Effect of pFSH dose reduction on in vivo embryo production in Dorper ewes

To evaluate the effect of pFSH dose on the in vivo embryo production of Dorper ewes in the semi-arid northeast of Brazil, 40 sheep females were distributed into two groups of 20 animals that received intravaginal CIDR for 14 days, and two days before device removal, they received one of the following treatments: in the FSH200 group, the ewes received 200 mg of pFSH; and in the FSH128 group, the ewes received a total of 128 mg in decreasing doses every 12 h. Beginning 12 h after the conclusion of the treatments, estrus detection was performed every four hours using two Dorper rams of proven fertility. The ewes were mated at estrus onset and 24 hours later. Seven days after intravaginal device removal, the superovulatory response was evaluated, and embryo collection was performed using the laparotomy method. The recovered flushings were subjected to embryo searches under a stereomicroscope and classified according to their qualities. Analyses of variance (ANOVAs) and LSD tests were used to compare the different parameters. The data expressed as percentages were analysed by chi-square test. The ovulation rate was higher in the FSH200 group, which had 16.3 ± 0.3 corpora lutea (CL), than in the FSH128 group, which had 11.3 ± 0.3 CL (P<0.05). However, higher fertilization rate (83.6% vs. 62.4%) and higher transferable (86.0% vs. 71.6%) and freezable (67.9% vs. 40.8%) embryo rates were observed in the FSH 128 group compared with the group that received 200 mg. Furthermore, no significant differences in the remaining parameters were observed between the experimental groups (P>0.05), demonstrating that pFSH dose reduction promoted a greater production of freezable and transferable embryos in Dorper ewes subjected to MOET.

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Effect of pFSH dose reduction on in vivo embryo production in Dorper ewes

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6sup2p4215 ◽

2015 ◽

Vol 36 (6Supl2) ◽

pp. 4215

Author(s):

João Bosco Loiola Filho ◽

Alane Pains Oliveira do Monte ◽

Thais Thatiane Dos Santos Souza ◽

Mayara De Souza Miranda ◽

Lívia Correia Magalhães ◽

...

Keyword(s):

Dose Reduction ◽

Fertilization Rate ◽

Corpora Lutea ◽

Device Removal ◽

Embryo Production ◽

Chi Square ◽

Estrus Detection ◽

Chi Square Test ◽

Intravaginal Device

To evaluate the effect of pFSH dose on the in vivo embryo production of Dorper ewes in the semi-arid northeast of Brazil, 40 sheep females were distributed into two groups of 20 animals that received intravaginal CIDR for 14 days, and two days before device removal, they received one of the following treatments: in the FSH200 group, the ewes received 200 mg of pFSH; and in the FSH128 group, the ewes received a total of 128 mg in decreasing doses every 12 h. Beginning 12 h after the conclusion of the treatments, estrus detection was performed every four hours using two Dorper rams of proven fertility. The ewes were mated at estrus onset and 24 hours later. Seven days after intravaginal device removal, the superovulatory response was evaluated, and embryo collection was performed using the laparotomy method. The recovered flushings were subjected to embryo searches under a stereomicroscope and classified according to their qualities. Analyses of variance (ANOVAs) and LSD tests were used to compare the different parameters. The data expressed as percentages were analysed by chi-square test. The ovulation rate was higher in the FSH200 group, which had 16.3 ± 0.3 corpora lutea (CL), than in the FSH128 group, which had 11.3 ± 0.3 CL (P<0.05). However, higher fertilization rate (83.6% vs. 62.4%) and higher transferable (86.0% vs. 71.6%) and freezable (67.9% vs. 40.8%) embryo rates were observed in the FSH 128 group compared with the group that received 200 mg. Furthermore, no significant differences in the remaining parameters were observed between the experimental groups (P>0.05), demonstrating that pFSH dose reduction promoted a greater production of freezable and transferable embryos in Dorper ewes subjected to MOET.

