Absence of seminal plasma from sperm-rich fraction decreases boar sperm quality characteristics during the course of liquid storage.

Seminal plasma (SP), the fluid that surrounds the sperm cells, is known to exert substantial influence on sperm physiology. The SP has a pivotal role in sperm function in vivo, and due to its components, it functions in an ambiguous manner in vitro, simultaneously possessing deleterious and beneficial effects. This experiment aimed to describe the differences between the presence or absence of SP from the sperm-rich fraction on some spermatozoa characteristics (kinetics, plasma and acrosome membrane integrity, lipid peroxidation and capacitation-like changes). Furthermore, this experiment focused on distinguishing the effects of SP on the variables evaluated from the effects of centrifugation during SP removal. Total and progressive sperm motility, as well as integrity of plasma and acrosome membranes, were less (P < 0.05) in the absence of SP. Membrane lipid peroxidation (P < 0.05) and sperm membrane stability (P < 0.05) did not differ among treatments. The SP from the sperm-rich fraction is important for the maintenance of adequate structural and functional characteristics of extended liquid boar semen and should be present in seminal doses throughout storage. Furthermore, the detrimental effect on the variables evaluated was caused solely by the absence of SP and not by the process of removal through centrifugation at 500 x g for 10 min.

Low levels of exosomal-miRNAs in maternal blood are associated with early pregnancy loss in cloned cattle.

Nuclear reprogramming mediated by somatic cell nuclear transfer (SCNT) has many applications in medicine. However, animal clones show increased rates of abortion and reduced neonatal viability. Herein, we used exosomal-miRNA profiles as a non-invasive biomarker to identify pathological pregnancies. MiRNAs play important roles in cellular proliferation and differentiation during early mammalian development. Thus, the aim of this study was to identify exosomal-miRNAs in maternal blood at 21 days of gestation that could be used for diagnosis and prognosis during early clone pregnancies in cattle. Out of 40 bovine-specific miRNAs, 27 (67.5%) were with low abundance in the C-EPL (Clone - Early pregnancy loss) group compared with the C-LTP (Clone - Late pregnancy) and AI-LTP (Artificial Insemination - Late pregnancy) groups, which had similar miRNAs levels. Bioinformatics analysis of the predicted target genes demonstrated signaling pathways and functional annotation clusters associated with critical biological processes including cell proliferation, differentiation, apoptosis, angiogenesis and embryonic development. In conclusion, our results demonstrate decreased exosomal-miRNAs in maternal blood at 21 days of gestation in cloned cattle pregnancies that failed to reach term. Furthermore, the predicted target genes regulated by these 27 miRNAs are strongly associated with pregnancy establishment and in utero embryonic development.

Cellular and extracellular vesicular origins of miRNAs within the bovine ovarian follicle.

The ovarian follicle components must provide an ideal environment to ensure the success of reproductive processes, and communication between follicular cells is crucial to support proper oocyte growth. Recently, it has been demonstrated that the presence of extracellular vesicles (EVs) carrying microRNAs (miRNAs) in follicular fluid represents an important autocrine and paracrine communication mechanism inside the ovarian follicle. In this study, we tested the hypothesis that the miRNA content of EVs isolated from ovarian follicular (granulosa and cumulus-oocyte complexes) cell-conditioned culture media is dependent upon cell type. We initially screened bovine granulosa cells (GCs) and cumulus-oocyte complexes (COCs), as well as their derived EVs for 348 miRNAs using real-time PCR, and detected 326 miRNAs in GCs and COCs cells and 62 miRNAs in GCs and COCs EVs. A bioinformatics analysis of the identified cell-specific and differentially expressed miRNAs predicted that they likely modulate important cellular processes, including signalling pathways such as the PI3K-Akt, MAPK and Wnt pathways. By investigating the origins of miRNAs within the follicular fluid, the results of this study provide novel insights into follicular miRNA content and intercellular communication that may be of invaluable use in the context of reproductive technologies, diagnostic of ovarian-related diseases and/or the identification of biomarkers for oocyte and embryo quality.

Genotypic and allelic frequencies of gene polymorphisms associated with meat tenderness in Nellore beef cattle.

