽  
Embryo Production ◽  
Cell Counts

Development and testing of new embryo production components is important to improve the outcome following in vitro production of bovine embryos. The objective of this study was to compare media used in two bovine embryo production systems (control and EmbryoTrans Biotech: ETB). In Exp. 1, abattoir-derived cumulus-oocyte complexes were randomly assigned and in vitro matured (IVM) in either control [Medium 199 with Earles salts (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 1% penicillin/streptomycin (Invitrogen), 0.2 mM sodium pyruvate, 2 mM L-glutamine (Sigma Chemical Co., St. Louis, MO, USA), and 5.0 µg mL–1 of Folltropin®-V (Vetoquinol, Pullman, WA, USA)] or ETB BO-IVM medium for 21 to 24 h. IVF was conducted in 500 µL of pre-equilibrated modified Tyrode-lactate medium for control (Pryor et al. 2011 Theriogenology 75, 24–33) or ETB BO-IVF in Nunclon® 4-well multi-dishes (VWR Scientific, Pittsburgh, PA, USA). Seventeen hours post-insemination, presumptive zygotes were cleaned of cumulus cells and cultured in either Bovine Evolve (Zenith Biotech, Guilford, CT, USA) supplemented with 4 mg mL–1 of Probumin BSA (EMD Millipore, Norcross, GA, USA), under oil (Irvine Scientific, Santa Ana, CA, USA) or ETB BO-IVC medium under BO-oil for 7 days (8 days post-IVF). All cultures were performed at 38.5°C in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 using BT37 incubators (Planer Plc, Sunbury, UK). For Exp. 2, all conditions were maintained except a modified ETB BO-IVCA medium was used. On Day 8 of IVC, grade 1 and 2 blastocysts (BL) through hatching blastocysts (HBL) were counted and used to calculate total viable rates. In Exp. 2, these embryos were fixed in cold methanol, washed in PBS/0.1% Tween 20, mounted in 10 μg mL–1 Hoechst 33342/glycerol, and viewed under UV light to count cells (n = 49 and 107 for control and ETB, respectively). Each experiment was replicated 3 times with a total of 425 oocytes in Exp. 1 and 430 in Exp. 2, divided equally between treatments. Percentage data were transformed using arcsine square root function before analysis and means compared using a paired Student’s t-test. For Exp. 1, there were no differences in rates of cleavage or viable embryos between control and ETB systems (81.3% and 42.9% v. 80.5% and 48.4%, respectively). In Exp. 2, ETB was superior to control for percent viable, HBL, and combined HBL/expanded BL (51.9, 23.9, 45.8% v. 29.2, 5.8, 20.5, respectively; P < 0.05). Differences between mean cell counts for viable embryos were significant (control = 127.0 ± 6.7 s.e.m. and ETB = 162.7 ± 5.7; P < 0.0001). Embryo viability decreased in control media between Exp. 1 and 2 (42.9 v. 29.2%; P < 0.05). Seasonal differences may have contributed via heat stress with temperatures ranging from 23.8°C for Exp. 1 to 33.8°C for Exp. 2. Interestingly, embryo development in the ETB media did not decrease under the same conditions. In conclusion, ETB media produced more high-quality embryos than control under varying conditions experienced by commercial IVF companies.

scholarly journals A shorter equilibration period in the VitTrans in-straw bovine embryo vitrification method improves post-warming outcomes

2020 ◽  
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario-Hidalgo ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Field Conditions ◽  
Bovine Embryo ◽  
Direct Transfer ◽  
Treatment Groups ◽  
Cell Counts ◽  
Hatching Rates

Abstract Background VitTrans is a device that enables the vitrification and warming/dilution of in vitro produced bovine embryos followed by their direct transfer to recipient females in field conditions. This study sought to improve the VitTrans method by comparing two equilibration times: short (SE: 3 min) and long (LE: 12 min). Outcome measures recorded in vitrified D7 and D8 expanded blastocysts were survival and hatching rates, differential cell counts, apoptosis rate and gene expression. Results While survival rates at 3 h and 24 h post-warming were reduced (P < 0.05) after vitrification, hatching rates of D7 embryos vitrified after SE were similar to those obtained in fresh non-vitrified blastocysts. Hatching rates of vitrified D8 blastocysts were lower (P < 0.05) than of fresh controls, regardless of treatment. Total cell counts, and inner cell mass and trophectoderm cell numbers were similar in hatched blastocysts derived from D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values, regardless of treatment. The rate of apoptotic cells was significantly higher in both treatment groups when compared to fresh controls, although apoptosis rates were lower using the SE than LE protocol. No differences emerged in expression of the genes BAX, AQP3, CX43 and IFNτ between blastocysts vitrified after SE or LE, whereas a significantly higher abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE compared to LE. Conclusions The VitTrans device combined with a shorter exposure to the equilibration medium improves vitrification/warming outcomes facilitating the direct transfer of vitrified embryos under field conditions.

