Production of transgenic cattle by somatic cell nuclear transfer (SCNT) with the human granulocyte colony-stimulation factor (hG-CSF)

The hG-CSF (human Granulocyte Colony-Stimulating Factor) is a growth and stimulation factor capable of inducing the proliferation of bone marrow cells, several types of leukocytes, among other hematopoietic tissue cells. hG-CSF is used in used to treat anomalies that reder a small number of circulating white blood cells, which may compromise the immune defenses of the affected person. For these reasons, the production of hG-CSF in a bioreactor system using the mammary gland of genetic modified animals is a possibility of adding value to the bovine genetic material and reducing the costs of hG-CSF production in pharmaceutical industry. In this study, we aimed the production of transgenic hG-CSF bovine through the lipofection of bovine primary fibroblasts with an hG-CSF expression cassette and cloning these fibroblasts by the somatic cell nuclear transfer (SCNT) technique. The bovine fibroblasts transfected with the hG-CSF cassette presented a stable insertion of this construct into their genome and were efficiently synchronized to G0/G1 cell cycle stage. The transgenic fibroblasts were cloned by SCNT and produced 103 transferred embryos and 2 pregnancies, one of which reached 7 months of gestation

Bovine epididymal spermatozoa treatment for in vitro fertilization: Heparin accelerates fertilization and enables a reduction in coincubation time.

This study aimed to establish a protocol for in vitro embryo production using epididymal sperm (EP). Samples were obtained from ejaculated sperm (EJ) and the epididymis of 7 Gir bulls. First, the effect of heparin (+) on the viability, longevity (Experiment 1) and fertilization rates (Experiment 2) of the EP was evaluated. In experiment 2, a pool of EP and EJ sperm (n = 7) was coincubated with cumulus-oocyte complexes (COCs) for 0, 3, 6, 12 and 18 h, and the fertilization rate (FR) was evaluated. A third experiment was performed to test sperm treatments for IVP using the Percoll (P) or PureSperm (PS) gradients or a spTALP wash for sperm selection. Cleavage, blastocyst rate (BR) and embryo sex were evaluated. In experiment 4, embryos were produced using 6, 12, and 18 h of sperm-oocyte coincubation. The cleavage, BR, and total number and percentage of apoptotic cells were determined. Heparin affected EP viability, longevity and FR. After 6 h, 82% of the oocytes were fertilized in the EP+ group, a higher value (P0.05). No differences (P>0.05) were observed among the groups that were coincubated for 6, 12 and 18 h with respect to embryo production, kinetics of development, total cell number and percentage of apoptotic cells. In conclusion, IVF time can be reduced to 6 h without affecting embryo production and quality. In addition, EP sperm selection can be performed by either a PS or P gradient

Blastocyst development of oocytes recovered from slaughterhouse ovaries (CONT) and by ovum pick-up (OPU) from the ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that received an ovulation inducer (MII) that were vitrified (VIT) at the metaphase II stage.

<p>^a,b,c,d Values with different superscripts in the same column are different at P<0.05.</p><p>* Hatched blastocyst at D8 as a percentage of oocyte number.</p><p>Blastocyst development of oocytes recovered from slaughterhouse ovaries (CONT) and by ovum pick-up (OPU) from the ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that received an ovulation inducer (MII) that were vitrified (VIT) at the metaphase II stage.</p

Experimental design flow diagram.

<p>Flow diagram of experimental design for the different treatments. Oocytes recovered from slaughterhouse ovaries (CONT), obtained by OPU from non-superstimulated females (IMA) and from superstimulated females (FSH) were matured in vitro. In vivo-matured oocytes were obtained by OPU from superstimulated females that received an ovulation inducer 24 hours previously (MII). A sample of matured oocytes from each of four groups was used to study the composition of plasma membrane phospholipids using MALDI-TOF. The remaining oocytes were divided in half, one half consisting of non-vitrified fresh oocytes (CONT, IMA, FSH and MII) and other of vitrified/ warmed oocytes (CONT Vit, IMA Vit, FSH Vit and MII Vit). At the end of the warming process, the eight groups were used for in vitro fertilization and culture. Cleavage at D2 and blastocyst development at D7 and D8 were evaluated. At D8, all of the blastocysts were measured, and those larger than 160 μM in diameter were stained for total cell number counting.</p

Comparison of the relative intensity of most dispersed ions after mass spectrometry (MALDI-TOF) analyses, of oocytes from different maturation systems.

