Distribution of sexes within the left and right uterine horns of cattle.

In cattle, limited data are available regarding the sex ratio of the offspring in relation to the horn of gestation. Therefore, the objective of this study was to evaluate the sex ratio of fetuses gestated in the left and right uterine horns of cattle (Bos taurus, Bos indicus and crosses). The distribution of male and female fetuses in the left and right uterine horn was analyzed on gravid, abattoir-derived reproductive tracts and artificially inseminated crossbred cows. The total number of fetuses/calves and the sex of the fetuses/calves gestated in each uterine horn were used as the end point for side comparisons using the Glimmix Procedure. Of 64 gravid reproductive tracts evaluated, 29 (45.3%) pregnancies occurred in the left uterine horn, whereas 35 (54.7%) occurred in the right. The sex ratio (% males) of fetuses in the left uterine horn (37.9%) was significantly lower than the sex ratio detected in the right uterine horn (65.7%). Of 113 pregnancies evaluated in artificially inseminated heifers, 53 (46.9%) occurred in the left uterine horn, whereas 60 (53.1%) occurred in the right uterine horn. The sex ratio of calves gestated in the left uterine horn (35.8%) was significantly lower than the sex ratio of calves gestated in the right uterine horn (63.3%). In conclusion, in these experiments, a significantly greater proportion of males were gestated in the right uterine horn of cattle and a greater proportion of females in the left uterine horn. Further investigation is needed to determine the mechanisms underlying the observed disparity of the expected sex ratio within the uterine horns of cattle.

Effects of culture medium and protein supplementation on mRNA expression of in vitro produced bovine embryos.

Numerous studies have reported aberrant gene expression levels attributed to suboptimal in vitro culture conditions. This study investigated the effects of different culture systems and protein sources on the developmental competence of in vitro production (IVP) embryos measured by cleavage and blastocyst rates, cell number, and relative abundance of POU5F1 (OCT4), nanog, GJA1 (connexin 43), and SLC2A1 (GLUT1) transcripts when compared to in vivo embryos. Experiment 1 compared IVP embryos cultured in either synthetic oviductal fluid (SOFaa) or potassium simplex optimized medium supplemented with amino acids (KSOMaa). Experiment 2 compared the same two culture systems with and without the addition of calf serum (CS). Results from both experiments indicated that despite similar developmental rates, significant differences were observed at the mRNA level. In Experiment 1, OCT4 was the only transcript to have a mean abundance level significantly higher in KSOMaa blastocysts when compared with both SOFaa and in vivo embryos. The same pattern of upregulation of OCT4 mRNA was noted in Experiment 2. There were no significant alterations of the ICM specific transcript nanog in either experiment. In contrast to reports by others, connexin 43 mRNA was not expressed at detectable levels in in vivo embryos analyzed in our studies. Blastocysts cultured in SOFaa with CS or KSOMaa had a significant upregulation of GLUT1 mRNA when compared with other treatments and in vivo embryos. Until differences between IVP and in vivo embryos are minimized, aberrations in IVP will continue to arise.

The influence of dietary selenium on common indicators of selenium status and liver glutathione peroxidase-1 messenger ribonucleic acid.

The objective of this research was to determine the influence of dietary Se on various indicators of Se status and relative liver glutathione peroxidase 1 (GPx-1) messenger RNA (mRNA) levels in growing Holstein bull calves. Calves (n = 14, 7/diet) were started 28 d after birth on a Se-adequate (SeA) or Se-deficient diet (SeD) and maintained on the diet until 180 d of age. Blood samples were taken from each calf for determination of erythrocyte GPx-1 and plasma GPx-3 activities and plasma Se concentration on d 28 of age, every 28 d thereafter, and at 180 d of age. To assess liver Se and GPx-1 mRNA, 3 calves were first killed at d 21 of age for baseline (BSL) measurements, and 4 calves from each treatment were killed at trial conclusion. Feed intake and ADG were not affected (P = 0.62) by dietary Se concentrations. However, liver Se concentration was greater (P < 0.05) for BSL calves and SeA calves than SeD calves, but no difference (P = 0.68) was observed between BSL calves and SeA calves. Plasma Se was greater for SeA calves (P < 0.01) than for SeD calves by d 56 of age. The GPx-1 activity was greater in SeA calves (P < 0.01) by d 84 of age, whereas GPx-3 activity was greater in SeA calves, but not until d 180 of age (P < 0.01). There was a 50% decrease in GPx-1 mRNA for the SeD calves (P < 0.05) compared with SeA calves. Thus, relative GPx-1 mRNA transcript level is reflective of Se status in the bovine. Furthermore, 152 d on a semi-purified, SeD diet is adequate to create a Se deficiency in growing Holstein bull calves started on a SeD diet at 28 d of age.