334 EFFECT OF INSULIN ON IN VITRO MATURATION OF CANINE OOCYTES

2006 ◽  
Vol 18 (2) ◽  
pp. 275
Author(s):  
H. S. Lee ◽  
Y. I. Seo ◽  
X. J. Yin ◽  
S. G. Cho ◽  
I. H. Bae ◽  
...  
Keyword(s):  
In Vitro Maturation ◽  
Uv Light ◽  
Cumulus Cells ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Oocyte Competence ◽  
Treatment Groups ◽  
Maturation Medium ◽  
Oviduct Fluid

In spite of our increased knowledge of in vitro oocyte maturation techniques, the success rate of obtaining mature canine oocytes in vitro remains very low compared with that for other domestic animals. The inefficient rate of meiotic resumption of canine oocytes is probably due to both the unique reproductive cycle and inappropriate in vitro maturation (IVM) medium. In an unpublished experiment, we found that the concentration of insulin was higher in estrus bitch serum (EBS; 8833 pg/mL) than in dog follicular fluid (DFF; preovulatory follicle, 122 pg/mL), which implies its possible role in the acquisition of oocyte competence. Therefore, in the present study we investigated the effects of supplementing the IVM medium with insulin on the incidence of maturation to metaphase II. Ovaries were collected from various stages of the estrous cycle by ovariohysterectomy, and oocytes with two or more intact cumulus layers and with a diameter >110 �m were selected and used for IVM. Oocytes were cultured in modified synthetic oviduct fluid (2004 Reprod. Nutr. Dev. 44, 105-109) supplemented with 10% EBS, 20 �g/mL estradiol, and different concentrations of insulin (0, 10, 100, or 1000 ng/mL) at 38.5�C, 5% CO2 in air. After 72 h, cumulus cells were removed from around oocytes using a small glass pipette. Denuded oocytes were fixed in 3.7% paraformaldehyde supplemented with 10 �g/mL Hoechst 33342 at room temperature for 40 min. Nuclear status was observed under UV light using a fluorescence microscope. The percentage of oocytes at the metaphase II stage was not different among the four groups 6.8, 1.8, 5.4, and 2.1% in the control, 10, 100, and 1000 ng/mL insulin groups, respectively. The incidence of oocytes with pronuclear-like structures or cleaving beyond the two-cell stage was not significant higher in the 10 and 100 ng/mL insulin treatment groups than in the control and 1000 ng/mL insulin groups 20.0 and 19.6% vs. 6.8 and 6.4%, respectively. These results indicate that the addition of insulin to the in vitro maturation medium of dog oocytes had no effect on the incidence of meiotic maturation to metaphase II, nor did it affect the frequency of occurrence of spontaneous oocyte activation.

107 FACTORS THAT INFLUENCE FERTILITY IN AN IVF EMBRYO TRANSFER PROGRAM IN DAIRY HEIFERS

2016 ◽  
Vol 28 (2) ◽  
pp. 183 ◽  
Author(s):  
L. C. Carrenho-Sala ◽  
R. V. Sala ◽  
M. Fosado ◽  
D. C. Pereira ◽  
S. Garcia ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Pregnancy Loss ◽  
Embryo Quality ◽  
Prostaglandin F2α ◽  
Transfer Program ◽  
Dairy Heifers ◽  
Embryo Transfer Program ◽  
Embryo Stage ◽  
Major Factors

