22 IN VITRO FERTILITY ESTIMATES USING SPERM KINETICS VARIABLES AND CONCEPTION RATES IN BEEF HEIFERS

2010 ◽  
Vol 22 (1) ◽  
pp. 169
Author(s):  
M. V. C. Ferraz Jr ◽  
R. S. Macedo ◽  
J. B. Barreto Filho ◽  
T. F. Silva ◽  
R. A. Braga Jr ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Ultrasound Images ◽  
Beef Heifers ◽  
Fast Evaluation ◽  
Spermatozoa Motility ◽  
Frozen Semen ◽  
Conception Rates ◽  
Inertial Moment

Sperm motility is a physical parameter evaluated in semen samples of the bull and is thought to be related to the fertility in the male. Despite being a characteristic of simple and fast evaluation, motility estimates involve subjective components when analyzed by light microscopy (LM) that might restrain their evaluation in some conditions. Moreover, in some species, poor correlations were observed between spermatozoa motility and semen fertility. The incidence of coherent light in a semen sample generates a phenomenon called biospeckle (BSL) that is capable of measuring the kinetics activity of the spermatozoa. In this work the relationship among sperm kinetics variables evaluated by LM and BSL and conception rates in beef heifers was investigated, with the purpose to predict frozen semen fertility when laser light is used. Sixty semen samples of 6 mature AI donor bulls (Bos taurus indicus) herein named A, B, C, D, E, and F were thawed at 37°C for 30 s in a water bath and evaluated by LM and BSL. In LM evaluation, an index (IND) was proposed to group together, in a single mean estimate, the spermatozoa motility (M: % scale) and velocity (V: 1 to 5 scale) according to the equation IND = [V × 20 + M]/2. In the BSL evaluation, each sample was illuminated (n = 10 per bull) by a laser beam (He-Ne, 632 nm and 10 MW) for 40 s and a mean inertial moment (IM) was obtained for each donor bull. These semen samples were used in an AI program of beef heifers (n = 166) clinically examined for reproductive health, body condition, and weight. Pregnancy diagnosis was done by ultrasound images (Falco 100, 6 MHz, Pie Medical, Crawley, UK) 28 days after insemination. Fertility estimates were done by the generalized linear model using logistic regression (stepwise methodology), generating an equation to predict the conception rate of the semen, the variables of which were IM and IND. Results of the predicted conception rates (pCR) using IM and IND and the observed conception rates (oCR) were A (0.5490; 0.57), B (0.6483; 0.68), C (0.7108; 0.71), D (0.4552; 0.28), E (0.4797; 0.54), F (0.3825; 0.47), respectively. Positive correlations (P < 0.05) were observed between pCR and oCR (r = 0.79) showing a similar behavior between in vitro and in vivo estimates. Results of this work showed that there is a high correlation between spermatozoa kinetics and semen fertility in the bull and that BSL motility analysis could be used as an approach to evaluate the fertility of semen samples. Financial support: FAPEMIG grant EDT 94/07, CNPq.

26 SPERM MOTILITY DECREASING AND SEMEN FERTILITY IN THE BULL EVALUATED BY BIOSPECKLE

2010 ◽  
Vol 22 (1) ◽  
pp. 170 ◽  
Author(s):  
R. S. Macedo ◽  
J. B. Barreto Filho ◽  
R. A. Braga Jr ◽  
G. F. Rabelo
Keyword(s):  
Sperm Motility ◽  
Laser Light ◽  
Bos Taurus ◽  
Test Group ◽  
Mean Value ◽  
Ultrasound Images ◽  
Group I ◽  
Conception Rates ◽  
Inertial Moment ◽  
Group Ii