Isolation, culture and characterisation of somatic cells derived from semen and milk of endangered sheep and eland antelope.

Semen and milk are potential sources of somatic cells for genome banks. In the present study, we cultured and characterised cells from: (1) cooled sheep milk; (2) fresh, cooled and frozen-thawed semen from Gulf Coast native (GCN) sheep (Ovis aries); and (3) fresh eland (Taurotragus oryx) semen. Cells attached to the culture surface from fresh (29%), cooled (43%) and slow-frozen (1 degrees C/min; 14%) ram semen, whereas no attachment occurred in the fast-frozen (10 degrees C/min) group. Proliferation occurred in fresh (50%) and cooled (100%) groups, but no cells proliferated after passage 1 (P1). Eland semen yielded cell lines (100%) that were cryopreserved at P1. In samples from GCN and cross-bred milk, cell attachment (83% and 95%, respectively) and proliferation (60% and 37%, respectively) were observed. Immunocytochemical detection of cytokeratin indicated an epithelial origin of semen-derived cells, whereas milk yielded either fibroblasts, epithelial or a mixture of cell types. Deoxyribonucleic acid microsatellite analysis using cattle-derived markers confirmed that eland cells were from the semen donor. Eland epithelial cells were transferred into eland oocytes and 12 (71%), six (35%) and two (12%) embryos cleaved and developed to morulae or blastocyst stages, respectively. In conclusion, we have developed a technique for obtaining somatic cells from semen. We have also demonstrated that semen-derived cells can serve as karyoplast donors for nuclear transfer.

Transfer of inner cell mass cells derived from bovine nuclear transfer embryos into the trophoblast of bovine in vitro-produced embryos.

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.

Expression of the human alpha1,2-fucosyltransferase in transgenic pigs modifies the cell surface carbohydrate phenotype and confers resistance to human serum-mediated cytolysis.

Hyperacute rejection (HAR) is the first critical immunological hurdle that must be addressed in order to develop xenogeneic organs for human transplantation. In the area of cell-based xenotransplant therapies, natural antibodies (XNA) and complement have also been considered barriers to successful engraftment. Transgenic expression of human complement inhibitors in donor cells and organs has significantly prolonged the survival of xenografts. However, expression of complement inhibitors without eliminating xenogeneic natural antibody (XNA) reactivity may provide insufficient protection for clinical application. An approach designed to prevent XNA reactivity during HAR is the expression of human alpha1, 2-fucosyltransferase (H-transferase, HT). H-transferase expression modifies the cell surface carbohydrate phenotype of the xenogeneic cell, resulting in the expression of the universal donor O antigen and a concomitant reduction in the expression of the antigenic Galalpha1,3-Gal epitope. We have engineered various transgenic pig lines that express HT in different cells and tissues, including the vascular endothelium. We demonstrate that in two different HT transgenic lines containing two different HT promoter constructs, expression can be differentially regulated in a constitutive and cytokine-inducible manner. The transgenic expression of HT results in a significant reduction in the expression of the Galalpha1,3-Gal epitope, reduced XNA reactivity, and an increased resistance to human serum-mediated cytolysis. Transgenic pigs that express H-transferase promise to become key components for the development of xenogeneic cells and organs for human transplantation.

The bladder as a bioreactor: urothelium production and secretion of growth hormone into urine.

Uroplakin genes are expressed in a bladder-specific and differentiation-dependent fashion. Using a 3.6-kb promoter of mouse uroplakin II gene, we have generated transgenic mice that express human growth hormone (hGH) in their bladder epithelium, resulting in its secretion into the urine at 100-500 ng/ml. The levels of urine hGH concentration remain constant for longer than 8 months. hGH is present as aggregates mostly in the uroplakin-delivering cytoplasmic vesicles that are targeted to fuse with the apical surface. Using the bladder as a bioreactor offers unique advantages, including the utility of all animals throughout their lives. Using urine, which contains little protein and lipid, as a starting material facilitates recombinant protein purification.

Transgenic dairy cattle: genetic engineering on a large scale.

Amid the explosion of fundamental knowledge generated from transgenic animal models, a small group of scientists has been producing transgenic livestock with goals of improving animal production efficiency and generating new products. The ability to modify mammary-specific genes provides an opportunity to pursue several distinctly different avenues of research. The objective of the emerging gene "pharming" industry is to produce pharmaceuticals for treating human diseases. It is argued that mammary glands are an ideal site for producing complex bioactive proteins that can be cost effectively harvested and purified. Consequently, during the past decade, approximately a dozen companies have been created to capture the US market for pharmaceuticals produced from transgenic bioreactors estimated at $3 billion annually. Several products produced in this way are now in human clinical trials. Another research direction, which has been widely discussed but has received less attention in the laboratory, is genetic engineering of the bovine mammary gland to alter the composition of milk destined for human consumption. Proposals include increasing or altering endogenous proteins, decreasing fat, and altering milk composition to resemble that of human milk. Initial studies using transgenic mice to investigate the feasibility of enhancing manufacturing properties of milk have been encouraging. The potential profitability of gene "pharming" seems clear, as do the benefits of transgenic cows producing milk that has been optimized for food products. To take full advantage of enhanced milk, it may be desirable to restructure the method by which dairy producers are compensated. However, the cost of producing functional transgenic cattle will remain a severe limitation to realizing the potential of transgenic cattle until inefficiencies of transgenic technology are overcome. These inefficiencies include low rates of gene integration, poor embryo survival, and unpredictable transgene behavior.

Glycine and alanine supplementation of culture medium enhances development of in vitro matured and fertilized cattle embryos.

One-cell cattle embryos were prepared by in vitro oocyte maturation and fertilization (IVM/IVF) and cultured with or without oviductal cells. Embryos were evaluated after 7 days in culture to determine the percentage developing from the 1-cell stage to the morula or blastocyst stage. The combination of glycine (2 mM) and alanine (1 mM) with oviductal cells (experiment 1) improved embryo development over that in control culture (29 vs. 13%; p < 0.05). An optimum response was obtained with 10 mM glycine and 1 mM alanine in coculture (experiments 2 and 3). In experiment 4, the effects of glycine (0 or 10 mM), alanine (0 or 1 mM), and the presence or absence of oviductal cells were tested. In the absence of oviductal cells, the addition of glycine, alanine, or glycine and alanine combined improved embryonic development over that in control medium (45, 33, 42 vs. 24%, p < 0.001, p < 0.05, p < 0.001, respectively). However, the effect of the combination of glycine and alanine was not different from that of glycine alone when oviductal cells were absent (42 vs. 45%, p > 0.10). In the presence of oviductal cells, glycine or the combination of glycine and alanine improved embryonic development over that in the control medium with cells (47, 55 vs 37%, p < 0.01, respectively). However, supplementation with alanine alone gave no improvement over controls when oviductal cells were present (40 vs. 37%, p > 0.10). These results indicate that glycine and alanine, when used independently, directly affect cattle embryo development, but in combination affect embryo development indirectly, possibly by altering oviductal cell function.(ABSTRACT TRUNCATED AT 250 WORDS)

Viable embryos and normal calves after nuclear transfer into Hoechst stained enucleated demi-oocytes of cows.

