A dose-dependent response to MEK inhibition determines hypoblast fate in bovine embryos.

BACKGROUND: The segregation of the hypoblast and the emergence of the pluripotent epiblast mark the final stages of blastocyst formation in mammalian embryos. In bovine embryos the formation of the hypoblast has been partially studied, and evidence shows that MEK signalling plays a limited role in the segregation of this lineage. Here we explored the role of different signalling pathways during lineage segregation in the bovine embryo using immunofluorescence analysis of NANOG and SOX17 as readouts of epiblast and hypoblast, respectively. RESULTS: We show that SOX17 starts to be expressed in 16-32-cell stage embryos, whereas NANOG is first detected from 8-cell stage. SOX17 is first co-expressed with NANOG, but these markers become mutually exclusive by the late blastocyst stage. By assessing the expression kinetics of NANOG/SOX17 we show that inhibition of MEK signalling can eliminate SOX17 expression in bovine blastocysts, without altering NANOG expression. Modulation of WNT, PKC and LIF did not affect NANOG expression in the epiblast when used in combination with the ERK inhibitor. CONCLUSIONS: This study shows that SOX17 can be used as a reliable early marker of hypoblast in the bovine, and based on its expression profile we show that the hypoblast segregates in day 7 blastocysts. Furthermore, SOX17 expression is abolished using 1 µM of PD0325901, without affecting the NANOG population in the epiblast. Modulation of WNT, PKC and LIF are not sufficient to support enhanced NANOG expression in the epiblast when combined with ERK inhibitor, indicating that additional signalling pathways should be examined to determine their potential roles in epiblast expansion.

Detection of recombinant human lactoferrin and lysozyme produced in a bitransgenic cow.

Lactoferrin and lysozyme are 2 glycoproteins with great antimicrobial activity, being part of the nonspecific defensive system of human milk, though their use in commercial products is difficult because human milk is a limited source. Therefore, many investigations have been carried out to produce those proteins in biological systems, such as bacteria, yeasts, or plants. Mammals seem to be more suitable as expression systems for human proteins, however, especially for those that are glycosylated. In the present study, we developed a bicistronic commercial vector containing a goat ß-casein promoter and an internal ribosome entry site fragment between the human lactoferrin and human lysozyme genes to allow the introduction of both genes into bovine adult fibroblasts in a single transfection. Embryos were obtained by somatic cell nuclear transfer, and, after 6 transferences to recipients, 3 pregnancies and 1 viable bitransgenic calf were obtained. The presence of the vector was confirmed by fluorescent in situ hybridization of skin cells. At 13 mo of life and after artificial induction of lactation, both recombinant proteins were found in the colostrum and milk of the bitransgenic calf. Human lactoferrin concentration in the colostrum was 0.0098 mg/mL and that in milk was 0.011 mg/mL; human lysozyme concentration in the colostrum was 0.0022 mg/mL and that in milk was 0.0024 mg/mL. The molar concentration of both human proteins revealed no differences in protein production of the internal ribosome entry site upstream and downstream protein. The enzymatic activity of lysozyme in the transgenic milk was comparable to that of human milk, being 6 and 10 times higher than that of bovine lysozyme present in milk. This work represents an important step to obtain multiple proteins or enhance single protein production by using animal pharming and fewer regulatory and antibiotic-resistant foreign sequences, allowing the design of humanized milk with added biological value for newborn nutrition and development. Transgenic animals can offer a unique opportunity to the dairy industry, providing starting materials suitable to develop specific products with high added value.

Bovine campylobacteriosis in heifer: pathogenesis study and insights in the conventional and molecular diagnosis in an experimental bovine model and field cases.

Campylobacter fetus spp. is a bacterium associated to reproductive losses in cattle worldwide. It is a venereal infectious disease known as bovine campilobacteriosis, with high impact mainly in countries with extensive production systems. Here, we show pathogenesis and diagnostic methods for Campylobacter fetus detection in cervico-vaginal mucus (CVM) samples from heifers experimentally infected and field cases from herds with low reproductive performance by campylobacteriosis infection. Bacterial culture, direct immunofluorescence test and qPCR were used as diagnostic methods to evaluate detection of C. fetus. In the experimental model 30 Aberdeen Angus and crossbred heifers and 4 Aberdeen Angus bulls for natural mating were assigned to 3 groups experimentally challenged with C. fetus subsp. fetus (Cff), C. fetus subsps venerealis (Cfv) and C. fetus subsp venerealis biovar intermedius (Cfvi), respectively, and a negative control group, all followed for 9 months. Also, field samples of CVM and aborted fetuses were recollected from seven beef cattle farms. Bacteriological culture had the higher C. fetus detection rate in CVM being the most appropriate, followed by qPCR (with commercial extraction DNA kit), direct immunofluorescence test and qPCR (with in-house extraction DNA method), in both, experimental model and field cases. From experimental model after natural mating, 62.5% and 25% heifers got pregnant from Cff and Cfvi groups, respectively, while from Cfv no pregnancy was detected. The strain more frequently detected was Cfvi, followed by Cff and Cfv. Colonization of Cff in female genital tract with high number of carriers and presence in aborted fetuses was evidenced, suggesting a high risk to bovine reproductive health. Bacteriemia was not detected after genital infection. Given the low detection rate of either test, we suggest the use of both, PCR based methods and bacterial culture could result in higher detection rate in farms with endemic campylobacteriosis.