Sperm motility decreasing in a semen sample over time is an indirect approach to assess spermatozoa viability and should be related to the ejaculate fertility. The biospeckle (BSL) is an interacting phenomenon between laser light and biological specimens that allows measurement of sperm kinetic activity by means of an index called the inertial moment (IM). The aim of this work was to evaluate sperm motility diminishing in frozen bovine semen with BSL and analyze semen fertility in relation to the motility decreasing behavior patterns showed by different semen samples. Fertility was assessed by beef heifer conception rates after AI. Semen of 6 mature IA donor bulls (Bos taurus indicus) was previously divided in 2 groups (group I: motility ≥50%; group II: motility <50%), each group comprised 3 animals. Semen was thawed at 37°C for 30 sin a water bath. One aliquot of 10 μL was placed in a warmed slide, covered with a slip, and evaluated by light microscopy and BSL just after thawing. Then, samples were kept at room temperature to induce a decrease in sperm motility. Each sample was illuminated 6 times by laser light at intervals of 2 min (n = 36) and IM values were obtained. The semen samples were used in an AI program of beef heifers (n = 166) under the same nutrition and management conditions. All inseminations were done by the same technician, and donor bulls were used consecutively throughout the breeding season. Pregnancy diagnosis was done by ultrasound images (Falco 100, 6 MHz, Pie Medical, Crawley, UK) 28 days after insemination. The SISVAR software was used in variance analysis and comparison of means by the Tukey test. Group I showed greater (P < 0.05) IM mean value (153.5 ± 27.48) compared with group II (107.94 ± 30.61), which means that this group had higher sperm motility during the time of evaluation. In addition, conception rates (0.63 ± 0.49) for group I were also higher (P < 0.05) compared with group II. Apparently semen fertility is related to the sperm kinetics measured by the IM. Mean IM values (134.0 ± 22.65 and 96 ± 22.34, groups I and II, respectively) obtained in the last illumination for each group did not differ (P > 0.05), but a tendency to differ was observed when a greater number of illuminations was done. Our data suggest that the BSL was capable of identifying spermatozoa surviving between high and low motility groups, and the sperm motility decreasing measured by the IM might be an objective approach to evaluate semen fertilization potential. Financial support: FAPEMIG, grant EDT 94/07, CNPq.

257 DIFFERENCES BETWEEN BOS TAURUS AND BOS INDICUS EMBRYOS: TRANSCRIPTS AMOUNTS

2010 ◽  
Vol 22 (1) ◽  
pp. 285
Author(s):  
S. Wohlres-Viana ◽  
M. M. Pereira ◽  
A. P. Oliveira ◽  
J. H. M. Viana ◽  
M. A. Machado ◽  
...  
Keyword(s):  
Production System ◽  
Production Systems ◽  
Bos Taurus ◽  
Bos Indicus ◽  
In Vitro Production ◽  
Embryo Production ◽  
Peroxiredoxin 1 ◽  
Vitro Production

The Zebu breeds (Bos indicus) are different from European breeds (Bos taurus) in some aspects of their reproductive physiology, including follicle recruitment, number of follicular waves, and oocyte ultrastructure. On the other hand, embryos produced in vivo and in vitro show morphological and developmental differences, which can be related to culture environment. The aim of this study was to evaluate the effect of breed (Gyr v. Holstein) within embryo production system (in vivo and in vitro), as well as effect of production systems within breeds on relative abundance of transcripts related to formation, survival, and subsequent development of blastocysts, such as those involved in water and small solutes transport (Aquaporins 3 and 11), blastocoel formation (Na+/K+-ATPase a1 and |52), and cellular stress response (Peroxiredoxin 1). For in vivo embryo production, donors were superstimulated with FSH and inseminated, and embryos were recovered 7 days after AI. For in vitro embryo production, oocytes recovered by ovum pickup were in vitro matured and fertilized and then cultured for 7 days in culture medium under 5% CO2 at 38.5°C. For each group, blastocysts (n = 15) distributed in 3 pools were used for RNA extraction (RNeasy MicroKit, Qiagen, Valencia, CA, USA), followed by RNA amplification (Messageamp II amplification kit, Ambion-Applied Biosystems, Foster City, CA, USA) and reverse transcription (SuperScript III First-Stand Synthesis Supermix, Invitrogen, Carlsbad, CA, USA). The cDNA were submitted to real-time PCR, using the H2a gene as endogenous control, and analyzed by REST© software. To evaluate breed effect within the production systems, 2 comparisons were performed: (1) in vivo: Gyr v. Holstein and (2) in vitro: Gyr v. Holstein, considering Holstein data as 1.00. To evaluate production system effect within breeds, 2 comparisons were performed: (1) Gyr: in vivo v. in vitro and (2) Holstein: in vivo v. in vitro, considering in vivo produced embryo data as 1.00. The results are shown as mean ± SEM. For in vivo comparison between breeds, Aquaporin 3 (1.66 ± 0.77), Na+/K+-ATPase a1 (1.61 ± 0.56), and Peroxiredoxin 1 (1.61 ± 0.66) were up-regulated (P < 0.05) in Gyr embryos when compared with Holstein embryos, whereas for in vitro comparison, no differences (P > 0.05) were found. For comparisons between production systems within breeds, only Peroxiredoxin 1 (0.31 ± 0.39) was down-regulated (P < 0.01) in in vitro produced Gyr embryos when compared with in vivo counterparts. No differences (P > 0.05) were found between production systems for the Holstein breed. In conclusion, these data suggest that there is a difference on gene expression between Bos taurus and Bos indicus blastocysts, but such difference between breeds can be attenuated by the in vitro production system, indicating an embryo adaptation to the in vitro culture conditions. The data also suggest that the in vitro production system can influence the amount of transcripts in Gyr embryos. Other genes should be evaluated for a better understanding of these differences. Financial support was provided by CNPq and FAPEMIG.

