153 EFFECT OF DIFFERENT HOLDING AND TRANSPORT MEDIA ON CONCEPTION RATES FOLLOWING TRANSFER OF IN VIVO AND IN VITRO FERTILIZATION-DERIVED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 180
Author(s):  
C. A. Zanenga ◽  
C. M. Martins ◽  
N. C. Rodovalho ◽  
F. Aidar ◽  
J. F. Hasler ◽  
...  
Keyword(s):  
Animal Health ◽  
Conception Rate ◽  
Bovine Embryos ◽  
Chi Square ◽  
Mato Grosso ◽  
Conception Rates ◽  
Chi Square Test ◽  
Donor Cows

Two experiments were conducted to compare conception rates following embryo transfer (ET) of bovine embryos held and transported in Syngro® holding medium (Bioniche, Belleville, Ontario, Canada) with other 2 holding media: Emcare® (ICPbio, Auckland, New Zealand) for in vivo-derived embryos and HEPES-buffered synthetic oviduct fluid (H-SOF) for IVF-derived embryos. The first trial was performed in the period from October through December 2006 at the Curitiba farm in Poços de Caldas, Minas Gerais, Brazil. A total of 140 in vivo-derived embryos were produced from 20 Nelore donor cows and transferred fresh at the same farm. After each donor recovery, embryos were equally separated per stage (morula or blastocyst) and classification (grades 1, 2, and 3) into 2 Petri dishes, each containing either Syngro or Emcare. The embryos were held for an average of 3 h after recovery, loaded into 0.25-mL straws, and transferred fresh into recipients heifers, which were all previously synchronized with the same hormonal protocol treatment and presented a corpus luteum on the day of transference. Conception rate was checked at approximately 60 days of conception by rectal palpation. The chi-square test was used for statistical analysis. The conception rate of embryos maintained in Syngro was significantly higher than those in Emcare: 64.2% (43/67) v. 47.9% (35/73; P < 0.05). A second experiment was performed between September and December 2008 at Embriza Biotechnology Laboratory, Campo Grande, Mato Grosso do Sul, Brazil. A total of 1689 IVF-derived embryos (stage = 7, quality = 1), produced from Nelore donor cows, were randomly assigned to be held and transported in either Syngro (769) or H-SOF transport medium (920). Transportation time ranged from 1 to 9 h, and the recipient farms ranged from 100 to 1200 km in distance from the Embriza Laboratory. Crossbred recipient heifers (Bos taurus × Bos indicus) were synchronized with prostaglandin or vaginal progesterone device protocols. Pregnancy diagnosis was performed by ultrasonography approximately 60 days after ET. Statistical comparisons were performed using the chi-square test. Conception rates resulting from embryos transported in Syngro (45.1%, 347/769) and in H-SOF (42.0%, 386/920) were not different (P = 0.19). Financial support from Embriza Biotecnology, Tecnopec LTDA, and Bioniche Animal Health

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

134 CONCEPTION RATES OF IN VITRO-PRODUCED BOVINE EMBRYOS CRYOPRESERVED IN 6% GLYCEROL AND TRANSFERRED BY THE DIRECT METHOD

2007 ◽  
Vol 19 (1) ◽  
pp. 184
Author(s):  
N. Takada ◽  
S. Hayasaka ◽  
K. Chiba
Keyword(s):  
Ethylene Glycol ◽  
Conception Rate ◽  
Hatching Rate ◽  
Glycerol Solution ◽  
Direct Transfer ◽  
Bovine Embryos ◽  
Conception Rates ◽  
Chi Square Analysis