A retrospective study was performed to evaluate factors that influence pregnancy per embryo transfer (P/ET) in an IVF-embryo transfer program. A total of 5026 fresh in vitro-produced embryos were transferred during 2014 and evaluated for effects of embryo quality, embryo stage, size of corpus luteum (CL; 18–19.9 mm or ≥20 mm), interval from GnRH to embryo transfer, number of previous embryo transfer (0, 1, 2, 3, ≥4); and interaction of embryo stage and interval from GnRH to embryo transfer. One group (n = 850) had detection of oestrus after prostaglandin F2α application but most heifers (n = 4176) received fixed timed embryo transfer after a 5-day CIDR-Synch protocol: Day –8 CIDR inserted; Day –3 CIDR removed and prostaglandin F2α; Day –2 prostaglandin F2α; Day 0 GnRH. Ultrasound was performed on Day 6 after GnRH or oestrus to measure CL size and on Day 32 and 60 to determine pregnancy. Data for P/ET were analysed by logistic regression (LOGISTIC procedure, SAS 9.4). Embryo quality influenced P/ET at Day 32 [Grade 1 48.4% (1273/2631) v. Grade 2 37.6% (900/2395); P < 0.01] and at Day 60 [Grade 1 38.9% (1023/2631) v. Grade 2 29.0% (694/2395); P < 0.01], and altered pregnancy loss [Grade 1 19.6% (250/1273) v. Grade 2 22.9% (206/900); P = 0.03]. Stage of the embryo also had an effect on P/ET at Day 32 [Stage 6 35.5%a (582/1641), Stage 7 46.3%b (1431/3092), and Stage 8 54.6%c (160/293); P < 0.01] and at Day 60 [Stage 6 28.2%a (462/1641), Stage 7 36.6%b (1131/3092), and Stage 8 41.6%b (122/293); P < 0.01], but did not affect pregnancy loss (P = 0.22). Interestingly, interval from GnRH (or oestrus) until embryo transfer did not affect P/ET at Day 32 (P = 0.10), 60 (P = 0.23), or pregnancy loss (P = 0.3), nor was there an interaction between interval and embryo stage at Day 32 (P = 0.77), 60 (P = 0.96) or pregnancy loss (P = 0.55). As shown in Table 1, embryo stage 6 was always the lowest and stage 8 always the greatest P/ET regardless of interval from GnRH to embryo transfer. Size of CL also did not affect P/ET at Day 32 (P = 0.09), 60 (P = 0.21), or pregnancy loss (P = 0.90). Number of previous embryo transfer also did not alter P/ET at Day 32 [0 = 43.3% (886/2046), 1 = 44.1% (639/1450), 2 = 43.4% (444/1024), 3 = 42.6% (146/343), and ≥4 = 35.6% (58/163); P = 0.33] or 60 (P = 0.51) or pregnancy loss (P = 0.12). In conclusion, embryo stage and quality are the major factors that impacted P/ET in this study, with surprisingly little effect of interval from GnRH to embryo transfer, size of the CL, and number of previous embryo transfer. Thus, recipient programs for IVF-embryo transfer can be designed with substantial flexibility. Table 1.Effect of embryo stage and recipient synchrony on pregnancies per embryo transfer on Day 32 in recipient dairy heifers

160 CONCEPTION RATES FOLLOWING TREATMENT OF LACTATING HOLSTEIN RECIPIENTS WITH A GONADOTROPIN RELEASING HORMONE (GnRH) ANALOGUE AT THE TIME OF TRANSFER OF OVUM PICK-UP/IVF-DERIVED EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 228
Author(s):  
T. L. C. Pinto ◽  
J. N. S. Sales ◽  
R. R. Carvalho ◽  
T. M. França ◽  
J. C. Souza
Keyword(s):  
Dairy Cows ◽  
Embryo Transfer ◽  
Management Tool ◽  
Gnrh Analogue ◽  
Gnrh Analogues ◽  
Treatment Groups ◽  
Lactating Dairy Cows ◽  
Conception Rates ◽  
Day Of Embryo Transfer