335 CYTOPLASMIC MICROINJECTION OF EXOGENOUS DNA IN IN VITRO AND IN VIVO DERIVED SHEEP EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 263
Author(s):  
F. Pereyra-Bonnet ◽  
A. Gibbons ◽  
M. Cueto ◽  
R. Bevacqua ◽  
L. Escobar ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Genetically Modified ◽  
Fold Increase ◽  
Egfp Expression ◽  
Control Group ◽  
Corpora Lutea ◽  
Exogenous Dna ◽  
Frozen Semen

Microinjection of DNA into the male pronucleus is a commonly used method to generate transgenic animals. However, it is only moderately efficient in several species because it requires proper male pronuclear visualisation, which occurs only in a narrow window of time in mice. The cytoplasmic microinjection of exogenous DNA (eDNA) is an alternative method that has not been fully investigated. Our objective was to evaluate if cytoplasmic microinjection of eDNA is capable of producing genetically modified embryos. In vitro and in vivo derived sheep embryos were cytoplasmically microinjected with pCX-EGFP previously incubated (5 min in a PVP droplet) with oolemma-cytoplasm fragments obtained from donor oocytes by microsurgery. A control group using microinjected plasmid alone was included in the in vivo procedure. For in vitro microinjection, IVF embryos were microinjected with circular plasmid with promoter (50 or 500 ng μL–1) or without promoter (50 ng μL–1) at 6 h after fertilization. The IVF was performed following (Brackett and Olliphant 1975 Biol. Reprod. 12, 260–274) with 15 × 106 spermatozoa mL–1, and presumptive zygotes were cultured in SOF. The expression of enhance green fluorescent protein (EGFP) was determined under blue light. For in vivo microinjection, embryos from superovulated sheep (by standard procedures) were recovered and microinjected with 50 ng μL–1 of linearized plasmid without promoter at 12 h after laparoscopic insemination with frozen semen (100 × 106 spermatozoa per sheep). Plasmid without promoter was used to avoid any possible cytotoxic effect produced by EGFP expression. The microinjection of IVF embryos with 50 ng μL–1 of plasmid was the best condition to produce embryos expressing eDNA (n = 96; 46.9% cleaved; 12.2% blastocysts; 53.0 and 4.1% of green embryos and blastocysts, respectively). Variables between the groups with or without promoter IVF were not statistically different (Fisher test: P < 0.05); however, when 500 ng μL–1 was microinjected, no blastocysts were obtained. In the in vivo embryo production group, 111 presumptive zygotes were microinjected (n = 37; with plasmid alone) from 16 donor sheep (11.5 ± 4.0 corpora lutea; 8.4 ± 4.8 presumptive zygotes recovered; 74.3% recovery rate). The mean time from injection to cleavage was 18.0 ± 4.5 h, and the percentage of cleavage and damage (due to the embryo injection) were >70% and <10%, respectively. Fifty-eight good quality embryos were transferred into the oviducts of 19 surrogate ewes; 12 of them are pregnant (63.1%). The presence of green IVF embryos demonstrates that eDNA was transported to the nucleus after cytoplasmic injection. We believe that the multi-fold increase (50- to 100-fold) in plasmid concentration compared with that used by others was the key step to our successful cytoplasmic microinjection. Accordingly, the new/old methodology described in this study provides an easy DNA construct delivery system of interest for the implementation of early reprogramming events. In addition, results obtained in the near future using in vivo cytoplasmic microinjection with high concentrations of eDNA could revalidate this technique for producing genetically modified large animals.