Ethylene glycol has been used as the standard cryoprotectant for direct transfer of bovine embryos due to its high permeability. But Merton et al. reported that cryoprotectivity of glycerol for bovine embryos was superior to that of ethylene glycol (2001 Theriogenology 55, 312 abst). We previously reported that nonsurgical transfer of in vivo-derived bovine embryos cryopreserved in a lower concentration (5%) of glycerol and thawed by stepwise method resulted in a 55.4% conception rate, whereas direct transfer without removal of cryoprotectant showed only a 45.1% conception rate (Takada et al. 2005 Jpn. J. Embryo Transfer 27, 59–64). In this experiment, survival and conception rates of in vitro-produced (IVP) bovine embryos cryopreserved in 6% glycerol solution (GLY) were compared to those of embryos cryopreserved in 10% ethylene glycol plus 0.1 M sucrose solution (EG). Cumulus–oocyte complexes were matured and fertilized according to Numabe et al. (2000 Theriogenology 54, 1409–1420). Presumed zygotes were cultured in mSOF supplemented with 5% calf serum (CS) and 0.25% linoleic acid albumin at 38.5�C under 5% CO2, 5% O2, 90% N2 for 7 days. At the expanded blastocyst stage, embryos were placed in GLY or EG in PBS supplemented with 20% CS for 15 min at room temperature and loaded into 0.25-mL straws. Straws were placed directly into an alcohol freezer. When the cryoprotectant was GLY, straws were seeded at -4.0�C, held for 10 min, cooled at 0.5�C min to -30.5�C, and then plunged into liquid nitrogen. When the cryoprotectant was EG, the seeding point was -7.5�C, and the plunging point was -34.0�C, but the rest of the protocol was the same as for GLY. In Exp. 1, thawing in both groups was done in a 30�C water bath, and the contents were directly rehydrated in PBS with 20% CS. Thawed embryos were cultured in mSOF with 5% CS for 24 h to assess embryo survival rate, based on the re-expansion of the blastcoele and on their hatching ability. In Exp. 2, embryos in both groups were thawed and transferred to synchronous recipients without removing the cryoprotectant. Data were analyzed using chi-square analysis. In Exp. 1, the developmental rates of post-thaw embryos were similar in GLY (46/52, 88.5%) and EG (45/52, 86.5%); however, the hatching rate was significantly higher (P &lt; 0.05) in embryos cryopreserved in EG (26/52, 50.0%) than in GLY (15/52, 28.8%). In Exp. 2, the conception rates of embryos were similar in both groups, GLY (7/15, 46.7%) and EG (6/15, 40.0%). In conclusion, after direct rehydration of embryos, the developmental ability of IVP bovine embryos cryopreserved in EG was superior to that of embryos cryopreserved in GLY in vitro. However, conception rates in vivo were similar in both groups. These results suggest that a lower concentration of glycerol might be still useful as a cryoprotectant for direct transfer of IVP bovine embryos.

23 EFFECTS OF THE NUMBER OF SERVICES FERTILITY IN DAIRY COWS

2011 ◽  
Vol 23 (1) ◽  
pp. 118 ◽  
Author(s):  
M. Yamaguchi ◽  
M. Tanisawa ◽  
H. Koyama ◽  
S. Takahashi ◽  
O. Dochi
Keyword(s):  
Dairy Cows ◽  
Conception Rate ◽  
Low Fertility ◽  
Chi Square ◽  
The Third ◽  
Total Percentage ◽  
Conception Rates ◽  
Service Conception ◽  
Chi Square Test ◽  
Rectal Palpation

In recent years, reproductive performance of dairy cows has been declining worldwide, especially among cows of high genetic merit for milk production. The cause of the low fertility may considerably vary across countries and is probably multifactorial. This problem remains unsolved. Further, the first-service conception rate of dairy cows has remarkably decreased worldwide. The number of services required for conception has increased due to the low fertility in dairy cows. However, there are few reports about the relationship between conception rates and the number of services in the current dairy cows. The objective of this study was to investigate whether the number of services affects the conception rates of dairy cows. Data concerning the conception rates was obtained for 8386 Holstein cows from 40 commercial dairy herds in eastern Hokkaido, Japan, from 2006 to 2009. The diagnosis of pregnancy was confirmed by rectal palpation between 30 and 45 days after insemination. The average interval between calving and the first-service was 87.3 days. The average milk yield was 8500 kg. Number of conducting services was from the first to the seventh service. The conception rate was analysed using chi-square test. The results are presented in Table1. The conception rate from the first to the seventh service was 40.2 to 54.0%. The conception rate at the first service was significantly low (40.2%); however, the conception rates did not significantly differ after the second-service. A similar tendency was observed during each year. Moreover, the total percentage of conceptions from the first to the third service was 83.8%. The average number of services per conception was 2.2. The results of this study indicate that the first service yielded the lowest conception rates. The conception rates after the second service did not significantly differ. Moreover, ∼84% of the dairy cows became pregnant between the first service and the third service. However, this result shows that ∼16% of the dairy cows in the herds were repeat breeding. In order to improve fertility, it is necessary to study the factors that affect the first-service conception rates of dairy cows. Moreover, to improve the conception rate of dairy cows, it is important to elucidate the cause of these problems. Table 1.The number and percentage of dairy cows conceiving at each of the services for consecutive services