Bovine oocytes were bisected, stained with Hoechst 33342 and observed under a fluorescent microscope to identify nucleated and enucleated demi-oocytes. Other oocytes were bisected but not stained, or bisected and only half of each oocyte stained, and viewed under a fluorescent microscope. The oocytes were then used for nuclear transfer by fusing them with embryonic blastomeres from a 5-6 day bovine embryo. The fusion rate and proportion developing into compact morulae or blastocysts was compared among different types of demi-oocytes. Expt 1 examined the effect of staining and indicated no effect on either fusion rate or embryonic development whether or not the oocytes were stained. In Expt 2, stained and unstained nucleated and enucleated oocytes were compared. As in the first experiment, there were no differences between stained and unstained demi-oocytes. There was no difference between fusion rates of nucleated and enucleated oocytes. However, there was a significant difference in embryonic development between nucleated (10.4%) and enucleated (22.6%) demi-oocytes (P less than 0.05). In a final experiment, stained and unstained enucleated oocytes were used for nuclear transfer and the resulting embryos transferred into recipient cows. There was no difference in pregnancy rates or in the number of normal calves born whether stained or unstained recipient oocytes were used. Results from these experiments indicate that Hoechst staining and fluorescent microscopy can be used to identify enucleated demi-oocytes, and that these can be used for nuclear transfer, and result in viable embryos and normal calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormone secretion by preimplantation embryos in a dynamic in vitro culture system.

Bovine embryos recovered from superovulated donors on Days 8-18 postestrus were cultured in vitro in a tissue perifusion system to quantify hormone secretion. Embryos were cultured for 24 h at 37 degrees C in Ham's F-10 medium supplemented 5% v/v with heat-treated, charcoal-stripped calf serum; 100 IU/ml penicillin; and 100 micrograms/ml streptomycin. The medium was saturated with 5% CO2 in air and perifused at 50 microliters/min (3 ml/h). Estrone (E1) estradiol (E2), progesterone (P4), prostaglandin E2 (PGE2), and prostacyclin (PGI2) were quantified by RIA in 6-h pools of perifusate fractions. Estrone was measurable (pg/h/embryo; mean +/- SE) on Days 13 (10.80 +/- 4.56) and 15 (34.80 +/- 9.80); E2 on Days 11 (36.80), 12 (81.28 +/- 29.80), 13 (11.75 +/- 4.09), 15 (157.20 +/- 112.60), and 16 (30.26 +/- 8.76); and P4 (ng/h/embryo) on Days 13 (0.5-1.0) and 17 (approximately 1.5). PGE2 was secreted by Day 10 bovine embryos during the last 6 h of culture (19-24 h) and throughout culture for Day 11-18 embryos. The rate of PGE2 secretion increased (p less than 0.05) over the previous days(s) at Days 13 and 17. The mean (+/- SE) secretion rates (pg/h/embryo) for the 24-h culture by embryonic ages were as follows: Day 11 (63.39 +/- 14.61), 12 (172.10 +/- 30.90), 13 (3094.08 +/- 283.35), 14 (1633.89 +/- 49.98), 15 (3739.23 +/- 1082.79), 16 (4955.37 +/- 1381.83), 17 (11893.23 +/- 1188.48), and 18 (13827.99 +/- 3587.88).(ABSTRACT TRUNCATED AT 250 WORDS)

Nuclear transfer in the bovine embryo: a comparison of 5-day, 6-day, frozen-thawed, and nuclear transfer donor embryos.

Micromanipulation and electrofusion were utilized for nuclear transfer in bovine embryos. Embryonic blastomeres from 5-day (estrus = day 0), 6-day, frozen-thawed 5-day, and first-generation nuclear transfer embryos (embryos were themselves a product of nuclear transfer with the original donor being a 5-day embryo) were transferred into bisected bovine oocytes by electrofusion. The percentage of donor cells fusing with the recipient oocytes was compared between different types of donor embryos. The percentage of embryos developing normally into morula or blastocysts following 6 days culture in the sheep oviduct was also recorded and compared between different donor embryo types. No significant differences were found between donor blastomeres for the percent successfully fused to oocytes: 5-day, 294 of 513 (57.3%); 6-day, 252 of 405 (62.2%); frozen-thawed 5-day, 111 of 144 (77.1%); nuclear transfer, 142 of 223 (63.7%); or the percent developing normally following nuclear transfer: 5-day, 92 of 444 (20.7%); 6-day, 84 of 357 (23.5%); frozen-thawed 5-day, 32 of 127 (25.2%); nuclear transfer, 31 of 199 (15.6%). These data suggest that a variety of donor embryos can successfully be utilized for bovine embryo cloning. Also, development of blastomeres from frozen-thawed 5-day donors and from donors that are themselves the product of nuclear transfer suggest that the production of multiple identical offspring is possible by frozen storage of seed stock and serial recloning.