121 DIFFERENTIAL mRNA EXPRESSION BETWEEN IN VIVO AND IN VITRO-DERIVED BOS INDICUS AND BOS TAURUS EMBRYOS

2009 ◽  
Vol 21 (1) ◽  
pp. 160
Author(s):  
L. Nasser ◽  
P. Stranieri ◽  
A. Gutiérrez-Adán ◽  
M. Clemente ◽  
L. Jorge de Souza ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Repeated Measures ◽  
Bos Taurus ◽  
Receptor Gene ◽  
Expression Patterns ◽  
Bos Indicus ◽  
Growth Factor Receptor ◽  
P53 Gene

Brazil is a leading country in the world of commercial use of in vitro-produced bovine embryos with 200 000 transfers per year. The majority of in vitro-produced embryos are pure breed Nelore and are transferred fresh with 40% pregnancy rate. However, pregnancies are drastically reduced with frozen in vitro embryos. This experiment is part of our effort to learn more about molecular composition and morphology of in vitro-derived embryos that may be responsible for such discrepancy. We examined molecular expression of mRNA transcripts of 6 selected genes; apoptosis Bax,TP53(p53), SHC1SHC(p66), insulin growth factor receptor (IGF2R), stabilization of the plasma membrane PLAC8 and glucose conversion H6PD in in-vivo (control) and in-vitro Nelore and Bos taurus embryos. In vivo embryos were collected from superovulated cows at Day 7. In vitro embryo was produced from oocytes aspirated from live cows. A total of 284 oocytes (4 replicates) were matured and fertilized by standard IVF procedures. Presumptive zygotes were cultured in CR2 medium with 5% BSA in 50 μL drops (25 zygotes per drop) at 39°C under paraffin oil and 5% CO2 in humidified air. Embryos that developed on Days 7 to blastocyst were transferred to recipients, and 10 blastocysts from each replicate were frozen for evaluation of gene expression patterns. Poly(A) mRNA was prepared from 3 groups of pools of 10 in vitro embryos and 10 of control in vivo-derived embryos. The quantification of all gene transcripts was carried out by real-time quantitative RT-PCR using the comparative CT method. Data on mRNA expression were normalized to the endogenous H2a.z and was analyzed by one-way repeated-measures ANOVA. The cleavage rates at Day 2 and number of blastocysts developed at Day 7 were 80.3 ± 3.2 and 42.2 ± 6.4, respectively. The level of expression of IGF2R was significantly (P < 0.05) higher in in vivo-derived embryos than in both groups of in vitro embryos. The expression of all 3 apoptosis genes were lower (P < 0.05) in in vivo than in vitro embryos with exception of p53 gene that was not different between Nelore in vitro and in vivo embryos but was significantly higher (P < 0.05) in Bos taurus in vitro embryos. There was no difference in expression of PLAC8 gene among any tested group of embryos and in expression of H6PD gene between Nelore in vitro and in vivo embryos. We concluded that significant differences in molecular makeup between in vitro and in vivo-derived Nelore embryos exist. Of particular importance seems to be pattern of expression of IGF2R receptor gene known as a good indicator of embryo quality, which promotes proliferation and differentiation. Similarly, higher expression of 2 BAX and p66 genes of apoptosis in in vitro embryos seems to be a further indication of inferior quality of Nelore in vitro-derived embryos that showed to be more profound in Bos taurus in vitro-derived embryos.