72 USE OF C57BL/6/EGFP MOUSE TESTICULAR CELLS TO TRAIN PERIVITELLINE SPACE MICROINJECTION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
D. M. de Souza ◽  
H. Fernandes ◽  
P. V. Silva ◽  
B. Cazari ◽  
P. D. Moço ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Training Model ◽  
Perivitelline Space ◽  
Chi Square ◽  
Testicular Cells ◽  
Chi Square Test ◽  
Cellular Components

The production of embryonic chimeras has been studied as a tool for in vivo pluripotency validation in embryonic stem cells (ESC) as well as to produce transgenic mice. Among the techniques to produce chimeras, one of the most used is microinjection (MI) of ESC into blastocysts or in the perivitelline space (PVS) of the embryos with 4 to 8 cells. A well-established training model for this technique could be very useful when ESC are not available, in which injected cells could be easily identified and their subsequent fate could be tracked. Hence, we aimed to test, in mice, a training model for MI in embryos (Swiss Webster, SW) using a pool of EGFP cells derived from testes of the C57BL/6/EGFP strain. Embryos were recovered from prepubertal female SW (n = 20), superstimulated and mated according to a previously described treatment. The MI was performed in the PVS of 4- to 8-cell embryos (collected at 2.5 dpc). When possible, embryos from the same female were randomly allocated to 3 groups: control (C, n = 17), embryos not subjected to MI; perforated (P, n = 15), embryos submitted to perforation by micropipette, without cell injection; and microinjected (MI, n = 32), embryos perforated and submitted to PVS injection with 6 to 8 cells from EGFP testes. After manipulation, embryos from all groups underwent 24 h of in vitro culture (37°C, 5% CO2 and saturated humidity). The viability and quality of the embryos (according to the IETS Manual 1998) and, in group MI, the fluorescence of testicular cells, were evaluated pre- and post-culture. The results were analysed by chi-square test (total frequency observed) and ANOVA (considering the four replicates) with significance being considered when P < 0.05. There was no difference among mortality rates [i.e. % of viable embryos that died after 24 h of culture, of the groups (5.9, 26.7 and 25.0% for C, P and MI, respectively]. The percentage of embryos that maintained or improved quality after 24 h of culture, in comparison with quality evaluation pre-culture, was different (P < 0.01) among groups C, P and MI (94.1, 73.3 and 43.8%, respectively). One chimeric blastocyst was obtained in the MI group (3.1%, 1/32). Considering the proposed conditions, this model for training of MI of EGFP testicular cells in the PVS was feasible and practical to acquire skills, when ESC are not available. Moreover, the method allows easy identification of injected and, eventually, aggregated cellular components. Financial support was received from FAPESP of Brazil.

71 EFFECT OF CELL QUANTITY IN MICE CHIMERA PRODUCTION BY DEMI-BLASTOCYST AGGREGATION

2012 ◽  
Vol 24 (1) ◽  
pp. 148
Author(s):  
C. Pontes Godoi ◽  
P. D. Moço ◽  
B. Cazari ◽  
P. T. Mihara ◽  
P. V. Silva ◽  
...  
Keyword(s):  
Cell Mass ◽  
Farm Animals ◽  
Control Group ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Chi Square Test ◽  
Aggregation Technique