Bovine campylobacteriosis in bulls: insights in the conventional and molecular diagnosis.

Campylobacter fetus is a gram-negative motile bacterium, with two subspecies relevant to cattle health: C. fetus subsp. venerealis (Cfv) and C. fetus subsp. fetus (Cff). Both subspecies are associated with reproductive losses in cattle. In this study, we evaluated the identification of C. fetus for the diagnosis of bovine campylobacteriosis through bacteriological culture, direct immunofluorescence (DIF) and molecular tests in preputial smegma (PS) samples of three Angus bulls challenged with Cfv, Cfv biovar intermedius (Cfvi) or Cff, respectively, in an experiment imitating the natural infection. Two DNA extraction protocols were tested (in-house thermal extraction and commercial kit). Aspiration and scraping collection for PS were compared by conventional tests. Additionally, bacteremia was also evaluated in blood samples. Bulls were challenged by natural mating with heifers that had been experimentally infected with C. fetus subspecies; which led to infection. The Cfv- and Cfvi-bulls were positive for at least 9 months. Although Cff is not considered a venereal strain, in this study it was transmissible to bull from heifers experimentally infected, as evidenced by its colonization and persistence in the preputial cavity for 5 to 6 months. This finding suggests a potential risk of dissemination within herds. The results obtained by bacteriological culture or direct immunofluorescence (DIF) showed no significant differences, regardless the sampling device used (aspiration with Cassou pipette, metal and plastic scraper). C. fetus qPCR, on the other hand, yielded better results with an in-house DNA extraction method than with a commercial kit (75% vs 66.6%). Furthermore, qPCR diagnosis was more efficient than culture (66.6%) or DIF (56%). Bacteremia in whole blood samples was negative by qPCR and bacteriological culture in all samples. Altogether, this study demonstrated the transmission of Cff from heifers to bull and also showed that PCR-based methods are promising for the diagnosis of Bovine Genital Campylobacteriosis from clinical samples of PS.

Isolation of Campylobacter fetus subsp. venerealis from seminal vesicle of a naturally challenged bull.

Campylobacter fetus is a well-recognized pathogen that affects reproductive rate in cattle. In the present study, two Angus bulls were kept (39 days) separately with a group of heifers experimentally infected with Campylobacter fetus subsp. venerealis (Cfv) and Campylobacter fetus subsp. venerealis biovar intermedius (Cfvi), respectively. Each bull resulted infected post-mating by its respective strain (Cfv and Cfvi). Semen samples collected from each bull at days 39, 82, 132 and 269 resulted positive for C. fetus by bacteriological culture and/or direct immunofluorescence (DIF) test, and confirmed by polymerase chain reaction (PCR) from colonies isolated. Diagnosis resulted better with bacteriological culture (100%) compared to DIF (37,5%). Campylobacter fetus was isolated from seminal vesicle and preputial mucosa by bacteriological culture and confirmed by PCR and DIF test from colonies previously isolated from these tissues (day 276). Microscopic lesions detected in both bulls showed moderate diffuse subepithelial lymphoplasmacytic postitis. None of the seminal vesicle presented relevant microscopic lesions. To our knowledge this is the first report of isolation of C. fetus from seminal vesicles in a bull. The experimental model herein described, mimicks the natural infection and constitutes a promising alternative for future studies of campylobacteriosis in cattle.

Plasma steroid profiles and ovarian response in llamas treated with eCG for superovulation combined with exogenous progesterone during early luteal phase.