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11 TIME OF INSEMINATION RELATIVE TO OVULATION EXPLAINS FERTILITY VARIATIONS OFFROZEN - THAWED SPERMATOZOA BETWEEN FARMS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab11 ◽

2005 ◽

Vol 17 (2) ◽

pp. 155 ◽

Cited By ~ 1

Author(s):

A. Bolarín ◽

G. Carvajal ◽

M. Hernandez ◽

J.M. Vazquez ◽

E.A. Martinez ◽

...

Keyword(s):

Litter Size ◽

Intrauterine Insemination ◽

Female Genital Tract ◽

Egg Yolk ◽

Corpora Lutea ◽

Chi Square ◽

Limited Life ◽

Estrus Detection ◽

Chi Square Test ◽

Female Genital

Swine fertility after AI with frozen-thawed spermatozoa varies between trials. As thawed spermatozoa have an extremely limited life span in the female genital tract, fertility of frozen-thawed spermatozoa depends mainly on the time of insemination relative to ovulation. The objective of this study was to evaluate whether the time of insemination relative to ovulation could explain the farm differences in fertility when frozen-thawed spermatozoa are used. Pooled sperm-rich fractions collected from three mature Pietrain boars were diluted in lactose/egg-yolk/glicerol/Orvus-ES-Paste extender, loaded in 0.5-mL straws (1 × 109 cells/mL), and frozen under controlled conditions (Carvajal et al. 2003 J. Androl. 25, 389–396). Thawing was conducted in a waterbath at 37°C for 20 s. Inseminations were performed using the deep intrauterine insemination technique (Martínez et al. 2002 Reproduction 123, 167–170) with 1 × 109 thawed spermatozoa (post-thaw motility >50%) diluted in 5 mL of Beltsville thaw solution (BTS). Ninety-seven and 82 weaned sows (parity 2–7) in farms A and B, respectively, were twice inseminated at 30 and 36 h after onset of estrus (estrus detection was performed twice a day by allowing females nose-to-nose contact with a mature boar and by applying back pressure). At insemination time, both ovaries were checked for ovulation by transrectal ultrasonography and sows were classified into three groups: F sows (follicles visible during the two examinations), O sows (ovulation visible during at least one examination), and C sows (corpora lutea visible during both examinations). Data were analysed with ANOVA and chi-square test, and are reported as % or mean ± SEM. Overall farrowing rates differed (P < 0.01) between farms: 70.1% (68/97) and 51.22% (42/82) in farms A and B, respectively. Litter size did not differ (P > 0.05) between farms (9.18 ± 0.24 and 9 ± 0.39 in farms A and B, respectively). Distribution of sows among F, O, and C groups differed (P < 0.05) between farms. Seventeen (17.52%), 70 (72.16%), and 10 (10.31%) sows in farm A and 33 (40.24%), 24 (29.27%), and 25 (30.49%) sows in farm B were classified as F, O, and C, respectively. Fertility in F, O, or C sows did not differ (P > 0.05) between farms. Farrowing rates and litter size in O sows (82.98% and 9.45 ± 0.23) were higher (P < 0.05) than in F (48% and 8.67 ± 0.54) and C (48.57% and 7.55 ± 0.62) sows. We can conclude that time of insemination relative to ovulation explains fertility differences between farms when frozen-thawed spermatozoa are used. This work was supported by INIA (RZ01-019) and INFO (CARM).

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Addition of sucrose on cryoprotectant solution of vitrification enhances the quality of Dorper sheep embryos produced in vivo

Semina Ciências Agrárias ◽

10.5433/1679-0359.2015v36n6supl2p4257 ◽

2015 ◽

Vol 36 (6Supl2) ◽

pp. 4257

Author(s):

Alane Pains Oliveira do Monte ◽

João Bosco Loiola Filho ◽

Thais Thatiane Dos Santos Souza ◽

Mayara De Souza Miranda ◽

Lívia Correia Magalhães ◽

...

