9 SINGLE FIXED-TIME LAPAROSCOPIC INTRAUTERINE INSEMINATION IN PIGS TO PRODUCE LOW-DIVERSE EMBRYOS

2013 ◽  
Vol 25 (1) ◽  
pp. 152
Author(s):  
K.-P. Brüssow ◽  
H. Torner ◽  
J. Rátky
Keyword(s):  
Intrauterine Insemination ◽  
Fixed Time ◽  
Uterine Wall ◽  
Sperm Cells ◽  
Chi Square ◽  
Cell Stage ◽  
Stage Of Development ◽  
Sperm Migration ◽  
Chi Square Test

In vivo-derived embryos at a defined stage of development are often a necessary requirement for ongoing biotechnological applications. Because double fixed-time insemination after ovulation induction is commonly used in pigs to produce embryos, variations in the time of ovulation and fertilization of the ovulated oocytes by spermatozoa mainly of 1 of the 2 inseminations can cause, however, diversities in embryo development. To moderate embryo diversity and to realize a uniform outcome of porcine embryo stages, single laparoscopic fixed-time insemination can be used to minimize embryo diversity. The potential of laparoscopic intrauterine insemination (LIUI) has been demonstrated in sperm-mediated gene transfer (Fantinati et al. 2005) and evaluation of sperm migration (Brüssow et al. 2006, 2011). The aim of the present study was to analyze the development and possible diversity of embryos after LIUI. Forty-eight puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day Regu-Mate® (Intervet, Millsboro, DE, USA) feeding and follicle development was stimulated with 850 IU of eCG 24 h after Regu-Mate® treatment. Ovulation was induced by 500 IU of hCG 80 h after eCG treatment. The LIUI was performed 31 h after hCG treatment. To that, ketamine/azaperone-anaesthetized gilts were fixed in a dorsal position, a pneumoperitoneum was produced and 3 trocar cannulas were inserted into the abdomen for optics and instruments. Laparoscopic handling was observed on a television monitor. Each uterine horn was carefully fixed with an atraumatic forceps 10 to 15 cm caudal from the utero-tubal junction and the uterine wall was punctured with a 2.5-mm diameter trocar. A 2.2-mm catheter connected to a syringe was inserted about 3 cm into the uterine lumen and 20 mL of extended, fresh boar semen (32.2 × 106 sperm cells mL–1; 65% motility) was deposited in the lumen. Embryos were surgically flushed from the genital tract on Day 2 and 3, respectively. Altogether, 778 oocytes were recovered (recovery rate 68 ± 17%); 45 of 48 gilts (93.8%) revealed fertilization and 76.1% of the recovered embryos (n = 592) were at the 2- and 4-cell stage. On Day 2 (n = 22 gilts), a higher percentage of gilts displayed only 2-cell embryos compared with both 2- and 4-cell, and only 4-cell embryos (72.2 v. 22.7 and 4.6%, P < 0.05; chi-square test). On Day 3 (n = 23 gilts), there was a shift regarding the embryo stage. The proportion of gilts with 2-cell, 2- and 4-cell, and only 4-cell embryos was 4.3, 0, and 95.7%, respectively (P < 0.05). Results of the present study demonstrate high rates of fertilization and of non-diverse developed embryos after single fixed-time LIUI in gilts. Additionally, these results were achieved after inseminating a 75% lower number of sperm cells per insemination dose. Laparoscopic intrauterine insemination can be suggested as an alternative for insemination of sex-sorted semen where the number of available sperm cells after the sorting procedure is restricted.

scholarly journals Single fixed-time laparoscopic intrauterine insemination as a tool to obtain low-diversity porcine embryos

2013 ◽  
Vol 58 (No. 8) ◽  
pp. 412-416 ◽  
Author(s):  
K-P Brüssow ◽  
A. Vernunft ◽  
B. Kempisty ◽  
J. Ratky
Keyword(s):  
Low Dose ◽  
Intrauterine Insemination ◽  
Fixed Time ◽  
Sperm Cells ◽  
Cell Stage ◽  
Porcine Embryo ◽  
Low Diversity ◽  
Boar Semen ◽  
Dorsal Position