7 THE EFFECT OF A NOVEL RECOMBINANT PROTEASE TREATMENT ON BOVINE SPERM FERTILIZING CAPACITY AND EMBRYO DEVELOPMENT IN VITRO

2009 ◽  
Vol 21 (1) ◽  
pp. 104
Author(s):  
J. T. Aaltonen ◽  
K. J. Mattson ◽  
N. M. Loskutoff
Keyword(s):  
Embryo Development ◽  
Disease Transmission ◽  
Bos Taurus ◽  
Gradient Centrifugation ◽  
Control Group ◽  
Bovine Embryo ◽  
Human Sequence ◽  
Bovine Sperm

As described in the IETS Manual (Stringfellow and Seidel, 1995), and endorsed by the OIE, trypsin can be used (for specific pathogens and livestock) to effectively remove certain infectious agents from in vivo-derived embryos for international transport. Because of the multimillion-dollar AI industry for livestock, the OIE has encouraged more research in developing similar decontamination techniques for semen as an added safeguard to animal quarantine for the prevention of disease transmission. Most or all of the earlier studies on embryos used a porcine pancreatic-derived trypsin. Because of more stringent guidelines from international regulatory agencies on the use of animal products, several serine protease recombinants are now available. Previous experiments comparing the porcine pancreatic extract with a recombinant bovine sequence trypsin developed in corn resulted in no statistical difference in cleavage or morula/blastocyst rates. (Mattson et al. 2008 Theriogenology 69, 724–727). An additional in vivo study treating bovine sperm with a yeast-derived human-sequence trypsin resulted in significantly more transferable-quality embryos after the AI of superovulated cows as compared with sperm not treated with trypsin (Blevins et al. 2008 Reprod. Fertil. Dev. 20, 84). The goal of this experiment was to examine the in vitro development of bovine embryos produced from sperm treated with a recombinant trypsin found in a commercially available density gradient centrifugation (DGC) product (Bovipure, Nidacon, Sweden) compared with DGC without trypsin. Oocyte aspiration, maturation, fertilization, and embryo culture were performed using standard methods in 5 replications (n = 2220 oocytes). Semen was collected and pooled from 2 Bos taurus bulls and frozen in an egg-yolk cryodiluent (Biladyl, Minitube). The semen was processed using Bovipure DGC composed of 2 mL of 40% colloid of silane-coated silica particles containing either a yeast-derived human sequence recombinant trypsin containing no animal by-products (n = 1126 oocytes) or the same colloid without trypsin as the control group (n = 1094 oocytes). Both 40% concentrations were layered over 2 mL of an 80% concentration of the same colloid without any additives. The density gradients were centrifuged at 300g for 20 min, after which time the pellets were washed in 5 mL of prewarmed TL Hepes solution (Cambrex) and centrifuged at 500g for 10 min. The resulting sperm pellets were then resuspended in a volume calculated to provide 1 × 106 sperm mL–1, to be used for in vitro inseminations. Results were compared using a 2-tailed unpaired t-test. Cleavage rates for the trypsin-treated sperm (n = 969, 35.8%) and the control (n = 950, 44.3%) groups were not statistically different (P = 0.20). Although more embryos reached the morula to blastocyst stages in the control group (n = 421, 61.0%) than in the trypsinized group (n = 347, 54.7%), these differences also were not statistically significant (P = 0.85). In conclusion, trypsinized Bovipure DGC of sperm before insemination showed no detrimental effects on IVF-derived bovine embryo development.

17 SPERM FERTILITY RATE ASSESSED BY EMBRYO PRODUCTION IN VIVO AND IN VITRO IN SOUTH AFRICAN BULLS

2017 ◽  
Vol 29 (1) ◽  
pp. 116
Author(s):  
M. H. Mapeka ◽  
F. V. Ramukhithi ◽  
C. M. Pilane ◽  
D. Norris ◽  
C. Banga ◽  
...  
Keyword(s):  
South African ◽  
Sperm Motility ◽  
Developmental Stages ◽  
Fertility Rate ◽  
Embryo Production ◽  
Vitro Fertilization ◽  
Frozen Semen ◽  
Recovery Methods