Keyword(s):

Embryo Quality ◽

Control Group ◽

Chi Square ◽

Mass Occurrence ◽

Chi Square Test ◽

Vitrification Solution ◽

Cryoprotectant Solution ◽

Pellucid Zone

This study aimed to evaluate the efficacy of adding sucrose in vitrification solution of ovine embryos produced in vivo. Forty Dorper ewes were selected and superovulated. Immediately prior to the embryo collection by laparotomy, a laparoscopy was performed to verify the superovulatory response. The recovered flushing was followed by embryo evaluation and embryos were divided in two experimental groups where embryos from Control group were submitted to a traditional vitrification protocol and embryos from Sucrose group to a modified vitrification protocol with sucrose. After warming, embryos were again divided regarding cryoprotectant removal (Indirect) or not (Direct). The embryo quality was classified as embryos of degrees I (excellent or good), II (regular), III (poor) and IV (dead or degenerate). It was also verified the homogeneity of mass, occurrence of embryonic mass retraction and rupture of pellucid zone. The results were expressed as percentages and were subjected to Chi-square test with P < 0.05. The embryos vitrified in the presence of sucrose had lower proportions of lower-quality embryos after warming (22.20 vs. 44.50%), higher percentages of homogeneous embryos after warming (63.89 vs. 38.89 %) while concerning other parameters there was no difference between these groups. It can be concluded that the addition of 0.4 M sucrose during vitrification improves the embryo quality.

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252 COMPARISON OF TWO SPERM SEPARATION PRODUCTS FOR USE IN BOVINE IVF

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab252 ◽

2005 ◽

Vol 17 (2) ◽

pp. 276 ◽

Cited By ~ 1

Author(s):

J. Pryor ◽

S. Romo ◽

D.D. Varner ◽

K. Hinrichs ◽

C.R. Looney

Keyword(s):

Sperm Concentration ◽

Embryo Production ◽

Chi Square ◽

Vitro Fertilization ◽

Before And After ◽

Chi Square Test ◽

Group 2 ◽

Live Sperm ◽

Sperm Separation

In commercial bovine in vitro fertilization (IVF) companies, there is a continuous need to improve results. Efforts to maximize in vitro embryo production have included modifications in the use of sperm separation gradients. The development of commercially available sperm centrifugation gradients represents a new possibility of increasing the number of viable sperm that can be obtained from low concentration (fresh or frozen, sexed or unsexed) semen samples in order to improve the efficiency of the IVF system to make embryo production as efficient as possible. The objective of this study was to compare two different separation gradients, as follows: Group 1: Percoll (Sigma, St. Louis, MO, USA), in 45% and 90% gradients; Group 2: EquiPure (Nidacon, Gathenburg, Sweden), in top and bottom layers. Before and after separation, sperm were evaluated at 200× magnification for total motility, and then stained to assess viability at 400× with fast-green/eosin stain (Sigma). Sperm separation was performed using frozen/thawed semen from one bull. Semen was separated by centrifugation at 200g for 30 min in both density gradients. Results obtained from Groups 1 and 2 were compared by chi-square test. Sperm separation with Percoll yielded lower numbers of sperm (average sperm concentration after separation of 92 × 106, vs. 159 × 106 sperm/mL for EquiPure; P < 0.05) but resulted in higher motility (60% vs. 39%, respectively; P < 0.05) of separated sperm. Rates of live sperm cells were not significantly different between groups (69.5% vs. 70%, respectively; P > 0.1). These results indicate that the commercial separation medium EquiPure may be associated with higher sperm concentration levels but with lowered sperm motility when compared to Percoll for bovine sperm separation. However, Equipure provided similar percentages of live sperm when compared to Percoll, which is currently used in our laboratory.

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19 LOW-DOSE DEEP INTRAUTERINE INSEMINATION IN SOWS UNDER CONDITIONS: INCIDENCE OF UNILATERAL FERTILIZATIONS

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab19 ◽

2005 ◽

Vol 17 (2) ◽

pp. 159 ◽

Cited By ~ 1

Author(s):

E.A. Martinez ◽

J.M. Vazquez ◽

I. Parrilla ◽

C. Cuello ◽

M.A. Gil ◽

...