<p>The 760.6 ion corresponds to [PC (34:1) + H] ⁺ and 782.6 to [PC (34:6) + H] ⁺ or [PC (34:1) + Na], both phosphatidylcholines (PC).</p><p>^ab Different letters in the same column indicates statically differences among the treatments after ANOVA according to Tukey’s test (P<0.05).</p><p>CONT = oocytes from slaughterhouse ovaries and matured in vitro; IMA = OPU oocytes from non-stimulated animals and matured in vitro; FSH = OPU oocytes from FSH simulated animal and matured in vitro; MII = OPU oocytes after in vivo maturation.</p><p>The data are expressed as arbitrary unit intensity and standard deviation (±SD), and each ion represents a different phospholipid.</p

Total number (N), mean and standard deviation (±SD) of follicles per female evaluated and classified by the color Doppler, after nine replicates, as having intense, moderate or absent blood vascularization in the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).

<p>^a,b,c Values with different superscripts in the same column are significantly different by Kruskall-Wallis test (P < 0.05).</p><p>Total number (N), mean and standard deviation (±SD) of follicles per female evaluated and classified by the color Doppler, after nine replicates, as having intense, moderate or absent blood vascularization in the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).</p

Percentage, mean (μm) and standard deviation (SD) of size (μm) and total cell number of D8 blastocyst with diameters > 160 μm derived from oocytes of different maturation conditions: slaughterhouse ovaries (CONT) and by OPU, from ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that had received a ovulatory inducer (MII).

<p>^a,b,c,d Values with different superscripts in the same column are significantly different by Kruskall-Wallis test (P < 0.05).</p><p>CONT = oocytes from slaughterhouse ovaries that were matured in vitro; IMA = OPU oocytes from non-stimulated animals matured in vitro; FSH = OPU oocytes from FSH simulated animal and matured in vitro; MII = OPU oocytes after in vivo maturation.</p><p>*Represents to the quantity of embryos that was able to be evaluated in the counting of total cell numbers because some embryos were lost during staining or could not be observed.</p><p>Percentage, mean (μm) and standard deviation (SD) of size (μm) and total cell number of D8 blastocyst with diameters > 160 μm derived from oocytes of different maturation conditions: slaughterhouse ovaries (CONT) and by OPU, from ovaries of non-superstimulated females (IMA), superstimulated females (FSH) and superstimulated females that had received a ovulatory inducer (MII).</p

3D PCA plot for MALDI-TOF data of individual oocytes from different maturation systems.

<p>Shown is a 3D PCA plot for the MALDI-TOF data of single oocytes. a) Red (n = 12), blue (n = 13), pink (n = 13) and dark yellow (n = 10). Each point indicates the 3D PCA plot of an oocyte based on its phospholipid composition. The following four fresh oocyte experimental groups are represented: immature and in vitro-matured oocytes recovered from slaughter house ovaries (CONT), oocytes obtained by OPU from non-superstimulated females (IMA), superstimulated females (FSH) and in vivo-matured oocytes obtained by OPU from superstimulated females that received an ovulation inducer (MI). b) indicates the main ions represented, 760.6[PC (34:1) + H]⁺ and 782.6 [PC (34:6) + H]⁺ or [PC (34:1) + Na]⁺, are responsible for the most variability between the treatments. The three principal components explain >73% of the variability of the data.</p

Total number (N) and percentage (%) of viable and nonviable oocytes recovered by ovum pick-up, after nine replicates, from the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).

<p>^a,b,c Values with different superscripts in the same column are significantly different, by to Chi—square test (P < 0.05).</p><p>* Only those oocytes presenting less than three cumulus cell layers or heterogeneous cytoplasm were classified as non-viable for IMA and FSH groups. For the MII group, only oocytes with heterogeneous cytoplasm or without the first polar body extrusion and/or cumulus cell expansion were considered non-viable. The percentage is expressed as the ratio to the total number of recovered oocytes.</p><p>**Oocytes with heterogeneous cytoplasm and presenting vacuolization at the three experimental groups were classified as degenerated. The percentage is expressed as the ratio of the total number of non-viable oocytes.</p><p>Total number (N) and percentage (%) of viable and nonviable oocytes recovered by ovum pick-up, after nine replicates, from the ovaries of non-superstimulated (IMA), superstimulated (FSH) and superstimulated females that received an ovulation inducer (MII).</p

Effects of Different Maturation Systems on Bovine Oocyte Quality, Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]+), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification