scholarly journals 153 EFFECTS ON SEX RATIO AND PREGNANCY RATES OF IN VIVO-DERIVED BOVINE EMBRYOS USING LOOP-MEDIATED ISOTHERMAL AMPLIFICATION SEXING METHOD

2005 ◽  
Vol 17 (2) ◽  
pp. 227
Author(s):  
A. Ideta ◽  
S. Iwasa ◽  
T. Takedomi ◽  
M. Urakawa ◽  
M. Konishi ◽  
...  
Keyword(s):  
Sex Ratio ◽  
Developmental Stages ◽  
Pregnancy Rates ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Bovine Embryos ◽  
Chi Square ◽  
Loop Mediated Isothermal Amplification ◽  
Frozen Embryos

In this study, we examined the effects of developmental stages and quality grades on sex ratio of in vivo-derived bovine embryos. Furthermore, pregnancy rates of fresh frozen-thawed sexed embryos or intact (non-sexed) fresh and frozen-thawed embryos were compared in order to efficiently carry out the sexing of embryos in the field. Embryos were collected from donors at 7 days after estrus following a routine superovulation protocol, and classified into four stages (late morula, early blastocyst, blastocyst, and expanded blastocyst) and two quality grades (Grade 1 and Grade 2–3) by the IETS manual. Embryos were frozen by direct transfer method from 1 to 3 h post-collection in 0.25-mL straws as described previously (Aoyagi et al. 1996 Theriogenology 45, 165 abst). Frozen embryos were thawed in 30°C water for 20 s following 7 s in air. They were then squeezed out into PBS + 5% FCS (PBS), washed twice, and incubated in CR1aa + 5% CS (CR1aa) or PBS. Recently, a commercial embryo sexing program was performed at our laboratory using loop-mediated isothermal amplification (LAMP). The procedure takes 5 min to perform each embryo biopsy and only 40 min for the LAMP process. A few cells of fresh (F; n = 105) and frozen-thawed (Z; n = 143) embryos of Grade 1 (H), and fresh (F; n = 77) embryos of Grade 2–3 (L) were biopsied with a microsurgical blade, and sex was determined by the LAMP method. Embryos were transferred non-surgically into heifers on Day 7 of the estrus cycle. Pregnancies were determined by ultrasonography on Day 30. Data were analyzed by the chi-square test. The sexing of all 325 embryos yielded 148 female (46%), and only 2 embryos were indeterminant (1%). There was no evidence of any effect of developmental stage on sex ratio (female embryos: late morula 69/157 (44%), early blastocyst 42/94 (45%), blastocyst 29/53 (55%), and expanded blastocyst 10/21 (48%)). However, when the sex ratio was examined for embryos of different quality grades, significantly more females were found in the embryos appearing more degenerated (female embryos: FH + ZH vs. FL; 42% vs. 57%, P < 0.05). Pregnancy rates on Day 30 with FH embryos (38/45, 84%) were similar to rates obtained with non-sexed fresh (60/81, 78%) and frozen-thawed embryos (44/54, 82%). The pregnancy rates on Day 30 with ZH embryos incubated in CR1aa (18/40, 45%) were lower than those of FH, non-sexed fresh, and frozen-thawed embryos. However, pregnancy rates of ZH embryos incubated in PBS (13/16, 81.3%) were significantly higher than for those frozen embryos that were thawed and incubated in CR1aa (P < 0.05). After the transfer of embryos sexed by the LAMP method to recipient animals, all 55 calves born were of the predicted sex. In conclusion, the present results showed that with the LAMP method for sexing of the embryos, there were only a few samples for which sex could not be determined. Examination of in vivo-derived Day 7 embryos indicated that female embryos graded lower than male embryos. Furthermore, the removal of a few cells from a fresh or frozen-thawed embryo did not impact its subsequent viability.

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

204 RELATIONSHIP BETWEEN RESPIRATORY ACTIVITY AND THE PREGNANCY RATE OF BISECTED BOVINE EMBRYOS IN VIVO

2007 ◽  
Vol 19 (1) ◽  
pp. 219 ◽  
Author(s):  
S. Moriyasu ◽  
H. Hirayama ◽  
K. Sawai ◽  
S. Kageyama ◽  
S. Aoyagi ◽  
...  
Keyword(s):  
Oxygen Consumption ◽  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Respiratory Activity ◽  
Pregnancy Rates ◽  
Bovine Embryos ◽  
Chi Square ◽  
Important Indicator ◽  
Consumption Rates

Oxygen consumption is an important indicator of the metabolic activity of living cells, which may provide valuable information for evaluating embryo quality. We have found that the bovine embryos with high oxygen consumption possess stronger potential for further development. However, the relationship between respiratory activity and the pregnancy rate of embryos is still unclear. In this study, we investigated the respiration rates of bisected bovine embryos and the pregnancy rates of demi-embryos after embryo transfer. Compact morula-stage embryos were bisected evenly by micro glass needle. One hundred bisected embryos were incubated for 24 h in embryo culture medium (IVD101; Research Institute for the Functional Peptides, Yamagata, Japan) at 39�C under 5% CO2, 5% O2, 90% N2. After the incubation, demi-embryos were classified into 2 groups: blastocoel-formed (BC) and blastocoel-not-formed (CM) embryos. Oxygen consumption rates of demi-embryos were measured by scanning electrochemical microscopy (SECM; Hokuto Denko Corporation, Tokyo, Japan). Within 3 h after the measurement, 80 demi-embryos were transferred into recipient cows (one demi-embryo/one recipient) at 7–8 days after estrus. Recipient cows were diagnosed for pregnancy by ultrasonography approximately 40 days after estrus. Statistical difference was analyzed by Tukey's post-hoc test and chi-square test. A total of 27 recipient cows became pregnant; the pregnancy rates for cows with CM and BC demi-embryos were 40.6% (13/32) and 29.2% (14/48), respectively. Mean oxygen consumption rates (� 10-14 mol s-1) in pregnant and non-pregnant cows were 0.47 and 0.39 for CM demi-embryos and 0.63 and 0.52 for BC demi-embryos, respectively. Retrospective analysis showed that the respiratory activity of demi-embryos in the pregnant group was higher than those in the non-pregnant group. In particular, the pregnancy rates for demi-embryos with respiratory activity higher than 0.35 in CM and 0.40 in BC groups were 52.0% (13/25) and 35.9% (14/39), respectively. On the other hand, cows with demi-embryos having an oxygen consumption rate under 0.35 in CM (n = 7) and 0.40 in BC (n = 9) groups did not become pregnant. These results demonstrated that bovine demi-embryos with higher respiratory activity showed a high pregnancy rate after embryo transfer. It is generally known that the pregnancy rate after the transfer of bisected embryos is lower than that of whole embryos. The measurement of oxygen consumption by SECM procedures is a useful tool to assess the quality of pre-implantation embryos and may contribute to the improvement of the success rate for bisected embryo transfer.

147 PREGNANCY RATES OF RECIPIENT ANIMALS FOLLOWING APPLICATION OF A SELECTIVE PROSTAGLANDIN F2α RECEPTOR ANTAGONIST DURING EMBRYO RECOVERY

2008 ◽  
Vol 20 (1) ◽  
pp. 154 ◽  
Author(s):  
F. N. Scenna ◽  
J. L. Edwards ◽  
G. M. Schuenemann ◽  
D. A. Roper ◽  
F. N. Schrick
Keyword(s):  
Embryo Development ◽  
Prostaglandin F2α ◽  
Pregnancy Rates ◽  
Double Blind Study ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Initial Experiment ◽  
Frozen Embryos ◽  
Cayman Chemical