Keyword(s):

Artificial Insemination ◽

Rate Ratio ◽

Low Dose ◽

Intrauterine Insemination ◽

Fertilization Rate ◽

Chi Square ◽

Fold Reduction ◽

Significant Difference ◽

Chi Squared ◽

Chi Square Test

A new procedure for nonsurgical deep intrauterine insemination (DUI) in non-sedated sows has recently been reported (Martinez et al. 2002 Reproduction 123, 163–170). In comparison to traditional artificial insemination (AI), using this procedure, a 20-fold reduction in the number of spermatozoa inseminated can be used without a decrease in fertility when hormonally treated post-weaning estrous sows are used. The aim of the present study was to evaluate the effectiveness of DUI under field conditions. In Experiment 1, crossbred sows (2–6 parity) were weaned at 20.75 ± 0.06 days. Estrous detection was performed once per day, beginning 3 days after weaning. Sows with a weaning to estrus interval of 4–5 days were selected to be inseminated. A total of 190 sows were inseminated at 12, 24, and 36 h after onset of estrus using one of the following two regimes: (1) DUI with 150 × 106 fresh spermatozoa in 5 mL of BTS (n = 95) and (2) Traditional AI with 3 × 109 fresh spermatozoa in 100 mL of BTS (n = 95) prepared from the same semen samples used for the DUI group. Farrowing rates (FR) and litter sizes (LTS; mean ± SEM) from both groups were compared using chi-squared test and ANOVA, respectively. There was no significant difference in the FR between groups (83.2 and 86.3% for DUI and AI groups, respectively). However, a decrease (P < 0.001) in the LTS was observed in sows inseminated by the DUI procedure (9.8 ± 0.29 and 10.9 ± 0.17, respectively). In Experiment 2, seventy one natural post-weaning estrus sows were used. Fifty-five sows were DUI inseminated three times with 150 (n = 17), 300 (n = 19), or 600 (n = 19) × 106 spermatozoa in 5, 10, or 20 mL of BTS, respectively. The remaining sows (n = 16) were traditionally inseminated. On Day 6 after estrus, sows were subjected to laparotomy and the tips of both uterine horns were flushed in order to evaluate pregnancy rate (PR: percentage of sows with at least 4 viable embryos) and fertilization rate (ratio of viable embryos to the total number of embryos and oocytes). PR was similar in all the groups, ranging from 84.2% (DUI 300 × 106 spermatozoa group) to 94.7% (DUI 600 × 106 spermatozoa group). Fertilization rate and the percentage of bilateral fertilization after DUI with 600 × 106 spermatozoa did not differ from those of the AI group (97.8 and 100% vs. 98.4 and 100%, respectively), but a significant decrease in both parameters (P < 0.05; chi-square test) was observed in sows inseminated with 300 (94.3 and 87.5%) or 150 (84.4 and 66.7%) × 106 spermatozoa. In conclusion, DUI with 150 × 106 spermatozoa offers similar FR but a lower LTS in sows with natural estrus in comparison with those parameters obtained when traditional AI is used. The lower litter size could be related to the low percentage of bilateral fertilization observed in that group. This work was supported by CDTI 020003.

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Mechanical Testing and Reliability Analysis for 3D Printed Cubic Lattices

Volume 14: Safety Engineering, Risk, and Reliability Analysis ◽

10.1115/imece2020-23694 ◽

2020 ◽

Author(s):

Nitin Nagesh Kulkarni ◽

Stephen Ekwaro-Osire ◽

Paul F. Egan

Keyword(s):

3D Printing ◽

Average Length ◽

Input Parameter ◽

Chi Square ◽

Reliability Method ◽

Input Parameters ◽

Chi Square Test ◽

Length Width ◽

3D Printed

Abstract 3D printing has enabled new avenues to design and fabricate diverse structures for engineering applications, such as mechanically efficient lattices. Lattices are useful as implants for biological applications for supporting in vivo loads. However, inconsistencies in 3D printing motivates a need to quantify uncertainties contributing to mechanical failure using probabilistic analysis. Here, 50 cubic unit cell lattice samples were printed and tested with designs of 50% porosity, 500-micron beam diameters, and 3.5mm length, width, and height dimensions. The average length, width, and height measurements ranged from 3.47mm to 3.48mm. The precision in printing with a 95% confidence level was greater than 99.8%. Lattice elastic moduli ranged from about 270 MPa to 345 MPa, with a mean of 305 MPa. Probabilistic analyses were conducted with NESSUS software. The distributions of input parameters were determined using a chi-square test. The first-order reliability method was used to calculate the probability of failure and sensitivity of each input parameter. The elastic modulus was the most sensitive among all input parameters, with 57% of the total sensitivity. The study quantified printing inconsistencies and sensitives using empirical evidence and is a significant step forward for designing 3D printed parts for mechanical applications.