The use of lactating dairy cows as ovum pick-up (OPU)/IVF-derived embryo recipients in large-scale embryo transfer programs may be limited by the effects of low progesterone concentrations on the uterine environment and subsequent embryo development. It was hypothesized that the injection of a GnRH analogue on the day of embryo transfer would induce ovulation and formation of an accessory corpus luteum, increasing progesterone concentrations and, as a result, conception rates. An experiment was designed to evaluate the effects of two different GnRH analogues injected IM on the day of embryo transfer on conception rates of lactating Holstein recipients. Oestrus was not synchronized and was detected continuously by multiple observers during the day and in the evening until midnight. Recipient days in milk varied from 45 to 150, daily milk production ranged from 20 to 45 kg, body condition scores and age ranged from 2.5 to 4.5 and 2.5 to 5.5 years, respectively. Herd pregnancy rates after the first artificial insemination ranged from 15% to 70%, considering the period the experiment was conducted. Embryos were produced by standard in vitro techniques following OPU on Holstein donors. Lactating Holstein recipients (n = 224) were allocated randomly to one of three treatment groups 7 days after detected oestrus: Control (n = 77) = vehicle; Buserelin (Sincroforte®, Ouro Fino, Sao Paulo, Brazil; n = 75) = 10 µg; and Deslorelin (Sincrorrelin®, Ouro Fino; n = 72) = 750 µg. Embryos were transferred nonsurgically 6 to 8 days after observed oestrus. Pregnancy was detected by ultrasonography between 62 and 72 days after embryo transfer. Percent pregnant data were analyzed as a binomial distribution under the GENMOD procedure of SAS (SAS Institute Inc., Cary, NC, USA). Conception rates were similar (P = 0.13) between Control (28%, 22/77), Buserelin (24%, 18/75), and Deslorelin (38%, 28/72) treatment groups. These data demonstrate that conception rates from in vitro produced embryos transferred to lactating dairy cows are not improved by treatment with a GnRH analogue on the day of transfer. Although not critically tested, results indicate that adequate conception rates may be obtained in commercial dairy operations from OPU/IVF-derived embryos, adding an important management tool for reproduction and animal breeding enhancement.

100 EMBRYO SURVIVAL AND CONCEPTUS ELONGATION FOLLOWING ASYNCHRONOUS EMBRYO TRANSFER IN CATTLE

2015 ◽  
Vol 27 (1) ◽  
pp. 143
Author(s):  
F. Randi ◽  
B. Fernandez ◽  
M. McDonald ◽  
C. Johnson ◽  
N. Forde ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Prostaglandin F2α ◽  
Early Embryo ◽  
Embryo Survival ◽  
Treatment Groups ◽  
Length Data ◽  
Uterine Environment ◽  
To Receive ◽  
Intravaginal Device

Maternal progesterone (P4) regulates early conceptus growth and development in ruminants. Early embryo transfer studies in sheep and cattle demonstrated a need for close synchrony between the embryo and the uterine environment of the recipient. However, manipulating P4 may be one way of strategically regulating the temporal changes that normally occur in the uterine environment in order to allow flexibility in the timing of embryo transfer. For example, previous studies have demonstrated that P4 administration during the first few days of the oestrous cycle facilitates pregnancy establishment with older embryos. The aim of this study was to examine the effect of embryo-uterine synchrony on conceptus elongation in cattle. Oestrous cycles of crossbred beef heifers were synchronised using an 8-day P4-Releasing Intravaginal Device (PRID Delta®, CEVA, Mountain View, CA, USA) with administration of a prostaglandin F2α analogue (Enzaprost®, CEVA; 5 mL equivalent to 25 mg of dinoprost) given on the day before PRID removal. Heifers were checked for signs of oestrus 4 times per day commencing 30 h after PRID withdrawal. Only those seen in standing oestrus (n = 50) were randomly assigned to 1 of 5 treatment groups to receive Day 7 in vitro-produced blastocysts (n = 10 per recipient) (1) on Day 5 post-oestrus; (2) on Day 5, with P4 supplementation via PRID from Day 3 to 5 + 750 IU of eCG at PRID insertion; (3) on Day 5, PRID Delta from Day 3 to 5 plus 3000 IU of hCG at PRID insertion; (4) on Day 7, or (5) on Day 9. At embryo age Day 14, all heifers were slaughtered and the uterus was flushed to recover and measure conceptuses. Data are summarised in Table 1. Fewer recipients yielded conceptuses (P < 0.05) and fewer conceptuses overall were recovered (P < 0.05) following transfer on Day 5 compared with Day 7 or Day 9. Supplementation with P4 resulted in short cycles (evidenced by corpus luteum regression and/or a recent ovulation at slaughter) in 33.3 to 54.5% of recipients receiving embryos on Day 5. Mean conceptus length was greater (P < 0.05) following transfer to an advanced uterus. In conclusion, transfer of embryos to a retarded (Day 5) uterine environment results in poor embryo survival. Supplementation with P4 shortened the interoestrous period in a significant number of heifers. Transfer to an advanced uterine environment promotes conceptus elongation, presumably driven by P4. Table 1.Embryo survival and conceptus length data

