Search

The in vitro production of bovine embryos by in vitro fertilization or nuclear transfer procedures is a powerful tool in wide use for scientific, conservationist and commercial purposes. However, developmental abnormalities are unpredictable consequences of such in vitro embryo manipulations, which may interfere with the pattern of fetal and placental growth and life ex utero, in a set of symptoms collectively called Large Offspring Syndrome (LOS). The economical significance of the syndrome is associated with increased rates of pregnancy losses, placental and fetal aberrations that culminate in abortion, hydrops of the fetal membranes, prolonged gestation, diminished signs of parturition, dystocia, and birth of large calves with lower postnatal survival. Lower pregnancy rates with higher gestational and postnatal losses represent significant economical losses for a lower prolificacy. The understanding of mechanisms of prenatal growth in normal development and in those related to the syndrome will be of significance for prevention or attenuation of abnormalities of common occurrence in cattle, with potential direct scientific and economical implications.La producción in vitro de embriones por fertilización in vitro o por procedimientos de transferencia de núcleos es una herramienta poderosa utilizada ampliamente para propósitos científicos, conservacionistas y comerciales. Sin embargo, las alteraciones del desarrollo son consecuencias impredecibles de tales manipulaciones in vitro, que pueden interferir con el patrón de de crecimiento fetal y placentario y la vida ex utero, que se agrupan en unos síntomas conocidos colectivamente como el síndrome del ternero grande (LOS). La importancia económica del síndrome está asociada con un incremento en las pérdidas gestacionales, aberraciones placentarias y fetales que culminan en aborto, condiciones hidópicas de las membranas fetales, gestaciones prolongadas, disminución en los signos de parto, distocias, y nacimiento de terneros grandes con baja supervivencia postnatal. Las bajas tasas de preñez con las altas tasas de pérdidas gestacionales representan pérdidas económicas significativas por baja prolificidad. El entendimiento de los mecanismos de crecimiento prenatal durante el desarrollo normal y en aquellos relacionados con el síndrome sería muy significativo en la prevención, atenuación de las anormalidades de ocurrencia común en el ganado, con implicaciones directas a nivel científico y económico.

Abstract Background Enteroaggregative Escherichia coli (EAEC) causes diarrhea, malnutrition and poor growth in children. Human breast milk decreases disease-causing bacteria by supplying nutrients and antimicrobial factors such as lysozyme. Goat milk with and without human lysozyme (HLZ) may improve the repair of intestinal barrier function damage induced by EAEC. This work investigates the effect of the milks on intestinal barrier function repair, bacterial adherence in Caco-2 and HEp-2 cells, intestinal cell proliferation, migration, viability and apoptosis in IEC-6 cells in the absence or presence of EAEC. Methods Rat intestinal epithelial cells (IEC-6, ATCC, Rockville, MD) were used for proliferation, migration and viability assays and human colon adenocarcinoma (Caco-2, ATCC, Rockville, MD) and human larynx carcinoma (HEp-2, ATCC, Rockville, MD) cells were used for bacterial adhesion assays. Goats expressing HLZ in their milk were generated and express HLZ in milk at concentration of 270 μg/ml . Cells were incubated with pasteurized milk from either transgenic goats expressing HLZ or non-transgenic control goats in the presence and absence of EAEC strain 042 (O44:H18). Results Cellular proliferation was significantly greater in the presence of both HLZ transgenic and control goat milk compared to cells with no milk. Cellular migration was significantly decreased in the presence of EAEC alone but was restored in the presence of milk. Milk from HLZ transgenic goats had significantly more migration compared to control milk. Both milks significantly reduced EAEC adhesion to Caco-2 cells and transgenic milk resulted in less colonization than control milk using a HEp-2 assay. Both milks had significantly increased cellular viability as well as less apoptosis in both the absence and presence of EAEC. Conclusions These data demonstrated that goat milk is able to repair intestinal barrier function damage induced by EAEC and that goat milk with a higher concentration of lysozyme offers additional protection.

Abstract Background Placental and fetal growth requires high rates of cellular turnover and differentiation, which contributes to conceptus development. The trophoblast has unique properties and a wide range of metabolic, endocrine and angiogenic functions, but the proliferative profile of the bovine placenta characterized by flow cytometry analysis and its role in fetal development are currently uncharacterized. Complete understanding of placental apoptotic and proliferative rates may be relevant to development, especially if related to the pathogenesis of pregnancy losses and placental abnormalities. Methods In this study, the proliferation activity and apoptosis in different regions of normal bovine placenta (central and boundary regions of placentomes, placentomal fusion, microplacentomes, and interplacentomal regions), from distinct gestation periods (Days 70 to 290 of pregnancy), were analyzed by flow cytometry. Results Our results indicated that microplacentomes presented a lower number of apoptotic cells throughout pregnancy, with a higher proliferative activity by the end of gestation, suggesting that such structures do not contribute significantly to normal of placental functions and conceptus development during pregnancy. The placentome edges revealed a higher number of apoptotic cells from Day 170 on, which suggests that placentome detachment may well initiate in this region. Conclusion Variations involving proliferation and apoptotic rates may influence placental maturation and detachment, compromising placental functions and leading to fetal stress, abnormalities in development and abortion, as frequently seen in bovine pregnancies from in vitro fertilization and cloning procedures. Our findings describing the pattern of cell proliferation and apoptosis in normal bovine pregnancies may be useful for unraveling some of the developmental deviations seen in nature and after in vitro embryo manipulations.