The aim of this study was to determine the sperm fertility rate by embryo production in vivo and in vitro in South African bulls and further compare the embryo quality developed from different oocyte recovery methods. A total of 15 frozen semen straws (5 Bonsmara; 5 Nguni; 5 Boran) were thawed and evaluated for sperm motility characteristics using sperm class analyzer. The fertilizing ability of frozen–thawed semen was assessed by performing AI and in vitro fertilization. For AI, 6 cows were superovulated and inseminated with frozen–thawed semen followed by flushing on Day 7 post-insemination and then evaluated for embryo developmental stages. For IVF, oocytes were retrieved using two recovery methods namely ovum pick-up (OPU) and ovary aspiration. A total of 383 (106, OPU; 277, ovary aspiration) oocytes were matured in M199 + 10% fetal bovine serum (FBS) maturation medium at 38.5°C for 24h. Oocytes were washed in Bracket and Oliphant’s fertilization medium, co-incubated with frozen–thawed (Boran) semen at 38.5°C for 6 h, and then cultured in SOF-BSA medium, incubated at 38.5°C, 5% CO2 for 7 days, and further evaluated for embryo development. Data were analysed by ANOVA. Total sperm motility was >70% in all breeds. Boran had a significantly (P < 0.05) higher total post-thaw sperm motility (93.2 ± 3.6) compared with Nguni (75.1 ± 4.2) and Bonsmara (80.7 ± 6.9). Furthermore, Boran had higher (P < 0.05) progressive motility (39.7 ± 3.4) and rapid motility (36.1 ± 5.9) compared with other breeds. Interestingly, Boran produced significantly (P < 0.05) higher blastocyst rate (56.34%) compared with Bonsmara (38.03%) Nguni (31.08%). Superovulation and OPU resulted in a significantly higher (P < 0.05) number of blastocysts (10.5 ± 3.3 and 10.5 ± 3.3) respectively, compared with aspiration (1.3 ± 3.3). Moreover, the OPU method yielded a significantly higher (P < 0.05) number of grade 2 blastocyst (3.0 ± 0.1) compared with aspiration (0.50 ± 0.1). However, there was no significant (P > 0.05) difference in the number of grade 1 and grade 3 blastocysts obtained when the 3 recovery methods were used. In conclusion, the Boran breed showed better a sperm fertility rate following in vivo and in vitro embryo production. The superovulation and OPU methods resulted in higher numbers and better quality blastocysts compared with aspiration.

scholarly journals Phosphatidylcholine and Sphingomyelin Profiles Vary in Bos taurus indicus and Bos taurus taurus In Vitro- and In Vivo-Produced Blastocysts1

2012 ◽  
Vol 87 (6) ◽  
Author(s):  
Mateus J. Sudano ◽  
Vanessa G. Santos ◽  
Alessandra Tata ◽  
Christina R. Ferreira ◽  
Daniela M. Paschoal ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bos Taurus Indicus

Measuring esophageal distension by high-frequency intraluminal ultrasound probe

2002 ◽  
Vol 283 (4) ◽  
pp. G886-G892 ◽  
Author(s):  
Poong-Lyul Rhee ◽  
Jianmin Liu ◽  
James L. Puckett ◽  
Ravinder K. Mittal
Keyword(s):  
High Frequency ◽  
Healthy Subjects ◽  
Normal Subjects ◽  
Ultrasound Images ◽  
Agarose Gel ◽  
Ultrasound Probe ◽  
Cross Sectional ◽  
Esophageal Sphincter

Distension of the esophagus can cause heartburn and chest pain; however, none of the available techniques to study the esophagus measure esophageal distension. We evaluated the technique of high-frequency intraluminal ultrasound probe (HFIUS) to measure the esophageal cross-sectional area (CSA) during gastroesophageal reflux (GER). The following methods were used: 1) the CSA of agarose gel tubes of known dimensions were measured using ultrasound probes; 2) seven normal subjects were studied to evaluate the esophageal CSA during different bolus volumes (1, 5, 10, 15, and 20 ml) of water swallows (WS); and 3) simultaneous pressures, pH, and ultrasound images of the esophagus were recorded in healthy subjects. In vitro studies showed that the HFIUS measured the CSA of the tubes accurately. The maximal CSA of the distal esophagus during WS with boluses of 1, 5, 10, 15, and 20 ml were 54, 101, 175, 235, and 246 mm2, respectively. Esophageal contents during 62 episodes of transient lower esophageal sphincter relaxations, 29 pH positive, and 33 pH negative GER episodes revealed that reflux of air into the esophagus occurred more frequently than liquid. The median CSA and estimated diameter of the esophagus during liquid GER was 44.1 mm2 and 7.5 mm, respectively. We conclude that HFIUS is a valid technique to measure the CSA of the esophagus in vivo during GER. Distension of the esophagus during physiological GER is relatively small.