Double fixed-time insemination after ovulation induction is commonly used in pigs to obtain in vivo produced embryos at defined stages of development for downstream biotechnological applications. However, variations in the time of ovulation and fertilisation of the ovulated oocytes by spermatozoa, mainly in one of the inseminations, can cause diversities in embryo development. The aim of the present study was to reduce embryo diversity and to achieve a &lsquo;uniform outcome&rsquo; of porcine embryo stages using single laparoscopic fixed-time insemination (LIUI). Altogether, 48 puberal German Landrace gilts were included in the study. Estrus of gilts was synchronized by 15-day long altrenogest (Regumate<sup>&reg;</sup>) feeding and follicle development was stimulated with 850 IU eCG 24 h after the final altrenogest application. Ovulation was induced with 500 IU hCG 80 h after eCG. LIUI was performed 31 h after hCG treatment. Gilts under general anaesthesia were fixed in a dorsal position, a pneumoperitoneum was produced and three trocar cannulas were inserted into the abdomen for optics and instruments. Each uterine horn was carefully punctured 10&ndash;15 cm caudal from the utero-tubal junction with a 2.5&nbsp;mm trocar. A 2.2 mm catheter was inserted about 3 cm into the uterine lumen and 20 ml of extended fresh boar semen (32.2 &times; 106 sperm cells/ml) were injected. Embryos were surgically flushed from the genital tract two (Day&nbsp;2) and three (Day 3) days after insemination. Altogether, 778 oocytes/embryos were recovered (recovery rate 68 &plusmn; 17%); 45 of 48 gilts (93.8%) revealed fertilisation and 76.1% of the recovered embryos (n = 592) were at the 2- and 4-cell stage. On Day 2 (n = 22 gilts), a higher percentage of gilts (72.7%, P &lt; 0.05) displayed only 2-cell embryos compared with gilts which had 2- and 4-cell (22.7%), or only 4-cell embryos (4.6%). On Day 3 (n = 23 gilts), the proportion of gilts with 2-cell, 2- and 4-cell, and only 4-cell embryos shifted to 4.3%, 0% and 95.7%, respectively (P &lt; 0.05). The results of the present study demonstrate high rates of fertilisation and homogenously developed embryos after single fixed-time laparoscopic intrauterine insemination in gilts. Additionally, these results were achieved by inseminating a 60% lower number of sperm cells per insemination dose compared to usual doses used for transcervical insemination. In conclusion, LIUI can be recommended for the in vivo production of embryos in a homogeneous developmental stage, and also as an alternative method for low-dose insemination. &nbsp;

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.

10 EFFECTS OF INSEMINATION TIME, BREED, AND INSEMINATOR ON FERTILITY OF EWES INTRAUTERINALLY INSEMINATED WITH FROZEN - THAWED SEMEN IMPORTED FROM NEW ZEALAND

2007 ◽  
Vol 19 (1) ◽  
pp. 123 ◽  
Author(s):  
Y. Fukui ◽  
H. Kohno ◽  
T. Togari ◽  
T. Matsuoka ◽  
H. Imai
Keyword(s):  
New Zealand ◽  
Intrauterine Insemination ◽  
Fixed Time ◽  
Transfer Program ◽  
Chi Square ◽  
Frozen Semen ◽  
Embryo Transfer Program ◽  
Chi Square Test ◽  
Equine Chorionic Gonadotropin ◽  
Lambing Rate

Artificial insemination, especially with the use of frozen semen, is one of the important tools for embryo transfer program in sheep. The present study investigated the effects of insemination times, breeds, and two inseminators on the fertility of ewes intrauterinally inseminated with frozen–thawed ram semen imported from New Zealand. At 8 sheep farms located in Hokkaido, Japan, during the breeding season (October to December) in 2005, a total of 64 mature (1- to 6-year old) Suffolk (32 heads) and Polled Dorset (32 heads) ewes were used. The ewes were treated with controlled intravaginal drug release (CIDR containing 0.3 g progesterone; Pharmacia &amp; Upjohn, Ltd., Auckland, New Zealand) for 12 days and an injection of 500 IU equine chorionic gonadotropin one day before CIDR removal. The fixed-time intrauterine inseminations (early: 43–46 h; late: 47–50 h) after CIDR removal were performed using the frozen–thawed semen from a Suffolk and Polled Dorset ram by two inseminators. The effects of breeds (Suffolk and Polled Dorset), fixed-time insemination times (early and late phases), and two inseminators on pregnancy (number of pregnant ewes/number of ewes inseminated, 60 days after insemination) and lambing (number of lambed ewes/number of ewes inseminated) rates were analyzed by chi-square test. The prolificacy was compared by Student&apos;s t-test, and differences were also analyzed by Tukey&apos;s omega procedure. The effect of the different farms on fertility was not examined due to the small numbers of ewes per farm. Pregnancy (60.0 and 72.4%, respectively) and lambing (60.0 and 71.4%, respectively) rates were not significantly different between Suffolk and Polled Dorset ewes. The inseminators also did not affect pregnancy (62.6 and 68.8%) and lambing (62.6 and 67.7%) rates. For the insemination times, the lambing rate tended to be higher (P ≤ 0.10) in the early insemination than in the late insemination (76.7% and 53.6%, respectively). The present results show acceptable fertility in ewes inseminated with Suffolk and Polled Dorset frozen semen imported from New Zealand. The early intrauterine insemination (43–46 h after CIDR removal) tended to result in higher fertility than the late insemination (47–50 h after CIDR removal). From 38 lambed ewes, 60 newborn lambs were produced, and this has provided new blood lines of Suffolk and Polled Dorset sheep in Japan.