The objectives of this study were to characterize the allelic and genotypic frequencies of polymorphisms in the µ-calpain and calpastatin genes, and to assess their association with meat tenderness and animal growth in Nellore cattle. We evaluated 605 Nellore animals at 24 months of age, on average, at slaughter. The polymorphisms were determined for the molecular markers CAPN316, CAPN530, CAPN4751, CAPN4753, and UOGACAST1. Analyses of meat tenderness at 7, 14, and 21 days of maturation were performed in samples of longissimus thoracis obtained between the 12th and 13th rib and sheared using a Warner Bratzler Shear Force. Significant effects were observed for meat tenderness at days 7, 14, and 21 of maturation for the marker CAPN4751, at day 21 for the marker CAPN4753, and at days 14 and 21 for the marker UOGCAST1. For genotypic combinations of markers, the results were significant for the combination CAPN4751/UOGCAST1 in the three maturation periods and CAPN4753/UOGCAST1 at days 14 and 21 of maturation.

Generation of LIF-independent induced pluripotent stem cells from canine fetal fibroblasts.

Takahashi and Yamanaka established the first technique in which transcription factors related to pluripotency are incorporated into the genome of somatic cells to enable reprogramming of these cells. The expression of these transcription factors enables a differentiated somatic cell to reverse its phenotype to an embryonic state, generating induced pluripotent stem cells (iPSCs). iPSCs from canine fetal fibroblasts were produced through lentiviral polycistronic human and mouse vectors (hOSKM/mOSKM), aiming to obtain pluripotent stem cells with similar features to embryonic stem cells (ESC) in this animal model. The cell lines obtained in this study were independent of LIF or any other supplemental inhibitors, resistant to enzymatic procedure (TrypLE Express Enzyme), and dependent on bFGF. Clonal lines were obtained from slightly different protocols with maximum reprogramming efficiency of 0.001%. All colonies were positive for alkaline phosphatase, embryoid body formation, and spontaneous differentiation and expressed high levels of endogenous OCT4 and SOX2. Canine iPSCs developed tumors at 120 days post-injection in vivo. Preliminary chromosomal evaluations were performed by FISH hybridization, revealing no chromosomal abnormality. To the best of our knowledge, this report is the first to describe the ability to reprogram canine somatic cells via lentiviral vectors without supplementation and with resistance to enzymatic action, thereby demonstrating the pluripotency of these cell lines.

Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage / Conceptos bovinos (Bos indicus) produzidos por transferência nuclear de células somáticas e partenogênese apresentam variações morfológicas desde o estágio de blastocisto

In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles(AU)

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas distintas, o trofectoderma e a massa celular interna. Este último irá sofrer diferenciação para formar o disco embrionário, o qual consiste em epiblasto e hipoblasto. O objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino proveniente de diferentes técnicas de produção in vitro, com ênfase no trofectoderma e na massa celular interna. Embriões bovinos com sete dias de gestação (fecundação = D1), derivados de técnicas de produção in vitro, foram fixados para processamento de microscopia de luz e eletrônica de transmissão. Os resultados sugerem que os embriões produzidos por transferência nuclear de células somáticas e partenogênese apresentaram alterações significativas em suas estruturas macro e microscópica. O tamanho foi reduzido, e a massa celular interna não tinha uma forma definida. Além disso, organelas responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor número e tinham alterações na forma quando comparadas aos resultados em embriões produzidos por fertilização in vitro. Conclui-se que os embriões produzidos por SCNT e partenogênese apresentam diferenças morfológicas quando comparados aos embriões de fertilização in vitro, tais como blastocele pouco desenvolvida, massa celular interna pouco definida e diferenças morfológicas nas organelas(AU)

Bovine conceptus of Bos indicus produced by somatic cell nuclear transfer and parthenogenesis present morphological variations since the blastocyst stage / Conceptos bovinos (Bos indicus) produzidos por transferência nuclear de células somáticas e partenogênese apresentam variações morfológicas desde o estágio de blastocisto