The objective of this study was to evaluate the effects of plasma progesterone (P4) concentrations during eCG-ovarian follicular superstimulatory treatment performed in early luteal phase and estradiol concentrations during peri-ovulatory period on ovarian response, number and embryo quality. On Day -2, females (n = 75) having a follicle ≥7 mm were treated with GnRH to induce ovulation. On Day 0, females that had ovulations (n = 54) were treated with 1000 IU eCG and were assigned to one of two treatments: (1) intravaginal device (ID) containing 0.5 g P4 (P4 group) and (2) no ID (Control group). On Day 5, females were administered PGF2α and the ID was removed. On Day 7 and 8, females were mated and embryo recovery was performed 7 or 8 days later. Blood samples were collected from Day 0 to 9. Number (± SD) of follicles ≥7 mm on day of mating was greater (P =  0.04) in the control (9.7 ± 4.2) than P4-treated (6.7 ±â€¯4.9) group; number of corpora lutea did not differ (5.5 ±â€¯3.1 and 5.2 ±â€¯3.4 respectively). Ovulation rate was greater (P <  0.01) in the P4-group (77.4%; 130/168) than control group (53.3%; 135/253). Number of embryos with an excellent grade (grade 1) tended to be greater (P =  0.07) in the P4-group (82.4%; 42/51) than control group (65.4%; 36/55). It was concluded that supplementation with exogenous P4 during eCG treatment in early luteal phase inhibits excessive follicular growth, increases ovulation rate and improves embryo quality.

Effect of GnRH analogue administration on Day 7 after natural mating on formation accessory corpus luteum, progesterone concentration and conception rate in llamas (Lama glama).

The objectives of the present study were to determine the effects of exogenous GnRH administered 7 days after breeding on the formation of an accessory corpus luteum (ACL), plasma progesterone (P4) concentrations and pregnancy rates. Adult females (n = 71) having a follicle ≥ 7 mm in diameter in the ovary were naturally mated (Day 0). On Day 7, ultrasonic examination was performed to confirm the occurrence of ovulation as evidenced by presence of an induced corpus luteum (ICL). Females with an ICL plus a dominant follicle ≥ 7 mm (n = 56) were treated with saline solution (SS, n = 29) or GnRH analogue (n = 27). On Day 14, the formation of an ACL was observed by ultrasonography. Blood samples were collected on Days 7 and 14 to quantify plasma P4 concentrations. On Day 14, 21 of 27 (77.8%) females in the GnRH group developed an ACL, whereas females in the SS group did not. Progesterone concentrations on Day 7 and 14 in those llamas diagnosed as pregnant on Day 30 were not different (P > 0.05) between groups. In addition, P4 concentration was similar for GnRH-treated females having two CL to those with a single CL. Pregnancy rates were similar (P > 0.05) between SS and GnRH groups (55.2% compared with 74.1% respectively) and the pregnancy rate for the GnRH group was not affected (P > 0.05) by the number of CL observed at Day 14 (66.6% and 75.6% for females with one and two CL respectively). In conclusion, GnRH administration on Day 7 after breeding leads to ACL formation; however, neither the plasma P4 concentration nor pregnancy rate was affected by having an ACL.

Exogenous human OKSM factors maintain pluripotency gene expression of bovine and porcine iPS-like cells obtained with STEMCCA delivery system.

OBJECTIVES: The use of induced pluripotent stem (iPS) cells as an alternative to embryonic stem cells to produce transgenic animals requires the development of a biotechnological platform for their generation. In this study, different strategies for the generation of bovine and porcine iPS cells were evaluated. Lentiviral vectors were used to deliver human factors OCT4, SOX2, KLF4 and c-MYC (OKSM) into bovine and porcine embryonic fibroblasts and different culture conditions were evaluated. RESULTS: Protocols based on the integrative lentiviral vector STEMCCA produced porcine iPS-like cells more efficiently than in bovine cells. The iPS-like cells generated displayed stem cell features; however, expression of exogenous factors was maintained along at least 12 passages. Since inactivation of the exogenous factors is still a major bottleneck for establishing fully reprogrammed iPS cells, defining culture conditions that support endogenous OKSM expression is critical for the efficient generation of farm animals' iPS cells.

Fetal mortality diagnosis by ultrasound in the vicuña (Vicugna vicugna).

Ultrasonography is widely used in domestic species of camelids, but there is no information about the use of this technique for pregnancy diagnosis and determination of embryonic or fetal losses in the vicuña (Vicugna vicugna). The study was performed in 202 vicuñas (3-year-old females, n = 31; adult females, n = 171) mated during the summer months (January through March 2001) at the Abra Pampa Experimental Farm of Altitude in north-west Argentina. Transrectal ultrasound examination was performed in May (estimated 40-120 days of gestation) to determine the number of pregnant females. The pregnancy rate was 45.5% (92/202). No significant difference (P > 0.05) was observed between the pregnancy rate of 3-year-old females (41.9%) and adult females (46.2%). In December (estimated 250-330 days of gestation) of the same year, a second ultrasonographic study was performed on those vicuñas that were diagnosed as pregnant from the first ultrasound scan. Of 92 animals diagnosed as pregnant in May, only 84 were present in December, because eight females died in the period of study. Overall, 11.9% (10/84) of fetuses were lost during the period (18.1% in 3-year-old vicuñas and 10.9% in adult vicuñas). In conclusion, transrectal ultrasonography was found to provide a rapid and non-invasive means for pregnancy and fetal mortality diagnosis in vicuñas.