Eight-cell-stage to pre-compaction morula are the most used embryonic stages to aggregation, because the embryos, in these early stages, synthesise cell adhesion molecules that increase the aggregation chances among them (Vestweber et al. 1987 Develop. Biol. 124, 451–456). Although post-compaction embryos produce reduced aggregation rates, they are not refractory to this process (Nogueira et al. 2010 Transgenic Res. 19, 344–345). Based on the evidence of less permissive aggregation in post-compaction-stage embryos and the need to expose the inner surface of those embryos to improve aggregation rate, the aim of this study was to evaluate, in mice, the influence of cell quantity (i.e. the quantity of half-embryos put together to aggregate themselves) in the chimerism rate of split blastocysts. Embryos, with preferentially different phenotypes, were obtained from C57BL/6/EGFP and Swiss Webster strains. Females ranging from 21 to 45 days old were superstimulated and mated according to Mancini et al. (2008 Transgenic Res. 17, 1015). Eight-cell-stage embryos (8C) and pre-compaction morula (PCM) were recovered (2 to 2.5 days post coitum) and had their zona pellucida removed using pronase treatment (2 mg mL–1 for 15 min), whereas blastocysts (recovered 3.5 dpc) were split with a microblade controlled by micromanipulator in an inverted microscope (NK2; Eppendorf, Hamburg, Germany and Eclipse Ti; Nikon, Tokyo, Japan, respectively). The aggregation groups were a control (C) with 2 pre-compaction whole embryos (8C or PCM, or both) and 2 experimental with post-compaction embryos [i.e. 2 (2DB) or 4 (4DB) demi-blastocysts]. The structures (2 or 4) of the groups were stuck to each other with the use of phytohemagglutinin (1 mg mL–1) and cultured in vitro by 24 h (37°C, 5% CO2 and saturated humidity). After culture, the presence of chimeric embryos was verified by detection of a single, cohesive cell mass or a structure in an 8 shape with more than one-half of its total diameter aggregated. For the 4DB group, a successful aggregation was considered when, at least 2 of 4 DB had aggregated. The results were analysed using chi-square test, Fisher's exact test and Kruskal-Wallis (to compare among groups, between groups and among medians of group replicates, respectively) and significance was considered when P < 0.05. The aggregation rates for the groups C, 2DB and 4DB were, respectively, 77.3a; 8.3b and 36.4%c (P < 0.001). The increasing of the aggregation technique efficacy, in post-compaction stages, would be particularly interesting in farm animals (e.g. bovine species), where it is not feasible to obtain, in vivo, pre-compaction stages embryos (as 8 cells) and when only trophectoderm aggregation is wanted. It was concluded that cell increasing (from 2 to 4 DB) improved the chimerism rate, but not enough to be similar to the control group. Supported by FAPESP of Brazil.

86 Difficulty of Transfer of In Vivo-Derived Bovine Embryos and Route of Administration of Flunixin Meglumine at the Time of Transfer may Affect Pregnancy Rate

2018 ◽  
Vol 30 (1) ◽  
pp. 182
Author(s):  
J. Duran ◽  
D. Argudo ◽  
S. Bravo ◽  
C. Soria ◽  
G. Guevara ◽  
...  
Keyword(s):  
Embryo Transfer ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Prostaglandin F2α ◽  
Bovine Embryos ◽  
Chi Square ◽  
Evaluation Systems ◽  
Flunixin Meglumine ◽  
Chi Square Test

