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

In a 2 × 2 factorial experimental design, embryo development, cryotolerance and global gene expression of Nellore (Bos taurus indicus) and Simmental (Bos taurus taurus) blastocysts produced in vitro (IVP) and in vivo (multiple ovulation derived embryo, MODE) were assessed. Blastocyst production was higher in Nellore than in Simmental (47.7 ± 2.0% vs 27.0 ± 2.0%) cows. The total numbers of ova or embryos recovered (5.5 ± 0.9 vs 3.7 ± 0.8) and transferable embryos (3.8 ± 1.0 vs 2.3 ± 0.8) per cow were not different between breeds. Simmental and MODE (34.6% and 38.5%, n = 75 and 70) blastocysts had higher survival rates after cryopreservation compared with Nellore and IVP (20.2% and 18.1%, n = 89 and 94) embryos, respectively. Differences between transcriptomes were addressed by principal-component analysis, which indicated that gene expression was affected by subspecies (158 genes), origin (532 genes) and interaction between both subspecies and origin (53 genes). Several functional processes and pathways relevant to lipid metabolism and embryo viability involving differentially expressed genes were identified. The lipid metabolism-related genes were upregulated in Simmental (AUH and ELOVL6) and IVP (ACSL3 and ACSL6) blastocysts. The expression profiles of genes related to mitochondrial metabolism (ATP5B), oxidative stress (GPX4), apoptosis (DAD1, DAP, PRDX2), heat shock (HSPA5), pregnancy (IFNT2, PAG2) and cell differentiation (KRT18) varied between experimental groups.

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Fetal calf serum enhances in vitro production of Bos taurus indicus embryos

Theriogenology ◽

10.1016/j.theriogenology.2010.08.017 ◽

2011 ◽

Vol 75 (3) ◽

pp. 429-433 ◽

Cited By ~ 9

Author(s):

F.G. Leivas ◽

D.S. Brum ◽

S.S. Fialho ◽

W.P. Saliba ◽

M.T.T. Alvim ◽

...

Keyword(s):

Fetal Calf Serum ◽

Bos Taurus ◽

Calf Serum ◽

In Vitro Production ◽

Bos Taurus Indicus ◽

Vitro Production

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PROPORÇÃO DO SEXO NA PRODUÇÃO IN VITRO DE EMBRIÕES BOVINOS

Archives of Veterinary Science ◽

10.5380/avs.v20i2.37390 ◽

2015 ◽

Vol 20 (2) ◽

Author(s):

Marcus Vinícius Galvão Loiola ◽

Alexandra Soares Rodrigues ◽

Marcos Chalhoub ◽

Priscila Assis Ferraz ◽

Rodrigo Freitas Bittencourt ◽

...

Keyword(s):

Bos Taurus ◽

Bos Taurus Indicus

Objetivou-se avaliar a proporção do sexo utilizando espermatozoides provenientes de sêmen convencional ou sexado para fêmea em um programa de produção in vitro de embriões (PIVE) bovinos. Foram utilizados oócitos de 87 doadoras Bos taurus indicus da raça Nelore, obtidos por sistema de aspiração folicular transvaginal guiado por ultrassonografia. Os oócitos selecionados foram maturados in vitro, fertilizados com espermatozoides oriundos de sêmen convencional (grupo 1 – CONV) ou sêmen sexado para fêmea (grupo 2 – SEX) e os embriões produzidos foram então cultivados in vitro por sete dias. Após o cultivo, os mesmos foram envasados e inovulados em receptoras mestiças Bos taurus taurus x Bos taurus indicus. Foram transferidos 1.319 embriões, sendo 1.081 produzidos com sêmen convencional (CONV) e 238 com sêmen sexado de fêmea (SEX). O diagnóstico precoce de gestação e a sexagem fetal foram realizados por ultrassonografia transretal. A taxa de gestação total foi 37,38% (493/1.319), sendo 37,74% (408/1.081) no grupo CONV e 35,71% (85/238) no grupo SEX. A proporção do sexo no grupo CONV foi desviada (P=0,002) a favor dos machos (57,84% de machos e 42,16% de fêmeas) e no grupo SEX ocorreu um desvio na proporção (P=0,001) a favor das fêmeas (68,24% de fêmeas e 31,76% de machos). Concluiu-se que a utilização do sêmen convencional em PIVE bovinos elevou a proporção de produtos machos e apesar da utilização de sêmen sexado de fêmea ser uma alternativa para pré-selecionar o sexo dos produtos, ainda houve uma considerável taxa de machos quando se utilizou este tipo de sêmen.

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Follicular development, morphological integrity, and oxidative stress in bovine preantral follicles cultured in vitro with ascorbic acid

Zygote ◽

10.1017/s0967199421000903 ◽

2021 ◽

pp. 1-7

Author(s):

Larissa Zamparone Bergamo ◽

Denis Vinicius Bonato ◽

Camila Bizarro-Silva ◽

Francieli Gesleine Capote Bonato ◽

Tamires Korchovei Sanches ◽

...