Companion research presented at this meeting has indicated that addition of a prostaglandin2α (PGF2α) receptor (FPr) antagonist to culture medium prevented the detrimental action of PGF2α on embryo development. The aim of this study was to evaluate addition of an FPr antagonist to the collection medium on pregnancy rates after transfer of bovine embryos to recipient animals. An initial experiment was performed to determine in vitro development of in vivo-derived morula-stage frozen-thawed embryos cultured in KSOM-PVA medium with 1000 nm AL-8810 (Cayman Chemical Inc., Ann Arbor, MI, USA) (AL, n = 94), 1000 nm AL-8810 and 10 ng mL–1 PGF2α (Cayman Chemical Inc.) (AL+PGF, n = 94), 10 ng mL–1 PGF2α (PGF, n = 94), or serving as controls (CON, n = 91). Embryos remained in their treatment for a 30-h period until blastocyst development was recorded. In a subsequent experiment, embryos were recovered (n = 783) from superovulated donors on Day 7 after artificial insemination with medium containing 1000 nm AL-8810 (AL), 100 nM AL-8810 (AL100), or with vehicle (VEH: 1 mL DMSO; Sigma-Aldrich, St. Louis, MO, USA) in a double blind study. Following collection, embryos were classified by stage and quality, and then transferred fresh to recipients or frozen (ethylene glycol, direct transfer). Frozen embryos, following thawing, were transferred during the subsequent breeding period. Pregnancy rates were determined by ultrasonography (28–35 days post-transfer) and confirmed by calving date. Data were analyzed using the GLIMMIX procedure of SAS (SAS Institute, Inc., Cary, NC, USA). Results from the initial experiment indicated that culture of in vivo-derived bovine embryos in medium containing AL-8810 improved blastocyst development compared to PGF (58.5% v. 45.7%; P = 0.05). In addition, a strong tendency to increase embryo development was observed in AL+PGF compared to PGF treatment group (57% v. 45.7%; P = 0.07). Overall pregnancy rates of fresh and frozen embryos were increased in the AL and AL100 groups (55% and 58%, respectively) compared to VEH (43%; P = 0.009). Since AL treatments did not differ in pregnancy rates, subsequent analysis combined AL and AL100 data. Transfer of frozen embryos collected with medium containing AL-8810 (n = 238) increased pregnancy rates (AL, 45%) compared to embryos recovered without (n = 221) AL-8810 (VEH, 34%; P = 0.01). Transfer of fresh embryos collected with medium containing AL-8810 (n = 241) tended to have increased pregnancy rates (AL, 76%) compared to control (n = 83; VEH, 66%; P = 0.09). Although data collection continues, no abnormalities in calf health, birth weight, or weaning weight have been observed between any treatments. In conclusion, recovery of embryos with flushing medium containing an FPr antagonist improved pregnancy rates after transfer. Funding was provided by Ultimate Genetics and the Tennessee Agricultural Experiment Station for completion of these studies.

scholarly journals The effect of vitrification in open pulled straws on pregnancy rates after transfer of in vivoproduced bovine embryos

2012 ◽  
Vol 51 (No. 9) ◽  
pp. 454-460 ◽  
Author(s):  
M. Lopatarova ◽  
S. Cech ◽  
L. Holy ◽  
R. Dolezel
Keyword(s):  
Developmental Stage ◽  
Conventional Method ◽  
Developmental Stages ◽  
Pregnancy Rates ◽  
Control Group ◽  
Bovine Embryos ◽  
Group V ◽  
Freezing Method ◽  
Conventional Freezing

The aim of this study was to compare pregnancy rates after transfer of in vivo produced embryos cryopreserved using open pulled straw (OPS) vitrification (Group V) or conventional freezing method as a control (Group C). Bovine embryos (Day<sub>6.5&ndash;7.5</sub>) collected from superovulated cows were classified according to developmental stages and morphological qualities (Grade 1 and 2) before cryopreservation and they were transferred to synchronized heifers after thawing. Pregnancy rates after transfer of morulae, early blastocysts and expanded blastocysts in Group V compared to Group C (54.5%, 12/22 vs. 56.0%, 14/25; 53.3%, 16/30 vs. 58.1%, 18/31 and 57.7%, 15/26 vs. 48.3%, 14/29) were not different (P &gt; 0.05). Likewise, pregnancy rates after transfer of embryos of Grade 1 and 2 in Group V compared to Group C (55.1%, 43/78 vs. 54.1%, 46/85 and 36.4%, 12/33 vs. 32.9%, 23/70, respectively) were not different (P &gt; 0.05). The study demonstrated similar viability of embryos which were frozen by vitrification or conventional method irrespective of their quality and developmental stage after transfer into recipients.