126 THE EFFECT OF FORSKOLIN ON IN VITRO PRODUCED BRAHMAN-SIRED BOVINE EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 163 ◽  
Author(s):  
J. H. Pryor ◽  
J. A. Trant ◽  
C. B. Ponchirolli-Schneider ◽  
C. R. Looney ◽  
C. R. Long ◽  
...  
Keyword(s):  
Treatment Group ◽  
Uv Light ◽  
Nile Red ◽  
Total Cell ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Intracellular Lipids ◽  
Lipolytic Effect ◽  
Development Rates

Forskolin (Colforsin) is a lipolytic agent that can be used to reduce intracellular lipids within in vitro produced (IVP) porcine embryos (Men et al. 2006 Theriogenology 66, 2008–2016). Lowering lipids within bovine embryos has shown to decrease sensitivity to cryopreservation. The objective of this preliminary experiment was to determine developmental differences and lipolytic effect on bovine IVP embryos exposed to 10 or 40 μm forskolin. Bovine oocytes (obtained from an abattoir) were fertilized with frozen/thawed Brahman semen (Day 0) and cultured in G1.5/G2.5 medium supplemented with 8 mg mL–1 BSA (Vitrolife, Englewood, CO, USA). On Day 6 post-fertilization, a total of 352 viable embryos were randomly submitted to 3 treatment groups; Control (no treatment, n = 116), 10 μm (n = 122) and 40 μm (n = 114) forskolin (Sigma, St. Louis, MO, USA) in G2.5 medium for 24 h. On Day 7, embryos were washed in holding medium (Vigro Holding Plus, Bioniche, Pullman, WA, USA). From each treatment group, a randomized half was cultured 72 h in G2.5 and assessed for hatched blastocyst rates (development), with the balance stained on Day 7 in Hoechst 33342 (2.5 μg mL–1) under UV light for total cell counts before being fixed/stained in Nile red (1 μg mL–1). Nile red stained embryos were mounted on glass slides with cover slips gently applied over each embryo. Embryos were photographed under UV light using the proper excitation filters. The area measurements of compact morula and blastocysts did not include the zona pellucida. Nile red units (NRU) were measured with computer software Image J (www.rbsweb.nih.gov) from 0 to 255 shades/pixel (0 = no lipids; 255 = highest lipid accrual). Each image was adjusted for variation in embryo size. Student’s t-test was used for statistical analysis. There was no difference (P > 0.05) between development rates for control, 10 μm and 40 μm forskolin (62%, 67% and 65%, respectively). Mean NRU by total cell numbers were calculated per embryo for each treatment group: control, 10 μm and 40 μm with the resulting means being 72.4 ± 5.0, 63.1 ± 5.1 and 66.6 ± 4.8 respectively. With treatment groups combined by embryo stage, compact morula tended to exhibit a higher mean NRU than blastocysts (78.6 ± 6.8 to 64.7 ± 3.1, respectively, P = 0.07). In conclusion, forskolin did not significantly affect embryo development rates or lipid content as measured by Nile Red staining. Compact morula tended to yield a higher NRU mean than blastocysts indicating an elevation in intracellular lipids. Unlike pig IVP embryos exposed to forskolin, forskolin failed to show a significant decrease in intracellular lipids within bovine IVP embryos. However, further research is in progress to test forskolin’s lipolytic effect on cryopreservation of in vitro and in vivo produced bovine embryos. The authors acknowledge support from the American Brahman Breeders Association and Dr. Moisés Barceló Fimbres.

scholarly journals A Shorter Equilibration Period Improves Post-Warming Outcomes after Vitrification and in Straw Dilution of In Vitro-Produced Bovine Embryos

Biology ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 142
Author(s):  
Iris Martínez-Rodero ◽  
Tania García-Martínez ◽  
Erika Alina Ordóñez-León ◽  
Meritxell Vendrell-Flotats ◽  
Carlos Olegario Hidalgo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Survival Rates ◽  
Bovine Embryos ◽  
Treatment Groups ◽  
Apoptosis Rate ◽  
Cell Counts ◽  
Hatching Rates