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122 TIMED ARTIFICIAL INSEMINATION IN WOOD BISON USING FROZEN-THAWED SEMEN

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab122 ◽

2016 ◽

Vol 28 (2) ◽

pp. 191 ◽

Cited By ~ 2

Author(s):

G. P. Adams ◽

S. X. Yang ◽

J. M. Palomino ◽

M. Anzar

Keyword(s):

Animal Health ◽

Egg Yolk ◽

Ovulation Rate ◽

Chi Square ◽

Semen Cryopreservation ◽

Wood Bison ◽

The Mean ◽

Intravaginal Device ◽

Synchronization Procedure

Recent progress with methods to control ovulation and semen cryopreservation in Wood Bison was the impetus to test the feasibility of timed AI to facilitate reclamation of this threatened species. A 2 × 2 design was used to compare the efficacy of 2 ovulation synchronization techniques and 2 semen cryopreservation protocols. Female Wood Bison were assigned randomly to 2 groups (n = 24/group) in which ovarian synchronization was induced by ultrasound-guided ablation of follicles >5 mm or intramuscular treatment with 2.5 mg of estradiol 17B + 50 mg of progesterone (E+P) in canola oil. A progesterone-releasing intravaginal device (PRID) was placed at the time of follicle ablation (for 5 days) or E+P treatment (for 8 days) in the respective groups. A luteolytic dose of prostaglandin was given at the time of PRID removal, and 2500 IU of hCG was given IM 3 days later. Bison were inseminated 24 and 36 h after hCG treatment using frozen-thawed semen. The semen was collected by electro-ejaculation from 4 Wood Bison bulls, pooled, and divided into aliquots diluted in either egg-yolk extender (EY) or cholesterol-loaded cyclodextrin extender (CLC). Half the bison in each synchronization group were inseminated with either EY- or CLC-extended semen. Bison were examined by ultrasonography every 12 h beginning on the day of hCG treatment for 3 days or until ovulation was detected, whichever occurred first. Pregnancy diagnosis was made by ultrasonography 34–36 days after insemination. Two bison were excluded during the experiment because of handling difficulty; therefore, the total number of bison used was 46. Ovulation rate and interval to ovulation were compared between synchronization groups by chi-square and t-test, respectively. Pregnancy rates were compared among groups by 2-way ANOVA after transforming data to arcsin. The ovulation rate was not different between synchronization groups [combined mean, 37/46 (80%)], nor was the degree of synchrony, as assessed by the residuals (variation from the mean) in the respective groups. However, the diameter (mean ± standard error of the mean) of the dominant follicle at the time of hCG treatment was smaller in the follicle ablation group than in the E+P group (10.5 ± 0.6 v. 13.9 ± 0.6; P < 0.04), and the interval from hCG treatment to ovulation tended to be longer (35.3 ± 1.6 v. 31.8 ± 1.3 h; P ≤ 0.10). Pregnancy rate was not affected by synchronization procedure, but pregnancy was detected only in the EY-inseminated group (9/23 v. 0/23; P < 0.01). Despite that post-thaw sperm motility was similar for EY and CLC semen (41.7 ± 2.9 and 44.6 ± 3.3%; respectively), CLC-treated semen failed to impregnate bison in vivo. We concluded that synchronization and timed insemination with frozen-thawed semen is feasible in Wood Bison. Of the 23 bison inseminated with EY-extended semen, 21 ovulated (91%), and of those that ovulated 9 became pregnant (43%). Both synchronization schemes were effective, but the ablation protocol may be improved by an additional day between ablation and hCG treatment. We thank Vetoquinol Canada and Merck Animal Health for providing hormone treatments.

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

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab72 ◽

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab71 ◽

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.

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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

Reproduction Fertility and Development ◽

10.1071/rdv30n1ab86 ◽

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.

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