In cattle, embryo development is characterized by the appearance of two distinct cell layers, the trophectoderm and the inner cell mass. The latter will undergo differentiation to form the embryonic disc consisting of the epiblast and hypoblast. The aim of this study was to ultrastructurally characterize the bovine embryo from different in vitro production techniques, with emphasis on trophectoderm and inner cell mass cells. Bovine embryos on day 7 (conception = D1) of pregnancy, derived via in vitro production techniques, were fixed for light and transmission electron microscopy processing. Results suggested that embryos produced by nuclear transfer of somatic cells and parthenogenesis showed significant changes in macroscopic and microscopic structure. Size was reduced, and the inner cell mass had no defined shape. Furthermore, organelles responsible for the absorption processes, communication, growth, and cellular metabolism were fewer and had changes in shape, when compared to results in embryos produced by in vitrofertilization. We concluded that embryos produced by parthenogenesis and SCNT exhibit morphological differences when compared with IVF embryos, such as undeveloped blastocoel, poorly defined distribution of ICM, and morphological differences in organelles.

Em bovinos, o desenvolvimento embrionário é caracterizado pelo surgimento de duas camadas distintas, o trofectoderma e a massa celular interna. Este último irá sofrer diferenciação para formar o disco embrionário, o qual consiste em epiblasto e hipoblasto. O objetivo deste estudo foi caracterizar ultraestruturalmente o embrião bovino proveniente de diferentes técnicas de produção in vitro, com ênfase no trofectoderma e na massa celular interna. Embriões bovinos com sete dias de gestação (fecundação = D1), derivados de técnicas de produção in vitro, foram fixados para processamento de microscopia de luz e eletrônica de transmissão. Os resultados sugerem que os embriões produzidos por transferência nuclear de células somáticas e partenogênese apresentaram alterações significativas em suas estruturas macro e microscópica. O tamanho foi reduzido, e a massa celular interna não tinha uma forma definida. Além disso, organelas responsáveis por processos de absorção, comunicação, crescimento e metabolismo celular estavam em menor número e tinham alterações na forma quando comparadas aos resultados em embriões produzidos por fertilização in vitro. Conclui-se que os embriões produzidos por SCNT e partenogênese apresentam diferenças morfológicas quando comparados aos embriões de fertilização in vitro, tais como blastocele pouco desenvolvida, massa celular interna pouco definida e diferenças morfológicas nas organelas.

Mitochondrial DNA dynamics during in vitro culture and pluripotency induction of a bovine Rho0 cell line.

Large number of cellular changes and diseases are related to mutations in the mitochondrial DNA copy number. Cell culture in the presence of ethidium bromide is a known way of depleting mitochondrial DNA and is a useful model for studying such conditions. Interestingly, the morphology of these depleted cells resembles that of pluripotent cells, as they present larger and fragmented mitochondria with poorly developed cristae. Herein, we aimed to study the mechanisms responsible for the control of mitochondrial DNA replication during mitochondrial DNA depletion mediated by ethidium bromide and during the in vitro induction of cellular pluripotency with exogenous transcription factor expression in a bovine model. This article reports the generation of a bovine Rho0 mesenchymal cell line and describes the analysis of mitochondrial DNA copy number in a time-dependent manner. The expression of apoptosis and mitochondrial-related genes in the cells during mitochondrial DNA repletion were also analyzed. The dynamics of mitochondrial DNA during both the depletion process and in vitro reprogramming are discussed. It was possible to obtain bovine mesenchymal cells almost completely depleted of their mitochondrial DNA content (over 90%). However, the production of induced pluripotent stem cells from the transduction of both control and Rho0 bovine mesenchymal cells with human reprograming factors was not successful.

Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production

This paper provides basic concepts of genomic selection (GS) methods in beef and dairy cattle production in combination with assisted reproductive technologies (ART) such as ovum-pick up and in vitroproduction (OPU-IVP). We first introduce genomic tools and discuss main methods of GS as practiced to-date. The general benefit from GS is that it enables selecting animals accurately early in life using genomic predictions particularly those phenotypes that are very difficult or expensive to measure. While it is known that GS increases genetic gain and profit in conventional cattle breeding, GS is much more desirable when combined with OPU-IVP in cattle production. The expected benefits of GS-OPU-IVP far exceed the benefits achieved by either GS or OPU-IVP alone mainly due to tremendous reduction in generation interval. The genetic improvement will increase even further, if genetic merit of donor cows and bulls used in OPU-IVP for key economic traits are maximal. The paper also highlights some challenges particularly with regard to embryo biopsies and quantity and quality of embryo DNA for whole genome genotyping and ways to overcome difficulties. We briefly discuss the somatic cell nuclear transfer (SCNT) technique in the context of applying GS on fibroblast cell lines from fetuses obtained from OPU-IVP techniques and provide our perspectives on how it might pave way for even more rapid cattle improvement. Main conclusion is that employing genomic selection in ARTs such as OPU-IVP of embryos coupled with embryo sexing and SCNT will lead to rapid dissemination of high genetic merit animals on a scale never been seen before. Finally, the paper outlines current research activities on combined genomic selection and advanced reproductive technologies in the GIFT project consortium (www.gift.ku.dk). (AU)

Genomic selection of in vitro produced and somatic cell nuclear transfer embryos for rapid genetic improvement in cattle production

This paper provides basic concepts of genomic selection (GS) methods in beef and dairy cattle production in combination with assisted reproductive technologies (ART) such as ovum-pick up and in vitroproduction (OPU-IVP). We first introduce genomic tools and discuss main methods of GS as practiced to-date. The general benefit from GS is that it enables selecting animals accurately early in life using genomic predictions particularly those phenotypes that are very difficult or expensive to measure. While it is known that GS increases genetic gain and profit in conventional cattle breeding, GS is much more desirable when combined with OPU-IVP in cattle production. The expected benefits of GS-OPU-IVP far exceed the benefits achieved by either GS or OPU-IVP alone mainly due to tremendous reduction in generation interval. The genetic improvement will increase even further, if genetic merit of donor cows and bulls used in OPU-IVP for key economic traits are maximal. The paper also highlights some challenges particularly with regard to embryo biopsies and quantity and quality of embryo DNA for whole genome genotyping and ways to overcome difficulties. We briefly discuss the somatic cell nuclear transfer (SCNT) technique in the context of applying GS on fibroblast cell lines from fetuses obtained from OPU-IVP techniques and provide our perspectives on how it might pave way for even more rapid cattle improvement. Main conclusion is that employing genomic selection in ARTs such as OPU-IVP of embryos coupled with embryo sexing and SCNT will lead to rapid dissemination of high genetic merit animals on a scale never been seen before. Finally, the paper outlines current research activities on combined genomic selection and advanced reproductive technologies in the GIFT project consortium (www.gift.ku.dk).

Isolation and characterization of mesenchymal stem cells from the yolk sacs of bovine embryos.

The yolk sac (YS) represents a promising source of stem cells for research because of the hematopoietic and mesenchymal cell niches that are present in this structure during the development of the embryo. In this study, we report on the isolation and characterization of YS tissue and mesenchymal stem cells (MSCs) derived from bovine YSs. Our results show that the YS is macroscopically located in the exocoelomic cavity in the ventral portion of the embryo and consists of a transparent membrane formed by a central sac-like portion and two ventrally elongated projections. Immunohistochemistry analyses were positive for OCT4, CD90, CD105, and CD44 markers in the YS of both gestational age groups. The MSCs of bovine YS were isolated using enzymatic digestion and were grown in vitro for at least 11 passages to verify their capacity to proliferate. These cells were also subjected to immunophenotypic characterization that revealed the presence of CD90, CD105, and CD79 and the absence of CD45, CD44, and CD79, which are positive and negative markers of MSCs, respectively. To prove their multipotency, the cells were induced to differentiate into three cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (chondrogenic: Alcian Blue, osteogenic: Alizarin Red, and adipogenic: Oil Red O) to confirm differentiation. Gene expression analyses showed no differences in the patterns of gene expression between the groups or passages tested, with the exception of the expression of SOX2, which was slightly different in the G1P3 group compared to the other groups. Our results suggest that YS tissue from bovines can be used as a source of MSCs, which makes YS tissue-derived cells an interesting option for cell therapy and regenerative medicine.

