206 ALPHA6, BETA1, AND BETA3 INTEGRINS EXPRESSED BY SPERM MAY BE INVOLVED IN CATTLE FERTILIZATION

2009 ◽  
Vol 21 (1) ◽  
pp. 201 ◽  
Author(s):  
R. F. Gonçalves ◽  
R. P. Bertolla ◽  
R. A. Mortara ◽  
V. H. Barnabe
Keyword(s):  
Bos Taurus ◽  
Gradient Centrifugation ◽  
Bos Indicus ◽  
Integrin Β1 ◽  
Alexa Fluor ◽  
Integrin Β3 ◽  
Sperm Suspension ◽  
Generous Contribution ◽  
Human And Mouse

Sperm-egg interaction is a complex molecular process leading to gamete fusion mediated by a series of molecular interactions. Some integrin subunits, which are adhesion molecules, are expressed on human and mouse sperm, but major questions about the roles of integrins in sperm-oocyte fusion remain unsolved. This study was conducted to determine the presence of integrin subunits on cattle (Bos indicus and Bos taurus) sperm, and whether fertilization might be affected by treating sperm with antibodies to integrin subunits. Frozen–thawed sperm, donated by ABS Pecplan, were centrifuged at 700g for 10 min and washed once in warm PBS (Nutricell®, Campinas, São Paulo, Brazil). Sperm were resuspended in PBS and treated with an equal volume of cold 4% paraformaldehyde (prepared fresh from formaldehyde) for 15 min on ice. All subsequent steps were conducted at RT. Fixed sperm were washed twice with PBS (10 000 rpm, 5 min) in an IEC Micromax (Needham, MA), and the pellet was resuspended in PBS containing 5% BSA. Samples of 1 mL, each containing 5 × 106 spermatozoa, were incubated overnight at 4°C with a monoclonal anti-integrin α6 immunoglobulin G (IgG), monoclonal anti-integrin β1 IgG, and monoclonal anti-integrin β3 IgG (Chemicon®). The next day, sperm were resuspended in Alexa Fluor® 488 goat anti-mouse IgG (Invitrogen). After being washed 3 times in PBS-BSA as before, a drop of sperm suspension was smeared on a slide, air-dried, mounted with antifade reagent with 4′,6-diamidino-2-phenylindole (Invitrogen), and covered with a coverslip for analysis. Frozen–thawed spermatozoa were washed by a 45/90% layered Percoll gradient centrifugation and incubated for 1 h in fertilization medium (FM; 1), FM with a monoclonal anti-integrin α6 IgG (2), FM with monoclonal anti-integrin β1 IgG (3), and FM with monoclonal anti-integrin β3 (4). In vitro-matured cattle oocytes were incubated (39°C, 5% CO2 in air) with 10 × 104 washed pretreated spermatozoa per 25 oocytes for 18 h. The oocytes were fixed in acid alcohol, stained with 1% acetate-orcein, and observed to determine the presence of pronuclei. Immunoreactive α6, β1, and β3 were concentrated at the apical segment of the acrosome in all bulls tested. There was no detectable fluorescence in any of the controls, and not all the sperm in an ejaculate were immunoreactive. Addition of anti-integrin α6, anti-integrin β1, and anti-integrin β3 decreased fertilization (P < 0.05) compared with the control: (1) 91.2 ± 2.0%; (2) 17.4 ± 2.0%; (3) 14.3 ± 2.0%; (4) 24.3 ± 2.0%. These findings show that α6, β1, and β3 integrins are expressed by cattle spermatozoa and may be involved in sperm-oocyte fusion and fertilization. This study was supported by FAPESP grants (2007/00363-5 and 2006/06008-0, Brazil). We acknowledge Nutricell and ABS Pecplan for their generous contribution.

scholarly journals Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro

2013 ◽  
Vol 79 (2) ◽  
pp. 351-357 ◽  
Author(s):  
C.F. Silva ◽  
E.S. Sartorelli ◽  
A.C.S. Castilho ◽  
R.A. Satrapa ◽  
R.Z. Puelker ◽  
...  
Keyword(s):  
Heat Stress ◽  
Bos Taurus ◽  
Bos Indicus

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.

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.