162 EVALUATION OF THE DEMI-EMBRYOS AGGREGATION TECHNIQUE EFFICACY ON THE PRODUCTION OF CHIMERAS WITH IN VIVO PRODUCED MURINE EMBRYOS

2010 ◽  
Vol 22 (1) ◽  
pp. 239
Author(s):  
M. P. M. Mancini ◽  
B. C. S. Campanha ◽  
D. M. Souza ◽  
C. P. Godoi ◽  
F. Frei ◽  
...  
Keyword(s):  
Advanced Stage ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Stage Of Development ◽  
Embryo Cells ◽  
Genotype Composition ◽  
23 Gauge ◽  
Aggregation Technique

Mixing embryo cells coming from different fertilizations (i.e. embryonic chimera) have been used as a tool to understand embryogenesis, organo- genesis, and pluripotency, as well as a source to obtain transgenic mammals. The objectives of this work were to evaluate the potential of mice demi-embryos, in advanced stage of the development (morulae and blastocysts) to aggregate in chimeras; to compare the chimerism rate of those embryos with the rate of whole 8- to 16-cell-stage embryos; and to measure the genotype composition of the resultant chimera. One-month-old transgenic (C57/BL6/EGFP strain, GFP) or non-transgenic (Swiss Webster strain, SW) mice weighing approximately 35 g were superstimulated with 5 or 10IU of eCG (for GFP or SW mice, respectively) followed with hCG injection of 5 or 10IU (GFP or SW mice, respectively) 48 h later. Embryos were harvested at different stages of development and allocated in 3 groups for aggregation technique. Blastocysts and morulae were bisected (microblade mounted on TransferMan NK-2, Eppendorf), whereas 8- to 16-cell-stage embryos had their zona pellucida mechanically removed (23-gauge needle). Embryos were manipulated in M2 culture medium at room temperature, and aggregation groups consisted of G1 (2 demi-blastocysts, n = 28), G2 (demi-blastocyst and demi-morula, n = 20), and G3 (2 whole 8- to 16-cell-stage embryos, n = 25). All embryos were placed in wells (Embryo GPS dish, SunIVF) containing KSOMaa medium (EmbryoMax, Millipore) under oil (Sigma, St. Louis, MO, USA) and were incubated at 37°C, 5% CO2 in air saturated with humidity. After 24 h of incubation, the presence of chimera was verified, and the percentage of area (square pixel) occupied by each embryonic type (GFP or SW) from both G2 (n = 3) and G3 (n = 3) were measured by the ImageJ program (v. 1.42i, USA). General results of the chimerism rate were 3.6%, 15.0%, and 60.0% (G1, G2, and G3, respectively; P < 0.001, chi-square). The G3 group differed from others (G1, P < 0.001 and G2, P = 0.003), which appeared similar (P = 0.294; Fisher’s exact test). The mean percentage (±SD) of GFP cells in the resultant chimera were 51.3 ± 4.1% and 50.6 ± 10.0% (for G2 and G3, respectively; P = 0.91, t-test). Moreover, the percentages of GFP cells within the same group of G2 or G3 at 0 v. 24 h of culture were not statistically different (data not shown). It was concluded that in our conditions, the embryonic chimerism by aggregation of murine demi-embryos is a feasible procedure, even for embryos in an advanced stage of development (morulae and blastocysts). Nevertheless, the chimerism rate with whole pre-compaction embryos (G3) was higher than that of G1 and G2 groups. Furthermore, the phenotype of embryonic chimera was equally composed, with no effect of strain (GFP or SW cells) or culture (0 or 24 h) on its composition. Supported by FAPESP, Brazil: 2006/06491-2 and 2007/07705-9 (MFGN) and 2007/04291-9 (MPMM).