Impactos das biotécnicas reprodutivas no controle epigenético de genes imprinted / Impact of reproductive biotechniques on the epigenetic regulation of imprinted genes

Técnicas de reprodução assistida (TRAs) são utilizadas nas medicinas humana e veterinária com oobjetivo principal de corrigir infertilidades adquiridas ou herdadas. A transferência nuclear de célula somática(TNCS) ocupa um lugar de destaque na veterinária pela possibilidade de geração de indivíduos geneticamenteidênticos, o que permite a produção de rebanhos homogêneos de alto mérito genético, e serve como modelo deestudo para técnicas de reprogramação. Porém, a utilização de TRAs, e em especial da TNCS, é consideradaresponsável pelo aumento na geração de conceptos portadores de alterações durante e após os desenvolvimentosembrionário e fetal. A provável causa é a alteração na regulação da reprogramação epigenética devido àmanipulação de gametas e embriões no período inicial do desenvolvimento, que leva a alterações na regulaçãoepigenética de genes imprinted. Esta revisão discute como marcas epigenéticas e expressão de genes imprintedpodem influenciar no desenvolvimento de conceptos bovinos produzidos por TNCS ou inseminação artificial(IA). O entendimento dos mecanismos epigenéticos relacionados aos desenvolvimentos embrionário e fetal, emespecial daqueles relacionados à dinâmica das alterações epigenéticas envolvidas no imprinting genômico, devecontribuir para a elaboração de biotécnicas mais eficientes para a medicina regenerativa ou a produção animal.(AU)

Assisted reproductive technologies (ARTs) are usually used in both human and veterinary medicineaiming the correction of heritable or acquired infertilities. The somatic cell nuclear transfer technique (SCNT) isof particular importance in veterinary as it enables the generation of genetically identical organisms, allowingthe production of homogeneous genetically improved herds, and also serving as a model for reprogrammingstudies. However, the use of TRAs, SCNT in special, may be responsible for the increase of developmentalrelatedabnormalities in the conceptuses. Such phenotypes are probably caused by a disruption in the epigeneticreprogramming due to the manipulation of gametes and embryos during the early development period, andtherefore leading to disturbances in the epigenetic regulation of imprinted genes. The present review discusseshow the epigenetic marks and expression of imprinted genes may influence the developmental competence ofanimals generated by SCNT or artificial insemination (AI). The understanding of the epigenetic mechanismsrelated to embryonic and fetal development, and in special, of those related to the epigenetic dynamics duringgenomic imprinting may contribute to the generation of efficient ARTs to be used in both regenerative medicineand animal production.(AU)

Impactos das biotécnicas reprodutivas no controle epigenético de genes imprinted / Impact of reproductive biotechniques on the epigenetic regulation of imprinted genes

Técnicas de reprodução assistida (TRAs) são utilizadas nas medicinas humana e veterinária com oobjetivo principal de corrigir infertilidades adquiridas ou herdadas. A transferência nuclear de célula somática(TNCS) ocupa um lugar de destaque na veterinária pela possibilidade de geração de indivíduos geneticamenteidênticos, o que permite a produção de rebanhos homogêneos de alto mérito genético, e serve como modelo deestudo para técnicas de reprogramação. Porém, a utilização de TRAs, e em especial da TNCS, é consideradaresponsável pelo aumento na geração de conceptos portadores de alterações durante e após os desenvolvimentosembrionário e fetal. A provável causa é a alteração na regulação da reprogramação epigenética devido àmanipulação de gametas e embriões no período inicial do desenvolvimento, que leva a alterações na regulaçãoepigenética de genes imprinted. Esta revisão discute como marcas epigenéticas e expressão de genes imprintedpodem influenciar no desenvolvimento de conceptos bovinos produzidos por TNCS ou inseminação artificial(IA). O entendimento dos mecanismos epigenéticos relacionados aos desenvolvimentos embrionário e fetal, emespecial daqueles relacionados à dinâmica das alterações epigenéticas envolvidas no imprinting genômico, devecontribuir para a elaboração de biotécnicas mais eficientes para a medicina regenerativa ou a produção animal.