Recipient handling during embryo transfer (ET) induces prostaglandin F2α (PGF2α) production in 2 periods: an early transient and rapid increase around the time of ET, followed by another 2 to 4 h later. This PGF2α is associated with embryonic loss during early gestation by affecting both the embryo and the corpus luteum. To control this, antiprostaglandins such as flunixin meglumine (FM) have been applied IM at the time of ET with varying results. In such studies, the interaction of IM administration of FM and difficulty of transfer has not always been evaluated, possibly confusing the interpretation of the results. Furthermore, IV FM injection at ET and its relationship with pregnancy rates (PR) has not been determined. The objectives were (1) to determine the relationship between difficulty of ET and PR; and (2) to evaluate the efficacy of IM v. IV FM on pregnancy outcomes. One hundred and ten crossbred (Bos taurus × Bos indicus) heifers (18-24 months old) from 3 farms were used as recipients. Two evaluation systems of ET difficulty were used: (1) duration of transfer (objective determination of the elapsed time measured in seconds between the introduction of the catheter and embryo release), and (2) level of difficulty experienced by the practitioner (subjective determination; 1 = minimum and 2 = medium to extreme manipulation). Quality 1 and 2 fresh embryos from superovulated cows were transferred by the same practitioner. At ET, recipients were randomly divided into 3 groups: (1) Control (no treatment, n = 31); (2) FM-IM (n = 39): injected IM with 2.2 mg kg−1 FM at ET; and (3) FM-IV (N = 40): injected with 2.2 mg kg−1 FM IV at ET. Pregnancy was diagnosed at 30 to 40 and 60 to 90 days after ET. Spearman’s test was performed to determine the correlation between duration and difficulty at ET and Chi-square test was used to compare PR. The mean duration of transfer for all heifers was 62.3 ± 57.5 s (11 to 357 s; median: 44.5 s). There was a high correlation (0.8; P < 0.001) between the ET difficulty evaluation systems. Overall, ET difficulty 1 had higher PR than ET difficulty 2 (64.2 v. 40.7; P = 0.013). The PR was significantly improved (P < 0.01) in the FM-IV group (75 and 70% at 30 and 60 days after ET) compared with control (45.2 and 32.3%) and FM-IM (33.3 and 30.7%). In conclusion, results indicate that the difficulty of transfer affects PR achieved following the transfer of in vivo-derived bovine embryos. Treatment with FM-IV following transfer resulted in significantly higher PR compared with control and FM-IM recipients. The IV injection of FM may antagonize the very early and transient increase of PGF2α caused by genital tract manipulation (even gently performed) at embryo transfer. Further research is necessary to confirm the results of the present study.

363 FACTORS INFLUENCING PREGNANCY RATES FOLLOWING TRANSFER OF BOVINE IN VIVO EMBRYOS BIOPSIED FOR SEX DETERMINATION

2007 ◽  
Vol 19 (1) ◽  
pp. 297
Author(s):  
S. Li ◽  
W. Yu ◽  
J. Fu ◽  
Y. Bai ◽  
F. Jin ◽  
...  
Keyword(s):  
Pregnancy Rate ◽  
Embryo Transfer ◽  
Pregnancy Rates ◽  
Chi Square ◽  
Embryo Survival ◽  
Chi Square Test ◽  
First Time ◽  
Fresh Embryos

Data collected from commercial embryo transfer programs in 63 farms in China during June 2002 to December 2005 was analyzed to examine the effects of various factors (biopsy, freezing, sample size, embryo development and quality, in vitro culture, and recipient quality) on pregnancy rates of in vivo-biopsied embryos. Embryos were flushed from superovulated dairy cattle and subjected to a biopsy for sexing determination using protocols and sexing kits supplied by AB Technology Ltd. Fresh embryos were implanted on the same day or frozen with AG freeze medium (AB Technology Ltd., Pullman, WA, USA) for later transfer. Recipients were synchronized with CIDA + PG protocols. Embryos were cultured in 6-well dishes containing 1.3 mL of holding medium (AB Technology Ltd.) in each well at room temperature (20–25�C) for examination of embryo survival in vitro. The chi-square test was used in statistic analysis. The implantation of fresh embryos after biopsy did not affect pregnancy rates (49.6%, 257/518) compared to that of non-biopsied fresh and frozen–thawed embryo groups (52.9%, 47/140 and 46.6%, 177/380, respectively). However, for biopsied embryos subjected to frozen and thawed procedures before implantation, particularly for those subjected to the removal of a larger biopsy, a reduced pregnancy rate was observed (41.8%, 297/710; P &lt; 0.01). Pregnancy rates among biopsied embryos at 3 different development stages (morula-early blastocyst, blastocyst, and expanded blastocyst) were not different. Similar results were found between embryo groups of grade 1 and 2. A significant decrease in pregnancy rate (0/10) was observed with embryos held in vitro for a longer period of time (&gt;5 h), suggesting detrimental effects of in vitro conditions on embryo survival. The highest pregnancy rate (68.0%) was observed in recipients synchronized for the first time before being implanted with biopsied embryos. Significant decreases in such rates were found in recipients synchronized for the second or third times or those with an abortion history at the first or second synchronization-implantation treatment (P &lt; 0.01). Better pregnancy rates (45.6%, 41/90; 46.1%, 76/165; and 45.5%, 5/11) were obtained for recipients implanted with biopsied embryos at Days 7.5, 8.0, and 8.5 post-heat detection, respectively, compared to 16% at Day 7 (3/18, P &lt; 0.05). It is concluded that mechanical treatment (cutting) does not reduce the survival of biopsied embryos; however, cryopreservation reduces their ability to survive in vivo. The analyses also suggest that holding embryos in vitro should not be longer than 5 h unless more favorable in vitro conditions can be provided. To achieve better results of implantation of biopsied embryos, embryo transfer should be performed during 7.5–8.5 days post-estrus, and the healthy recipients synchronized for the first time should be used.