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

86 Comparison of in vivo and in vitro embryo production in Bonsmara donors

2019 ◽  
Vol 31 (1) ◽  
pp. 168
Author(s):  
B. H. Bernal ◽  
J. L. Barajas ◽  
J. A. Ortega ◽  
A. Cedeño ◽  
S. Andrada ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Gonadotropin Releasing Hormone ◽  
P Value ◽  
In Vitro Production ◽  
Embryo Production ◽  
Releasing Hormone ◽  
Gradient Technique ◽  
Vitro Production

A retrospective analysis of embryo production records from 2013 to 2017 was carried out to evaluate the in vivo and in vitro production (IVP) of embryos in donors of the Bonsmara breed (i.e. tropically adapted Bos taurus). Only donors with production records of both in vivo and in vitro embryos during the same period were used. A total of 127 superovulations and ova/embryo collections of 19 donors were evaluated. The donors were superstimulated with the following protocol: on Day 0 they received a device with 1g of progesterone (DIB, Zoetis, Argentina), 50mg of rogesterone (Progestar, Zoetis), and 5mg of oestradiol-17β (17ßOestradiol, Rio de Janeiro, Argentina) or 2mg of oestradiol benzoate (Gonadiol, Zoetis) intramuscularly (IM) at the same time. Superstimulatory treatments were initiated on the morning of Day 4 with Folltropin-V (Vetoquinol, France; total dose=240 to 340mg IM) in twice-daily decreasing doses over 4 days. All donors received 2 IM injections of 500µg of cloprostenol (Ciclase DL, Zoetis) on the morning and afternoon of Day 6 and; the intravaginal devices were removed on the morning of Day 7 and 100µg of Gonadorelin (gonadotropin-releasing hormone, Gonasyn gdr, Zoetis) was given on the morning of Day 8. Donors were inseminated using semen from 9 Bonsmara bulls, 12 and 24h after gonadotropin-releasing hormone. On Day 15, ova/embryos were collected and classified according to IETS standards. A total of 89 follicular aspirations (ovum pickup) of 19 donors for IVP were evaluated. The ovum pickups were performed at random stages of oestrous cycle, without superstimulation or other hormone treatments. A total of 1109 viable oocytes (12.5±0.9 per ovum pickup) were collected and matured for 24h in 100-µL drops of maturation medium (TCM-199, supplemented with hormones) under mineral oil and incubated at 38.5°C in 5.5% CO2 and humidity at saturation. Fertilization was performed using 3 Bonsmara bulls that were also used for in vivo embryo production. Viable sperm were obtained using the percoll gradient technique (45-90%). The sperm pellet was dissolved in TL-Sperm, centrifuged, and then diluted to a final concentration of 1.5×106 sperm/mL. Zygotes were stripped and placed in drops of 100µL of SOF medium supplemented with 0.4% BSA under oil at 38.8°C, 5.5% CO2, 7% O2, and humidity at saturation for 7 days. The culture medium was renewed on Days 3 and 5. The data were analysed using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA), a P-value &lt;0.05 was considered significant. The mean (±standard error of the means) number of CL, ova/embryos collected, fertilized ova, and transferable embryos were 12.9±0.6, 8.8±0.6, 6.6±0.5, and 4.7±0.4, respectively. A total of 662 oocytes (66.3±2.4%) cleaved 48h post-IVF. On Day 7, an average of 4.4±0.3 embryos were produced. No differences were detected in the number of transferable embryos produced in vivo v. those produced in vitro. Furthermore, no significant differences were found between the techniques or bulls on the proportion of embryos produced in relation to the ova/embryos or oocytes obtained (in vivo 51.5±3.2% v. in vitro 42.9±2.5%). In conclusion, the in vivo and in vitro production of embryos are both effective alternatives to increase the number of offspring from valuable Bonsmara donors.

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