Keyword(s):

Oxidative Stress ◽

Ascorbic Acid ◽

Culture Medium ◽

Bos Taurus ◽

Follicular Development ◽

Ovarian Follicles ◽

Bos Taurus Indicus ◽

Bonferroni Test ◽

And Oxidative Stress

Summary The aim of this study was to evaluate the follicular development, morphological integrity, and oxidative stress of preantral ovarian follicles from Bos taurus indicus females grown in vitro with ascorbic acid. Ovaries (n = 20) from Bos taurus indicus females were collected, fragmented, and were cultured in vitro for 6 or 12 days in minimum essential medium (MEM), or MEM supplemented with 50 or 100 ng/ml ascorbic acid, with an extracellular matrix of agarose gel, in an incubator at 38.5°C; every 2 days, 100% of the culture medium was replaced. The data were analyzed using the chi-squared test and/or Fisher’s exact test. In the event of a significant effect, the proportions were compared using a 2 × 2 proportion test. The oxidative stress analysis data were submitted to analysis of variance followed by the Bonferroni test. Values were considered significant when P ≤ 0.05. The addition of 100 ng/ml of ascorbic acid to the in vitro culture medium of preantral ovarian follicles from bovine females promoted follicular development, was efficient in maintaining morphological integrity, as well as the stability of reactive oxygen species, after 6 days of in vitro culture.

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

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab257 ◽

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab121 ◽

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.

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

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab7 ◽

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.

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Effects of scrotal insulation in Nellore bulls (Bos taurus indicus) on seminal quality and its relationship with in vitro fertilizing ability

Theriogenology ◽

10.1016/j.theriogenology.2008.07.005 ◽

2008 ◽

Vol 70 (9) ◽

pp. 1560-1568 ◽

Cited By ~ 34

Author(s):

C.E. Fernandes ◽

M.A.N. Dode ◽

D. Pereira ◽

A.E.D.F. Silva

Keyword(s):

Bos Taurus ◽

Bos Taurus Indicus ◽

Fertilizing Ability ◽

Scrotal Insulation

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

Ciencia y Tecnología Agropecuaria ◽

10.21930/rcta.vol10_num1_art:127 ◽

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

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 Brucella abortus. 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 (Bos taurus taurus) y cebú (Bos taurus indicus) y en muestras serológicamente positivas provenientes de animales cruzados (Bos taurus x Bos indicus). 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 (p = 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 Brucella abortus. Allelic frequencies for SNP variants in the gene Nramp1 in bovine infected with Brucella abortus or classified by resistance to the pathogen 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 (Bos taurus taurus) and zebu breeds (Bos taurus indicus) and serum samples belonging to positive crossbred animals (Bos taurus x Bos indicus). 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.

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A INTERFERÊNCIA DA PRODUÇÃO DE LEITE NAS TAXAS DE RECUPERAÇÃO OOCITÁRIA E CONVERSÃO EM EMBRIÕES DE DOADORAS DAS RAÇAS GIR E MEIO SANGUE

Archives of Veterinary Science ◽

10.5380/avs.v23i4.60351 ◽

2018 ◽

Vol 23 (4) ◽

Author(s):

Victor Henrique Miquelanti ◽

Carlos Renato Freitas Guaitolini ◽

Eduardo Trevisol ◽

Andre Maciel Crespilho ◽

Rosiara Rosária Dias Maziero

Keyword(s):

Bos Taurus ◽

Bos Taurus Indicus

Este estudo teve por objetivo avaliar a interação entre a produção de leite e a taxa de recuperação oocitária, produção in vitro de embriões e conversão oócito-embrião de doadoras da raça Gir, Girolando 1/2 sangue e Girolando 5/8. Para o estudo foram utilizadas 150 vacas doadoras de oócitos, selecionadas com base em seu mérito genético. Destas, 50 fêmeas da raça Gir (Bos taurus indicus), 60 fêmeas Girolando 1/2 sangue (1/2 Bos taurus taurus x 1/2 Bos taurus indicus) e 40 Girolando 5/8 (5/8 Bos taurus taurus x 3/8 Bos taurus indicus). A raça Gir apresentou recuperação oocitária inferior às demais avaliadas (Gir 17,5 ± 11,3 x Girolando 1/2 sangue 23,8 ± 15,0 x Girolando 5/8 24,8 ± 11,7) (P<0,05). Porém, o percentual de conversão em blastocistos mostrou ser superior, apesar de não apresentar diferença significativa na quantidade de embriões produzidos (Gir 29,1% x Girolando 1/2 sangue 27,7% x Girolando 5/8 18,5%) (P>0,05). Não houve interação entre a produção de leite e o genótipo da doadora (P>0,05). Entretanto, foi observado uma elevação na recuperação de oócitos e produção de embriões, com a progressão da curva de lactação nas raças Gir e Girolando 1/2 sangue. Por outro lado, as Girolando 5/8 apresentaram diminuição na recuperação de oócitos e conversão em embriões conforme aumento da produção leiteira. Assim, concluiu-se que produção de leite das matrizes não interferiu nos índices de recuperação de oócitos, tampouco na conversão em embriões. Entretanto, várias lacunas precisam ser preenchidas para melhor compreensão das características reprodutivas, principalmente de animais cruzados.

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