scholarly journals 194THE EFFECT OF OVIDUCTAL INCUBATION OF OOCYTES AND EMBRYOS ON ADHERENCE OF BOVINE VIRAL DIARRHEA VIRUS

2004 ◽  
Vol 16 (2) ◽  
pp. 218
Author(s):  
A. Bielanski ◽  
C.L. Lutze-Wallace ◽  
S. Nadin-Davis
Keyword(s):  
Embryonic Cells ◽  
Chi Square ◽  
Diarrhea Virus ◽  
Proteolytic Resistance ◽  
Chi Square Test ◽  
Pathogenic Agents ◽  
Induced Changes ◽  
Viral Diarrhea Virus

The zona pellucida (ZP) plays a major role as a protective shell against infection of embryonic cells and as a carrier of infectious agents in the spread of livestock diseases through embryo transfer practices. It has been demonstrated that pathogenic agents are more likely to adhere to the surface of ZP of IVF embryos than to that of in vivo fertilized embryos. It has been suggested that divergent conditions for the production of these two types of embryos may lead to changes in their morphology and to differences in the interaction of the ZP with pathogens. The objective of this study was to investigate whether or not experimentally induced changes in hardening ZP (HZP) affect adherence of BVDV to the ZP of oocytes and embryos produced in vitro. To induce HZP, the oocytes or IVF embryos in groups of 30–50 were incubated in the ligated, ampullar part of the oviduct at 38°C in 5%CO2, 5%O2 and 90% nitrogen for 5h. Following incubation, a proportion of oocytes/embryos was exposed to 1% pronase to determine HZP (the time of ZP lysis), while the remaining oocytes and embryos were incubated with 106 TCID50/mL of either noncytopathic (NY-1) or cytopathic (NADL) strain of BVDV at 38°C for 3h. Subsequently, oocytes and embryos were washed according to the method recommended by IETS and then tested for the presence of BVDV (virus isolation and PCR tests). At the end of experiments the oviductal tissues were tested by PCR and proven free of BVDV. For immature and matured oocytes and embryos not exposed to the oviduct, the ZP dissolution times were 3.6±0.24 (mean±SEM, n=20), 3.8±0.24, and 4.0±1.24min, respectively (Chi-square test; P&gt;0.05). Corresponding times for those incubated in the oviduct were 393±47, 431.0±50, and 467±61min, respectively (P&gt;0.05). There was no difference between the number of virus-positive oocytes and embryos (n=965 in 193 samples) following experimental exposure to BVDV regardless of whether or not they were previously incubated in the oviduct (P&gt;0.05). Lower, but not significant, differences in percentages of samples associated with the infectious cytopathic strain of BVDV as compared to a noncytopathic strain were detected (P&gt;0.05). It was concluded that the modification in proteolytic resistance properties of ZP during in vitro oviductal incubation did not influence the adherence of BVDV to ZP of oocytes or IVF embryos. Further studies are warranted to determine why IVF embryos are more prone to the adherence of pathogenic agents than are in vivo fertilized embryos.