This study was designed to the optimize vitrification and in-straw warming protocol of in vitro-produced bovine embryos by comparing two different equilibration periods, short equilibrium (SE: 3 min) and long equilibrium (LE: 12 min). Outcomes recorded in vitrified day seven (D7) and day eight (D8) expanded blastocysts were survival and hatching rates, cell counts, apoptosis rate, and gene expression. While survival rates at 3 and 24 h post-warming were reduced (p < 0.05) after vitrification, the hatching rates of D7 embryos vitrified after SE were similar to the rates recorded in fresh non-vitrified blastocysts. The hatching rates of vitrified D8 blastocysts were lower (p < 0.05) than of fresh controls regardless of treatment. Total cell count, and inner cell mass and trophectoderm cell counts were similar in hatched D7 blastocysts vitrified after SE and fresh blastocysts, while vitrified D8 blastocysts yielded lower values regardless of treatment. The apoptosis rate was significantly higher in both treatment groups compared to fresh controls, although rates were lower for SE than LE. No differences emerged in BAX, AQP3, CX43, and IFNτ gene expression between the treatments, whereas a significantly greater abundance of BCL2L1 and SOD1 transcripts was observed in blastocysts vitrified after SE. A shorter equilibration vitrification protocol was found to improve post-warming outcomes and time efficiency after in-straw warming/dilution.

scholarly journals Effect of the MS Bacteriophage on STEC O157:H7 Populations in Beef

2019 ◽  
Vol 3 (2) ◽  
Author(s):  
S. T. Laidler ◽  
E. L. Shebs ◽  
F. M. Giotto ◽  
M. J. Lukov ◽  
A. S. De Mello
Keyword(s):  
Uv Light ◽  
Meat Products ◽  
Bacterial Attachment ◽  
Contamination Level ◽  
Treatment Groups ◽  
Randomized Design ◽  
Peptone Water ◽  
Completely Randomized Design ◽  
Commercial Applications

ObjectivesThis research was conducted to evaluate the killing efficiency of the Mello-Shebs (MS) O157:H7 bacteriophage against three strains of STEC O157:H7 in vitro and on beef surface under aerobic conditions.Materials and MethodsKilling efficiency of bacteriophage MS O157:H7 (University of Nevada, Reno– Meat Science library) was tested against three strains including ATCC® 43895 (stx1 and stx2 positive), ATCC® 43894 (stx1 and stx2 positive), and Micreos 128. To determine the efficiency of the phage in vitro, each strain of STEC O157:H7 was incubated at 37°C overnight and then diluted to achieve 1500 CFU/mL. Subsequently, 0.1 mL of this dilution for each strain was plated onto 1.6% LB agar plates. The experimental plates received 0.1 mL of phage solution at 108 PFU/ml, in quadruplicate. The plates were incubated at 37°C for 24 h and the resulting colonies were counted and compared to controls to determine killing efficiency of the phage against each STEC strain. To evaluate the effect of bacteriophage application on beef, m. cutaneous trunci (rose meat, IMPS 194) were sourced from a USDA inspected facility, fabricated into 100 cm2 and randomly assigned to either control or treatment groups. Samples acclimated to 7°C were inoculated with a STEC cocktail to result in a contamination level of approximately 3 log CFU/cm2 on meat surfaces after swabbing. After bacterial attachment for 30 min at 7°C, control samples were treated with sterile buffered peptone water (BPW) and experimental samples were treated with the phage solution at 108 PFU/ml. After dwelling for 1 h at 7°C, samples were swabbed and the resulting 1 mL was thoroughly vortexed, serial diluted, and plated onto LB plates. The plates were incubated at 37°C for 24 h and the resulting colonies were counted. Data was analyzed as a completely randomized design in SAS. Means were separated by LSMEANS and differences were indicated by using DIFF functions.ResultsIn vitro killing efficiency of bacteriophage MS O157:H7 was determined to be 85%, 98.89%, and 97.16% for ATCC® 43894, ATCC® 43895, and Micreos 128 strains, respectively. On beef, bacteriophage application significantly decreased STEC loads by 0.626 log CFU/cm2 (P = 0.0184, SEM = 0.21).ConclusionBacteriophage MS-O157:H7 applications as an antimicrobial on beef reduces STEC O157:H7 populations on contaminated beef. Commercial applications of this bacteriophage may improve STEC control in meat products, however, having this treatment combined with other interventions such as organic acids or UV light treatment may increase the killing efficiency of STEC populations, warranting further research to determine industry applicability.

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.

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