142 CHIMERISM BY AGGREGATION OF BISECTED IN VITRO PRODUCED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 175
Author(s):  
E. M. Razza ◽  
I. P. Emanuelli ◽  
C. M. Barros ◽  
M. F. G. Nogueira
Keyword(s):  
In Vitro Culture ◽  
Cell Aggregation ◽  
Bovine Embryos ◽  
Chi Square ◽  
Cell Stage ◽  
Exact Test ◽  
Aggregation Rate ◽  
Stage Of Development

Aggregation is one of the main techniques used to obtain embryonic chimeras. This procedure can be performed with whole or demi-embryos, in different stages of development and produced by in vivo or in vitro systems. However, aggregation efficiency tends to be reduced when using embryos in advanced stages (e.g. morulae and blastocysts). The aim of this work was to evaluate the effect of the agglutinating agent phytohemagglutinin-L (PHA) in the percentage of chimeras produced with in vitro-produced (IVP) bovine embryos. Cumulus–oocyte complexes (COC; 445; quality I and II) were matured in drops of 90 μL of TCM-199 bicarbonate supplemented with 10% of FCS and incubated for 22 to 24 h. Fertilization was performed in TALP-IVF medium for 18 h. Presumptive zygotes were transferred to SOF medium for in vitro culture. Incubation conditions were 38.5°C and 5% CO2 in air. To conduct the manual bisection, embryos were placed into 3-μL microdrops of protein-free HEPES-buffered SOF medium. The bisection was executed with a microblade (Ultra-Sharp Splitting Blade, Bioniche, Bogart, GA, USA) under stereomicroscope (35× magnification). Half-structures were joined and transferred to an embryo reconstruction plate, where they were kept for 3 min in drops containing 500 μg mL–1 phytohemagglutinin-L, before the approximated pairs were transferred to SOF medium in cell aggregation well-of-the-well (WOW) micro-wells to in vitro culture. The structures were randomly allocated and the aggregation was performed between 2 whole (zona free) 8- to 16-cell stage embryos to construct aggregated chimeras in the presence [group (G)1, n = 32] or absence of PHA (G2, n = 34) and between demi-morula and demi-blastocyst with PHA (G3, n = 28) or without (G4, n = 29). The aggregation of structures was evaluated after 24 h. Aggregation rates among the 4 experimental groups and the main effects were analysed by Chi-square or Fisher’s exact test and significance was considered when P < 0.05. Embryo aggregation was higher in group G1 than G2 (75.0 and 50.0%, respectively; P = 0.045). Aggregation rate of demi-embryos was similar either in the presence (G3, 39.3%) or in the absence of PHA (G4, 20.7%; P = 0.16). The presence of PHA significantly increased the aggregation rates of the whole pre-compaction embryos (G1) compared with G3 (75.0 and 39.3%, respectively; P < 0.01). The use of PHA resulted in higher aggregation rates (58.3%) than non-use (36.5%; P = 0.03), whereas the embryonic stage of pre-compaction development (G1+G2) produced a higher rate of aggregation (62.1%) than post-compaction demi-embryos (G3+G4, 29.8%; P < 0.001). We could infer a positive effect of PHA on the aggregation rate of bovine IVP embryos only to the 8- to 16-cell stage of development. Financial support: FAPESP, Brazil (06/06491-2, 07/07705-9, 09/10679-5, and 09/04888-0).

Effect of GnRH injection timing in the production of pronuclear-stage zygotes used for DNA microinjection

Zygote ◽  
2004 ◽  
Vol 12 (3) ◽  
pp. 257-261 ◽  
Author(s):  
Hernan Baldassarre ◽  
Bin Wang ◽  
Melanie Gauthier ◽  
Nathalie Neveu ◽  
Anthoula Lazaris ◽  
...  
Keyword(s):  
Medroxyprogesterone Acetate ◽  
Treatment Protocol ◽  
Hormonal Treatment ◽  
Injection Timing ◽  
Chi Square ◽  
Cell Stage ◽  
Embryo Recovery ◽  
Stage Of Development ◽  
Production Programme ◽  
Dna Microinjection

This study was aimed at developing a hormonal treatment protocol in order to optimize the proportion of pronuclear-stage embryos to be used for DNA microinjection in a goat transgenic founder production programme. A total of 46 adult BELE® and 47 adult standard goats (1–5 years old) were used as donors and recipients, respectively. They were heat-synchronized using intravaginal sponges containing 60 mg medroxyprogesterone acetate for 10 days with an injection of 125 μg cloprostenol on the morning of the eighth day. Recipients were injected with 400 IU eCG at the time of sponge removal while donors received a total of 133 mg NIH-FSH-P1 (Folltropin-V) given twice daily in decreasing doses over 3 days starting 48 h before sponge removal. Ovulation was induced in donors by injecting 100 μg of GnRH at 24 h (GnRH24) or 36 h (GnRH36) after sponge removal. Embryo recovery was performed by oviduct flushing following a standard mid-ventral laparotomy procedure. The proportion of embryos in the pronuclear stage of development was higher in the GnRH36 group (90% vs 34%, p<0.01). Embryos were microinjected with a DNA expression cassette followed by transfer to the oviduct of synchronized recipients. A higher, yet not statistically significant, pregnancy rate was found in the recipients transferred with pronuclear-stage embryos compared with those transferred with 2-cell-stage embryos (64% vs 37%, chi-square p=0.06). One transgenic female founder was produced from the group of recipients transferred with pronuclear-stage microinjected embryos.