106 EFFECT OF GLYCEROL AND ETHYLENE GLYCOL ON THE DEVELOPMENT OF IN VITRO BOVINE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 161
Author(s):  
A. C. Nicacio ◽  
R. Simões ◽  
M. A. Peres ◽  
J. S. A. Gonçalves ◽  
M. E. O. D'Ávila Assumpção ◽  
...  
Keyword(s):  
Ethylene Glycol ◽  
Cell Monolayer ◽  
Control Group ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Embryo Viability ◽  
Chi Square ◽  
Chi Square Test ◽  
Hatching Rates

The aim of this study was to evaluate the viability of in vitro-produced bovine embryos after exposure to different cryoprotectant solutions and cryopreservation. Bovine ovaries were collected at slaughterhouse and oocytes were matured, fertilized, and cultured in vitro. The embryos were co-cultured on a granulosa cell monolayer in SOF + 5% FCS and nonessential amino acids. In Experiment 1, expanded blastocysts were exposed to 10% ethylene glycol (EG) solution for 10 min (Group EG) or to 10% EG solution for 10 min and to 20% EG + 20% glycerol (Gly) solution for 30 s (Group EG/Gly). Cryoprotectants were diluted with PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min, and the hatching rate was evaluated after culture. In Experiment 2, after exposure, EG Group was cryopreserved by slow freezing procedure (1.2�C/min) and EG/Gly Group was vitrified on nitrogen vapor for 2 min. After thawing, cryoprotectants were diluted using PBS + 0.2% BSA + 0.3 M sucrose and PBS + 0.2% BSA solutions, both for 3 min; hatching rate was evaluated after culture. As a control group for both experiments, non exposed embryos were cultured and evaluated for hatching rate. In Experiment 1, the hatching rates were 59.72% (43/72) for control, 62.38% (63/101) for EG, and 69.00% (69/100) for EG/Gly groups. In Experiment 2, hatching rates were 59.72% (43/72) for control, 15.22% (7/46) for EG, and 0.00% (0/46) for EG/Gly groups. Results were analyzed by chi-square test. In Experiment 1, no differences were observed among groups (P > 0.05) and in Experiment 2, differences were observed among control, EG, and EG/Gly groups (P < 0.05). In conclusion, the cryoprotectants were not deleterious to the development of in vitro bovine embryos until hatching, but the cryopreservation procedures decreased embryo viability. This work was supported by FAPESP 04/05335-1.

153 IN VITRO CULTIVATION OF CAPRINE EMBRYOS PRODUCED IN VIVO

2010 ◽  
Vol 22 (1) ◽  
pp. 235
Author(s):  
A. C. Martinez ◽  
R. C. M. Tramontini ◽  
M. A. Saito Jr ◽  
C. O. Abreu ◽  
C. R. Alcalde
Keyword(s):  
Culture Media ◽  
Animal Health ◽  
Poor Quality ◽  
In Vitro Cultivation ◽  
Chi Square ◽  
Fetal Serum ◽  
Bovine Fetal Serum ◽  
Grade Iii

Independent of embryo stage, when grade III embryos are transferred, they usually provide poor pregnancy rates. The aim of this study was to compare the effect of different in vitro cultivation media on the development of in vivo derived grade III caprine embryos. Twelve adult crossbreed goats from the Animal Breeding and Reproduction Laboratory of Maringá State University were used for this experiment. The goats were not pregnant or lactating. After embryo collection, embryos were classified regarding their development and quality, according to the International Embryo Transfer Society manual. Morulae classified as grade III were used in this experiment. The embryos were cultivated in 2 different media: Holding Plus medium (Bioniche Animal Health, Belleville, Ontario, Canada) or PBS plus 10% of bovine fetal serum (Nutricell, Campinas, Brazil) in 38.5°C waterbath with water circulation for 24 hours. After 24 h, the embryos were morphologically assessed. The percentage of embryos that developed during the cultivation period was calculated to evaluate the effectiveness of culture media. The embryos that produced new cell divisions and changed from grade III (poor quality) to grade II or grade I (excellent or good) were considered developed embryos. Chi-square was used to determine statistical differences between media. In the present work, the rate of embryos that developed in Holding Plus medium (Bioniche Animal Health) was 75%, and with PBS plus 10% bovine fetal serum was 40% of the total cultivated embryos. Table 1.Embryo development assessment after in vitro cultivation process

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