Assisted reproductive technologies (ARTs) are usually used in both human and veterinary medicineaiming the correction of heritable or acquired infertilities. The somatic cell nuclear transfer technique (SCNT) isof particular importance in veterinary as it enables the generation of genetically identical organisms, allowingthe production of homogeneous genetically improved herds, and also serving as a model for reprogrammingstudies. However, the use of TRAs, SCNT in special, may be responsible for the increase of developmentalrelatedabnormalities in the conceptuses. Such phenotypes are probably caused by a disruption in the epigeneticreprogramming due to the manipulation of gametes and embryos during the early development period, andtherefore leading to disturbances in the epigenetic regulation of imprinted genes. The present review discusseshow the epigenetic marks and expression of imprinted genes may influence the developmental competence ofanimals generated by SCNT or artificial insemination (AI). The understanding of the epigenetic mechanismsrelated to embryonic and fetal development, and in special, of those related to the epigenetic dynamics duringgenomic imprinting may contribute to the generation of efficient ARTs to be used in both regenerative medicineand animal production.

Generation of bovine (Bos indicus) and buffalo (Bubalus bubalis) adipose tissue derived stem cells: isolation, characterization, and multipotentiality.

Adult stem cells are known for their plasticity and their potential to differentiate into several different cell types; these characteristics have implications for cell therapy and reproductive biotechnologies. In this study, we report on the isolation and characterization of mesenchymal stem cells (MSC) derived from bovine and buffalo adipose tissue. Cells isolated using enzymatic digestion of bovine and buffalo adipose-tissue biopsy samples were grown in vitro for at least 15 passages, verifying their capacity to proliferate. These cells were also subjected to immunophenotypic characterization for the presence of CD90, CD105, and CD79, and the absence of CD45, CD34, and CD73, which are positive and negative markers of MSC, respectively. To prove their multipotency, the cells were induced to differentiate into three different cell types, chondrocytes, osteoblasts, and adipocytes, which were stained with tissue-specific dyes (Chondrogenic-Alcian Blue, Osteogenic-Alizarin Red, and Adipogenic-Oil-Red O, respectively) to confirm differentiation. Gene expression analysis of pluripotency-related genes was also conducted. Our results suggest that adipose tissue from bovines and buffalos can be used as a source of MSC, making adipose tissue-derived cells an interesting option for cell therapy and regenerative medicine. Additionally, these findings have implications for reproductive biotechnology because the use of MSC as nuclear donors has been linked to an increase in the efficiency of nuclear transfer.

The influence of morphology, follicle size and Bcl-2 and Bax transcripts on the developmental competence of bovine oocytes.

This study analysed two non-invasive oocyte selection methods in relation to in vitro embryo development capacity and expression of apoptosis-related genes. Selection was based on morphological quality of oocytes or follicle diameter. Oocytes were classified as grade I (GI ≥3 layers compact cumulus cells and homogeneous cytoplasm; grade II (GII ≤3 layers compact cells and homogeneous cytoplasm;, and grade III (GIII ≥3 layers, but cells with slight expansion and slightly granulated cytoplasm). Blastocyst development was lower for GII (28.5%) than for GIII (47.7%, p < 0.05), and GI was similar to both (36.9%, p > 0.05). Relative expression of Bcl-2 gene was lower in the GI (1.0, p < 0.05) than in the GII (1.8) and GIII (2.2), which were not different (p > 0.05). There was no difference (p > 0.05) between GI (1.0), GII (0.92) and GIII (0.93) regarding the Bax transcript. However, the Bax and Bcl-2 transcript ratios in GII (Bax; 0.92 and Bcl-2; 1.8) and GIII (Bax; 0.93 and Bcl-2; 2.2) were different (p < 0.05). Regarding oocytes from follicles of different sizes, cleavage and blastocyst rates for 1-3 mm (82.5; 23.7%) were lower (p < 0.05) than for 6-9 mm (95.6; 41.1%), but similar (p > 0.05) to 3-6 mm (93.7; 35.4%), which were not different (p > 0.05). Regarding Bax and Bcl-2 expression, the oocytes were similar (p > 0.05) for 1-3 mm (Bax; 1.0 and Bcl-2; 1.0), 3-6 mm (Bax; 1.0 and Bcl-2; 0.93) and 6-9 mm (Bax; 0.92 and Bcl-2; 0.91). In conclusion, oocyte selection based on morphological appearance does not guarantee the success of embryonic development. Additionally, the absence of apoptosis is not necessarily a benefit for the development of oocytes. Bovine COCs with initial signs of atresia may be used for the in vitro production of embryos, and COCs taken from follicles >3 mm in diameter are better suited to in vitro embryo development.