Rapid and sensitive diagnosis of adenoviral keratoconjunctivitis by loop-mediated isothermal amplification (LAMP) method

2004 ◽  
Vol 28 (6) ◽  
pp. 445-450 ◽  
Author(s):  
Taketoshi Wakabayashi ◽  
Ryoko Yamashita ◽  
Tetsuhiko Kakita ◽  
Mito Kakita ◽  
Tetsuro Oshika
Keyword(s):  
Isothermal Amplification ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification

scholarly journals Loop-Mediated Isothermal Amplification for Detection ofStaphylococcus aureusin Dairy Cow Suffering from Mastitis

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Zhang Tie ◽  
Wang Chunguang ◽  
Wei Xiaoyuan ◽  
Zhao Xinghua ◽  
Zhong Xiuhui
Keyword(s):  
Staphylococcus Aureus ◽  
High Sensitivity ◽  
High Specificity ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Specific Primers ◽  
Loop Mediated Isothermal Amplification ◽  
Reaction Tube ◽  
Specificity And Sensitivity ◽  
Detectable Concentration

To develop a rapid detection method ofStaphylococcus aureususing loop-mediated isothermal amplification (LAMP), four specific primers were designed according to six distinct sequences of thenucgene. In addition, the specificity and sensitivity of LAMP were verified and compared with those of PCR. Results showed that the LAMP reaction was completed within 45 min at 62.5°C, and ladder bands were appeared in LAMP products analyzed by gel electrophoresis. After adding 1x SYBR Green l, the positive reaction tube showed green color and the negative reaction tube remained orange, indicating that the LAMP has high specificity. The minimal detectable concentration of LAMP was1×102 CFU/mL and that of PCR was1×104 CFU/mL, indicating that the LAMP was 100 times more sensitive than the PCR. The LAMP method for detection ofStaphylococcus aureushas many advantages, such as simple operation, high sensitivity, high specificity, and rapid analysis. Therefore, this method is more suitable for the rapid on-site detection ofStaphylococcus aureus.

scholarly journals Rapid identification of transgenic cotton (Gossypium hirsutum L.) plants by loop-mediated isothermal amplification

2011 ◽  
Vol 47 (No. 4) ◽  
pp. 140-148 ◽  
Author(s):  
N. Rostamkhani ◽  
A. Haghnazari ◽  
M. Tohidfar ◽  
A. Moradi
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lamp Assay ◽  
Transgenic Cotton ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Detection Sensitivity ◽  
Lamp Method ◽  
Loop Mediated Isothermal Amplification ◽  
Leaf Tissues

In an attempt to speed up the process of screening of transgenic cotton (G. hirsutum L.) plants, a visual and rapid loop-mediated isothermal amplification (LAMP) assay was adopted. Genomic DNA was extracted from fresh leaf tissues of T<sub>2</sub> transgenic cotton containing chitinase (chi) and cry1A(b) genes. Detection of genes of interest was performed by polymerase chain reaction (PCR), LAMP and real-time PCR methods. In LAMP assay the amplification was performed after 30 min at 65&deg;C when loop primers were involved in the reaction. The involvement of loop primers decreased the time needed for amplification. By testing serial tenfold dilutions (10<sup>&ndash;1</sup> to 10<sup>&ndash;8</sup>) of the genes of interest, the detection sensitivity of LAMP was found to be 100-fold higher than that of PCR. The rapid DNA extraction method and LAMP assay can be performed within 30 min and the derived LAMP products can be directly observed as visually detectable based on turbidity in the reaction tube. The accuracy of LAMP method in the screening of transgenes was confirmed by PCR and real-time PCR. The developed method was efficient, rapid and sensitive in the screening of cotton transgenic plants. This method can be applied to any other crops.

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