Effects of slitting the zona pellucida and its subsequent sealing on freeze-thaw survival of Day 7 bovine embryos.

The effects of slitting the zona pellucida and its subsequent sealing by either embedding in agar or surrounding with an additional zona pellucida on the development of frozen/thawed Day 7 bovine embryos were investigated in vitro and in vivo. A total of 225 embryos was frozen and thawed rapidly as controls (Group 1), after slitting the zona pellucida (Group 2), after slitting and subsequent sealing of the zona pellucida with agar (Group 3), or after slitting the zona pellucida (Grothen transferring the embryo into an additional zona pellucida (Group 4). The survival rate (embryos classified morphologically as excellent, good, or poor) was 95.1, 95.4, 92.2, and 94.3% for Groupsl, 2, 3, and 4, respectively. Culture of 145 embryos in vitro for 60 h revealed that 57.1, 59.5, 47.4, and 57.1% developed to hatching and hatched blastocysts in Groups 1, 2, 3, and 4, respectively. Within Group 3, however, a significantly (P < 0.05) lower percentage of the embryos continued to develop when the agar was not removed after thawing (31.8%) compared with embryos from which the agar had been removed (68.8%). After nonsurgical transfer of 78 embryos, the pregnancy rate was significantly (P < 0.05) lower (8.3%) with embryos of Group 3 compared with controls (61.5%) or embryos of Group 2 (42.9%). No significant difference existed between controls and embryos of Group 2. We conclude that an intact zona pellucida prior to rapid freezing is not essential for the survival of Day 7 bovine embryos.

One-step sucrose dilution of frozen-thawed sheep embryos.

Sheep embryos of the late morula to early blastocyst stage were frozen, thawed and cultured to test several sucrose solutions for post-thaw dilution of the cryoprotective agent glycerol. Ewes of mixed breeding were superovulated and embryos were flushed from the uterus either surgically or at slaughter 5 d after estrus. Fifty-eight embryos were pooled in microdrops of modified Dulbecco's phosphate buffered saline (MDPBS) then randomly divided into four treatments. A 2 x 2 factorial design was used to compare 0.25 M sucrose in MDPBS as an in-straw cryoprotectant dilution with a standard step-wise dilution procedure within standard fast and slow freeze-thaw systems. After storage in liquid nitrogen for 6 to 8 d, the embryos were thawed and the cryoprotectant (1.4 M glycerol) removed before culture in microdrops of modified synthetic oviduct fluid under paraffin oil in water-saturated 5% CO(2) in air atmosphere at 37 C. No significant interaction was found between the freeze-thaw procedure and cryoprotectant + dilution procedures. Embryos in the fast freeze-thaw procedure had a mean development score of 1.3 +/- 0.3 and those in the slow freeze-thaw procedure had a mean score of 1.2 +/- 0.3. The mean development score 2.0 +/- 0.3 for the standard dilution procedure was superior (P<0.001) to the score of 0.6 +/- 0.2 for the 0.25 M sucrose dilution procedure. In a separate trial, 18 sheep morulae were collected and equilibrated with 1.4 M glycerol in MDPBS. A standard fast freeze-thaw procedure was used and, after 18 d of storage at -196 C, the glycerol was diluted from the embryo with 1.0 M sucrose. Culture was conducted in a similar manner and a mean development score of 1.0 +/- 0.3 was obtained. These results indicate standard cryoprotectant dilution procedures for sheep embryos are superior to dilution with 0.25 M sucrose. In a limited study, dilution with 1.0 M sucrose was also not as effective as standard dilution procedures.