179 COMPARISON OF OOCYTE AND EMBRYO PRODUCTION AMONG BOS TAURUS, BOS INDICUS, AND INDICUS-TAURUS DONOR COWS

2010 ◽  
Vol 22 (1) ◽  
pp. 248 ◽  
Author(s):  
J. H. F. Pontes ◽  
K. C. F. Silva ◽  
A. C. Basso ◽  
C. R. Ferreira ◽  
G. M. G. Santos ◽  
...  
Keyword(s):  
High Efficiency ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Cumulus Cells ◽  
Holstein Cows ◽  
Embryo Production ◽  
Total N ◽  
The Mean ◽  
Donor Cows

In recent years, Brazil has become the leading country in the world for the number of embryos produced in vitro (Thibier M 2009 IETS Embryo Transfer Newsletter 22, 12-19). This is partly due to the large numbers of Bos indicus animals in Brazil, making up about 80% of the total cattle. The mean oocyte production per ultrasound-guided follicular aspiration from Bos indicus is higher than those for European breeds (Pontes JHF et al. 2009 Theriogenology 71, 690-697). In the present study, we analyzed 5407 ovum pick ups (OPU) and compared the average production of total (n = 90,086) and viable (n = 64,826) oocytes and the number of embryos produced in vitro from Gir (Bos taurus indicus), Holstein (Bos taurus taurus), 1/4 Holstein × 3/4 Gir, and 1/2 Holstein-Gir crossbreed cows. To obtain oocytes, OPU was repeated from 4 to 7 times (mean = 5.7 ± 2.4) in each donor cow aged from 3 to 7 years (mean = 5.0 ± 2.3) during a 12-mo period. COCs (n = 90,086) obtained were classified according to the presence of cumulus cells and the oocyte cytoplasm aspect (homogeneous or heterogeneous/fragmented). The viable oocytes (n = 64,826) were in vitro matured for 24 h at 38.8°C in an atmosphere of 5% CO2 in air. Since this was a commercial programm, frozen sexed semen (2 × 106 mL-1) from Gir (n = 8) or Holstein (n = 7) sires previously tested for high efficiency was used for IVF. Fertilization was carried out (18-20 h) and the presumed embryos were cultured for 7 days in the same conditions as were used for IVM. Data were analyzed by ANOVA. On average, 16.7 ± 6.2 oocytes were obtained per OPU/IVF procedure and 71.96% were considered viable. The mean numbers of total oocytes per OPU/IVF procedure were 17.1 ± 4.4 for Gir cows (n = 617), 11.4 ± 3.9 for Holstein cows (n = 180), 20.4 ± 5.8 for 1/4 Holstein × 3/4 Gir (n = 44), and 31.4 ± 5.6 for 1/2 Holstein-Gir crossbreed females (n = 37, P < 0.01). The mean numbers of viable oocytes per OPU/IVF procedure were 12.1 ± 3.8 for Gir cows, 8.0 ± 2.6 for Holstein cows, 16.8, ± 5.0 for 1/4 Holstein × 3/4 Gir, and 24.3 ± 4.7 for 1/2 Holstein-Gir crossbreed females (P < 0.01). The average number of embryos produced by OPU/IVF were 3.2 (n = 12,243/3378) for Gir cows, 2.2 (n = 2426/1138) for Holstein cows, 3.9 (n = 1033/267) for 1/4 Holstein × 3/4 Gir, and 5.5 (n = 1222/224) for 1/2 Holstein-Gir. The average number of embryos produced per IVF session from 1/2 taurus × indicus donor cows was greater (P < 0.01) than from Bos indicus cows. The number of recoverable and viable oocytes and the number of embryos produced in vitro from Bos indicus donors were higher than from Bos taurus females. Therefore, the highest oocyte yield and the greatest embryo production were obtained from 1/2 taurus × indicus females. This work was supported by In Vitro Brasil.

scholarly journals Effects of a high-energy diet on oocyte quality and in vitro embryo production in Bos indicus and Bos taurus cows

2015 ◽  
Vol 98 (5) ◽  
pp. 3086-3099 ◽  
Author(s):  
J.N.S. Sales ◽  
L.T. Iguma ◽  
R.I.T.P. Batista ◽  
C.C.R. Quintão ◽  
M.A.S. Gama ◽  
...  
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Oocyte Quality ◽  
High Energy ◽  
Embryo Production ◽  
In Vitro Embryo Production

scholarly journals Frecuencias alélicas para variantes SNP en el gen Nramp1 en bovinos infectados con Brucella abortus o clasificados por resistencia al patógeno

2009 ◽  
Vol 10 (1) ◽  
pp. 43
Author(s):  
Fernando Cerquera M. ◽  
Rodrigo Martínez S. ◽  
Rubén Toro O. ◽  
Jaime Tobón C. ◽  
Jaime Gallego G. ◽  
...  
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Brucella Abortus ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Natural Resistance ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Allelic Frequencies ◽  
Bos Taurus Indicus