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

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

<p>This study aimed to evaluate the efficacy of adding sucrose in vitrification solution of ovine embryos produced <em>in vivo</em>. 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 &lt; 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.</p>

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

2005 ◽  
Vol 17 (2) ◽  
pp. 159 ◽  
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.

Mechanical Testing and Reliability Analysis for 3D Printed Cubic Lattices

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.

242 PROMISING TWO-STEP EVALUATION SYSTEM FOR SELECTING HIGH DEVELOPMENTAL COMPETENCE IN VITRO FERTILIZED EMBRYOS IN CATTLE USING WELL-OF-THE-WELL DISH

2015 ◽  
Vol 27 (1) ◽  
pp. 210
Author(s):  
M. Taniai ◽  
M. Takayama ◽  
O. Dochi ◽  
K. Imai
Keyword(s):  
Evaluation System ◽  
Evaluation Method ◽  
Calf Serum ◽  
Time Lapse ◽  
Developmental Competence ◽  
Bovine Embryo ◽  
Chi Square ◽  
Cell Stage ◽  
Chi Square Test

Bovine IVF embryos are evaluated morphologically using light microscopy just before transfer. However, this evaluation method is subjective, and an objective method with more certainty is needed. Sugimura et al. (PLoS ONE 2012 7, e36627) reported a promising system for selecting healthy IVF bovine embryo by using time-lapse cinematography and 5 prognostic factors. This study was to investigate the efficacy of a 2-step evaluation system of IVF embryos using microscopy for selecting high developmental competence IVF embryos. Cumulus-oocyte complexes (COC) were collected by ovarian follicular aspiration (2 to 5 mm diameter) obtained from a local abattoir. The COC (n = 488) were matured in TCM-199 medium supplemented with 5% calf serum (CS) and 0.02 IU mL–1 of FSH at 38.5°C for 20 h in an atmosphere of 5% CO2 (20 COC 100 µL–1 droplets). After 10 h of gametes co-culture (5.0 × 106 sperm cells mL–1), the presumptive zygotes were cultured in 125 µL of CR1 aa medium supplemented with 5% CS in well of-the-well culture dishes (AS ONE, Japan; 25 zygotes well–1) at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 for 9 days. Two-step evaluations of embryos were done at 27 and 55 h post-IVF (hpi). In the first step of evaluation, cleavage patterns at 27 hpi were categorized as mono-cell, 2-cell with even blastomeres and without fragments (normal cleavage), 2-cell with uneven blastomeres, and ≥3 blastomeres. During the second step of evaluation, embryos were classified by their number of blastomeres (2 to 5 cells, 6 to 8 cells, and >8 cells) and the absence or presence of multiple fragments (<20 or >20%) at 55 hpi. The data were analysed by chi-square test. The blastocyst rate (BL%) of embryos cleaved before 27 hpi (56.6%, n = 106) was higher (P < 0.01) than those of embryos cleaved after 27 hpi (37.0%, n = 235). A greater percentage (P < 0.05) of 2-cell embryos with normal cleavage (68.0%, n = 50) developed to blastocysts than from with =3 blastomeres at 27 hpi (40.6%, n = 32). Superior BL% (P < 0.01) was obtained from embryos categorized as 6- to 8-cell stage (58.6%, n = 140) and >8 cell stage (70.6%, n = 25) compared with those embryos at the 2- to 5-cell stage at 55 hpi (26.1%, n = 176). Embryos with no fragments (58.0%, n = 467) had higher BL% (P < 0.01) compared with those with <20% fragments (30.7%, n = 127) and having with >20% fragments (17.5%, n = 25) at 55 hpi. The highest of BL% was observed in embryos showing a normal cleavage to 2-cells with at 27 hpi and having >6 cells with no fragments at 55 hpi (95.2%, n = 21, P < 0.01). These results demonstrate that the 2-step evaluation system at 27 and 55 hpi using microscopy is an effective method for selecting IVF embryos with high developmental competence.

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