Protein synthesis and degradation gene SNPs related to feed intake, feed efficiency, growth, and ultrasound carcass traits in Nellore cattle.

We looked for possible associations of SNPs in genes related to protein turnover, with growth, feed efficiency and carcass traits in feedlot Nellore cattle. Purebred Nellore bulls and steers (N = 290; 378 ± 42 kg body weight, 23 months ± 42 days old) were evaluated for daily feed intake, body weight gain (BWG), gross feed efficiency, feed conversion ratio, partial efficiency of growth, residual feed intake (RFI), ultrasound backfat, rump fat, and ribeye area. Genotypes were obtained for SNPs in the growth hormone receptor (GHR-1 and GHR-2); calpain (CAPN4751); calpastatin (UoGCAST); ubiquitin-conjugating enzyme 2I (UBE2I-1 and UBE2I-2); R3H domain containing 1 (R3HDM1-1, -2, -3, and -4), ring finger protein 19 (RNF19); proteasome 26S subunit, non-ATPase, 13 (PSMD13); ribosomal protein, large, P2 (RPLP2); and isoleucine-tRNA synthetase 2, mitochondrial (IARS2) genes. Allelic substitution, additive and dominant effects were tested and molecular breeding values were computed. CAPN4751, GHR-1 and -2, IARS2, R3HDM1-4, and UoGCAST were found to be normally segregating polymorphisms. Additive and dominance effects were observed on BWG, feed efficiency and carcass traits, although dominant effects predominated. Significant allelic substitution effects were observed for CAPN4751, GHR-1 and -2, and UoGCAST on BWG, gross feed efficiency, RFI, and carcass traits, under single- or multiple-marker analyses. Correlations between molecular breeding values and phenotypes were low, excepted for RFI, based on allelic substitution estimates obtained by stepwise linear regression. We conclude that SNPs in genes related to protein turnover are related to economically important traits in Nellore cattle.

Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.

Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P < 0.05). The Bax and Bcl2 gene expression ratio in control and transgenic cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed with early or late-passage cells when fusion (63.1% and 49%), cleavage (67.7% and 69.9%), eight-cell embryo (36.4% and 44.4%) and blastocyst (21.6% and 20.8%) rates were compared. In conclusion, culture behavior was different between control and eGFP cells. However, when different in vitro culturing periods were compared, long-term cultured transgenic fetal fibroblasts remained competent for blastocyst production when used as nuclei donors in the nuclear transfer technique, a feature needed for the genetic manipulation of cell culture experiments aiming for transgenic animal production.

Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.

Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine ß-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

Strategies to increase in vitro embryo yield: lessons from cell and mocelular research

Numerous reproductive biotechnologies are commonly employed to enhance animal production mainly through multiplying animals with high-quality traits in a large-scale production system. There is however, several peculiarities during the process of embryo in vitro production that are still in need of further studies in order to obtain a higher efficiency. This present review discuss some of such particularities, as well as new models of embryo and gamete production, which will probably be part of a new era of reproductive biotechnologies in a near future.

Strategies to increase in vitro embryo yield: lessons from cell and mocelular research

Numerous reproductive biotechnologies are commonly employed to enhance animal production mainly through multiplying animals with high-quality traits in a large-scale production system. There is however, several peculiarities during the process of embryo in vitro production that are still in need of further studies in order to obtain a higher efficiency. This present review discuss some of such particularities, as well as new models of embryo and gamete production, which will probably be part of a new era of reproductive biotechnologies in a near future.(AU)