<p>La resistencia natural a la brucelosis en bovinos ha sido asociada a factores genéticos, principalmente a algunos polimorfismos de nucleótido simple ubicados dentro del gen Nramp1. La presente investigación evalúa el efecto de variantes tipo polimorfismos de nucleótido simple presentes en regiones codificantes y en la región 3’UTR del gen Nramp1, en la clasificación de los animales como resistentes o susceptibles; además se determinan los genotipos predominantes en animales naturalmente infectados y comprobados como positivos por la presencia de anticuerpos anti <em>Brucella abortus</em>. Se establecieron las frecuencias genotípicas y alélicas para cinco polimorfismos de nucleótido simple identificados dentro del gen Nramp1 en animales de las razas blanco orejinegro (<em>Bos taurus taurus</em>) y cebú (<em>Bos taurus indicus</em>) y en muestras serológicamente positivas provenientes de animales cruzados (<em>Bos taurus </em>x <em>Bos indicus</em>). La determinación de genotipos se realizó mediante la metodología polimorfismo conformacional de cadena sencilla. Se realizó un ensayo de desafío infeccioso in vitro, para estimar la capacidad de los macrófagos bovinos para controlar la sobrevivencia bacterial, lo que permitió definir los individuos como resistentes o susceptibles. Los resultados sugieren una asociación significativa del SNP4 (<em>p </em>= 0,0506) con la variación para el fenotipo de susceptibilidad, pues se encontró el genotipo homocigoto (BB) en alta frecuencia en animales catalogados como resistentes y el genotipo heterocigoto (AB) en alta frecuencia en animales catalogados como susceptibles y en animales con títulos de anticuerpos anti <em>Brucella abortus</em>.  </p><p> </p><p><strong>Allelic frequencies for SNP variants in the gene Nramp1 in bovine infected with </strong><strong><em>Brucella abortus </em></strong><strong>or classified by resistance to the pathogen</strong>  </p><p>The natural resistance to brucellosis in cattle has been associated to genetic factors mainly to some single nucleotide polymorphism (SNP), located within Nramp1 gen. The current research has studied the effect of nucleotide variants to be found in coding regions and other one located in 3 non translated region of Nramp1 gene, on the animal classification as resistant or susceptible, moreover was identified the main genotypes to be found on the infected animals, confirmed as positives by antibody antibrucella titles. Was established the genotypic and allelic frequencies for five single nucleotide polymorphism in animals from blanco orejinegro (<em>Bos taurus taurus</em>) and zebu breeds (<em>Bos taurus indicus</em>) and serum samples belonging to positive crossbred animals (<em>Bos taurus x Bos indicus</em>). The genotype was defined by the methodology known as “single strand conformational polymorphism”. To estimate the macrophage capacity to control the bacterial survival, an in vitro assay was performed, which allowed define the phenotype as resistant or susceptible. The results suggest a significant association for SNP4 (p = 0.0506) with the phenotypic variation for resistant or susceptibility, because was found the genotype (BB) at higher frequency in susceptible animals and naturally infected animals, than those resistant animals. </p>

scholarly journals 180EFFECT OF TIME OF ECG TREATMENT ON PREGNANCY RATES IN BOS INDICUS×BOS TAURUS RECIPIENTS SYNCHRONIZED WITH PROGESTERONE VAGINAL DEVICES AND TRANSFERRED WITHOUT ESTRUS DETECTION

2004 ◽  
Vol 16 (2) ◽  
pp. 212
Author(s):  
L.F. Nasser ◽  
E.L. Reis ◽  
A.M. Oliveira ◽  
G.A. Bo ◽  
P.S. Baruselli
Keyword(s):  
Bos Taurus ◽  
Bos Indicus ◽  
Fixed Time ◽  
Estradiol Benzoate ◽  
Pregnancy Rates ◽  
Bovine Embryo ◽  
Treatment Groups ◽  
Estrus Detection ◽  
Treatment Interaction

It has been shown recently that treatments with progesterone (P4)-releasing devices combined with estradiol benzoate (EB) plus P4 on Day 0, eCG and PGF on Day 5 and a second application of EB one day after device removal (Day 9) can be used successfully to transfer bovine embryos at a self-appointed time, without the necessity of estrus detection. Although the treatment solved one of the major problems in recipient management, estrus detection, it requires handling the recipients at least five times for treatments and embryo transfer. An experiment was designed to evaluate whether reducing one day of handling, by the administration of eCG and PGF at the time of removal of the P4 device (Day 8), results in comparable pregnancy rates than giving eCG on Day 5. A secondary objective was to determine the effect of injectable P4 at the time of device insertion plus EB treatment. Crossbred Bos taurus×Bos indicus beef heifers (n=301) were randomly assigned to 4 treatment groups in a 2 by 2 factorial design. All Heifers received a P4 device (DIB, Syntex, Argentina) plus 2mg EB i.m. (Syntex) at unknown stages of the estrous cycle (Day 0), with or without 50mg of P4 given i.m. at the same time. Heifers were further subdivided to receive PGF (0.150mg d-cloprostenol, Prolise, Tecnopec, Sao Paulo, Brazil) and 400IU of eCG (Novormon, Syntex) i.m. on Days 5 or 8. In all heifers, DIB devices were removed on Day 8 and 1 mg EB was administered i.m. on Day 9. Day 10 was arbitrarily considered as the day of estrus. On Day 17, heifers were bled for plasma P4 concentrations and examined by ultrasonography to determine the number of CL and their diameter. Heifers that had &gt;1 CL or a single CL with diameter ≥18mm received an in vitro-produced (IVP) embryo by nonsurgical transfer performed by the same veterinarian. Pregnancy rates were determined by ultrasonography 30 days later. The effects of Day of eCG administration (Day 5 or Day 8), P4 of treatment (E2 or E2+P4) and the day-by-P4 treatment interaction on the numbers of CL and plasma P4 were analyzed by ANOVA, and the proportion of recipients selected and pregnant were analyzed using non-parametric tests (NPAR1WAY, SAS). There was no significant effect of P4 treatment or the P4-by-day of eCG interaction in any of the parameters evaluated. However, there was a significant effect of day of eCG administration on plasma P4 concentrations (Day 5=2.4±0.3 v. Day 8=1.7±0.2; P=0.03) and the number of CL (Day 5=1.4±0.1 v. Day 8=1.1±0.0; P=0.02) on Day 17. Furthermore, the proportion of recipients pregnant/treated tended (P=0.1) to be higher in heifers in the Day 5 Group (71/151, 47.0%) than in those in the Day 8 Group (61/150, 40.7%). Although delaying the eCG and PGF administration from Day 5 to Day 8 saves one trip through the chute for treatments, it resulted in lower plasma P4 concentrations and tended to decrease pregnancy rates in bovine embryo recipients synchronized with DIB devices and EB and transferred at a fixed time. Furthermore, the administration of injectable P4 at the time of DIB insertion did not affect pregnancy rates.

272 DEVELOPMENTAL COMPETENCE OF OOCYTES OBTAINED FROM BOS TAURUS AND BOS INDICUS DAIRY COWS RAISED IN A TROPICAL CLIMATE

2006 ◽  
Vol 18 (2) ◽  
pp. 243
Author(s):  
L. S. A. Camargo ◽  
J. H. M. Viana ◽  
W. F. Sa ◽  
A. M. Ferreira ◽  
A. A. Ramos ◽  
...  
Keyword(s):  
Dairy Cows ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Calf Serum ◽  
Developmental Competence ◽  
Tropical Region ◽  
Humid Atmosphere ◽  
Vitro Fertilization ◽  
Developmental Capacity

The effects of heat stress on Bos taurus reproductive performance in tropical and subtropical regions are well known, and have been associated with lower oocyte developmental capacity. The aim of this study was to evaluate the developmental competence of oocytes from Bos taurus (Holstein) and Bos indicus (Gyr) dairy cows raised in a Brazilian tropical region, located at 21°35′S latitude, 43°51′W longitude, and 435 meters altitude. Cumulus–oocyte complexes (COCs) were recovered by oocyte pickup (OPU) from mature non-lactating Holstein (n = 9) and Gyr (n = 13) donor cows between the end of spring and the beginning of autumn, with at least two OPU sessions/cow. COCs were in vitro-maturated in TCM-199 (GIBCO, Grand Island, NY, USA) with 10% inactivated estrus cow serum for 24 h under 5% CO2 at 38.5°C in air. Bos taurus and Bos indicus semen with similar cleavage rates, previously evaluated by in vitro fertilization with oocytes obtained from slaughterhouse ovaries, were used to reduce bull effect. Holstein and Gyr spermatozoa were obtained through swim-up method and co-incubated with Holstein (n = 390) and Gyr (n = 505) oocytes, respectively, in Fert-TALP medium (Parrish et al. 1988 Biol. Reprod. 38, 1171–1180) supplemented with 10 μg/mL heparin (Sigma-Aldrich, Sao Paulo, Brazil) and 6 mg/mL fatty acid-free bovine albumin (Sigma) for 18 h in 5% CO2 at 38.5°C in air. Presumptive zygotes were co-cultured with their own cumulus cells in CR2aa medium (Wilkinson et al. 1996 Theriogenology 45, 41–49) supplemented with 10% fetal calf serum in humid atmosphere of 5% CO2 at 38.5°C in air. On Day 7 to 8 of co-culture, Gyr and Holstein blastocysts were assessed and those classified as grade 1 (IETS Manual) were transferred to synchronized Bos indicus × Bos taurus crossbred recipients managed under the same nutritional and environmental conditions. Pregnancy diagnosis was performed between 35 and 50 days after estrus. Cleavage, blastocyst, and pregnancy rates were analyzed by chi-square test. Cleavage and blastocyst rates were greater (P < 0.05) in Gyr than in Holstein (66.7% vs. 53.1% for cleavage and 19.6% vs. 10.8% for blastocyst, respectively), but the pregnancy rate was similar (P > 0.05; 44.5% vs. 60% for Gyr and Holstein, respectively). These results show that Gyr oocytes obtained in a tropical region have greater developmental capacity than Holstein oocytes, suggesting an interaction between genotype and environment that influences the success of an in vitro embryo production program; nevertheless, the blastocyst viability after transferring to recipients is similar for both breeds.

48 GLOBAL GENE EXPRESSION PATTERN OF BOS INDICUS AND BOS TAURUS VITRIFIED EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 117
Author(s):  
R. Cancian ◽  
M. Macelai ◽  
G. Tavares ◽  
R. S. Valente ◽  
E. S. Caixeta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Pattern ◽  
Cellular Response ◽  
Bos Taurus ◽  
Bos Indicus ◽  
Gene Expression Pattern ◽  
Global Gene Expression ◽  
Differentially Expressed ◽  
Canonical Pathways

The cryopreservation of in vitro-produced (IVP) bovine embryos is one of the most challenging areas of the assisted reproductive biotechnologies. The aim of the present study was to evaluate the global gene expression pattern of Bos indicus (Nellore) and Bos taurus (Simmental) IVP embryos after vitrification. Follicular aspiration was performed on Nellore (n = 14) and Simmental (n = 14) cows, and oocytes (n = 840 and 450; respectively) were submitted to in vitro maturation and in vitro fertilization. Presumptive zygotes were denuded and cultured in SOFaa with 0.5% BSA and 2.5% FCS during 7 days under standard culture conditions. Blastocysts (grade 1 and 2) were vitrified, warmed, and cultured for an additional 12 h under the same conditions. Nellore (n = 8) and Simmental (n = 8) IVP blastocysts considered viable after vitrification, with re-expanded blastocoel, were submitted to total RNA extraction (PicoPure, Arcturus, Applied Biosystems®, Foster Dity, CA, USA), DNAse I treatment (Qiagen®, Valencia, CA, USA), and amplification (RiboAmp, Applied Biosystems®). Fragmented cRNA were obtained through 3′IVT Express Kit (Affymetrix®, Santa Clara, CA, USA) to perform the hybridization using GeneChip Bovine Genome Array (Affymetrix®). Microarray data analysis was performed using the FlexArray 1.6.1.1 software. Genes with at least a 1.5-fold change and a P-value of less than 0.05 were considered differentially expressed. Of the 1278 genes differentially expressed between Bos taurus and Bos indicus vitrified embryos, 1108 were annotated, with 1193 genes up-regulated and 85 genes down-regulated in Bos taurus compared with Bos indicus IVP vitrified embryos. Differentially expressed genes were associated with the functional networks of cell cycle, cellular movement and DNA replication, recombination and repair; RNA post-transcriptional modifications; gene expression, protein synthesis; RNA damage and repair; cellular function and maintenance; and cell death and survival. The top 6 canonical pathways generated by Ingenuity Pathway Analysis® with the differentially expressed genes were ELF2 signalling, oxidative phosphorylation, tricarboxylic acid cycle, protein ubiquitination pathway, mTOR signalling, and IGF-1 signalling. In conclusion, Bos taurus IVP embryos seem to trigger different cellular response mechanisms to the vitrification stress in comparison with Bos indicus IVP embryos. Differential response is mainly represented by different expression profiles of genes regulating important canonical pathways involved in cellular response to stress that could be related with the higher post-cryopreservation survival capacity observed in Bos taurus embryos.Research was supported by FAPESP, CNPq, FAPERGS, and LNBio – National Laboratory of Biosciences/MCT.

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