Studies investigating physiological deviations from normality in newborn calves derived from in vitro fertilization procedures remain important for the understanding of factors that reduce calf survival after birth. The aim of this study was to investigate parameters affecting health and welfare of newborn Flemish calves derived from in vitro embryo production (IVP) in the first hours of life in comparison to in vivo‐derived calves. Physical traits of newborn calves and fetal membranes (FM) were recorded soon after birth. Newborn venous blood samples were collected at several time points within the first 24 h of life for analyses of energy substrates, electrolytes, blood gases, acid–base balance, blood chemistry, and haematology. A liver biopsy was taken within the first hour after birth for analysis of gene expression of key enzymes of the fructolytic and glycolytic pathways. Newborn IVP calves were heavier and larger at birth, which was associated with heavier FM. At several time points during the first 24 h of life, IVP‐derived calves had altered rectal temperature, blood gases, electrolyte concentrations, blood parameters for liver, kidney and muscle function, and acid–base balance, plasma lipid metabolism, and hemogram parameters. The relative mRNA abundances for triokinase and lactate dehydrogenase‐B were greater in IVP calves. In summary, IVP‐derived newborn calves were at higher risk of clinical problems after birth, which was markedly greater in heavier and larger calves. Such animals take longer to adapt to extrauterine life and should receive a special attention during the immediate neonatal period. [ABSTRACT FROM AUTHOR]

The urge for the control of reproductive processes in animals has propelled a great gain in knowledge, also setting off the development of four generations of assisted reproductive technologies (AR T) for humans and animals. The use of assisted reproductive techniques has been of great importance in livestock production. In general terms, the main first three generations of ARTs, including 1) artificial insemination (AI) and gamete and embryo freezing, 2) multiple ovulation and embryo transfer (MOET) and 3) in vitro fertilization (IV F) procedures, have matured into successful commercial applications, facilitating the increase in production through genetics, the reduction in generation intervals, the control of diseases, and the cutback in production costs. The fourth generation of AR T encompasses processes that are still more experimental, comprising cloning by nuclear transfer (NT) of embryonic or somatic cells, transgenesis, and stem cell biology. Such technologies are intertwined with one another and with currently available molecular tools, being completely dependent upon the previous generations of technologies. However, many reproductive challenges still hinder maximal livestock reproductive performance, affecting productivity and profitability. It is clear that the application of such technologies as lucrative activities will remain questionable if not associated with other components of animal production, such as animal health, nutrition and adequate animal husbandry practices.El afán por controlar los procesos reproductivos en animales ha llevado a una gran ganancia en conocimiento, impulsando el desarrollo de cuatro tecnologías reproductivas asistidas (AR Ts) para animales y humanos. El uso de AR Ts ha sido de gran importancia en la producción ganadera. En términos generales, las tres principales generaciones de AR T, incluyendo 1) inseminación artificial (AI) y congelación de gametos y embriones, 2) superovulación y transferencia de embriones (MOET) y 3) procedimientos de fertilización in Vitro, han madurado en aplicaciones comerciales exitosas, facilitando el incremento en la producción a través de la genética, reducción del intervalo generacional, control de enfermedades, y reducción de costos de producción. La cuarta generación de AR Ts incluye procesos que aún son muy experimentales, como transferencia de núcleos (NT) de células somáticas, transgénesis, y biología de células madre. Estas tecnologías se intercalan las unas con las otras y con las herramientas moleculares actuales, dependen completamente de las generaciones de tecnologías previas. Sin embargo, hay muchos retos reproductivos que no permiten alcanzar el potencial reproductivo máximo, afectando la productividad y la rentabilidad. Es claro que la aplicación de tales tecnologías como actividades lucrativas se mantendrán cuestionadas si no se asocian a otros componentes de la producción pecuaria, como la salud animal, nutrición, y prácticas de manejo adecuadas.

Background: The use of conventional artificial insemination (AI) in sheep production is usually associated with lower fertility rates when frozen semen is used. Cooled ram semen has been an alternative over frozen semen due to the higher viability, seminal quality and fertility rates following AI. The semen preservation process promotes sperm cell modifications similar to capacitation (capacitation-like) that causes cell damage affecting viability and seminal quality, but such effects are unclear for cooled semen. The aim of this study was to determine the status of sperm cell capacitation (CA) and acrosome reaction (AR) during ram semen processing and cooling under different extenders, dilution factors, and aerobiosis conditions as a function of storage time at 5o C. Materials, Methods & Results: Two consecutive ejaculates per day per male were collected from 2 adult rams by artificial vagina at 48-72 h intervals, in three replications. After macro- and microscopic evaluations, semen was segregated into groups under 3 extenders (Tris-egg yolk or TY, citrate-egg yolk or CY, skimmed milk or SM), 2 dilution factors (1 x 109 or Bi, 100 x 106 or Mi cells/mL), and 2 aerobiosis conditions (aerobic or A, semi-anaerobic or SA). Diluted semen was cooled to 5ºC and stored for up to 72 h, with evaluations every 24 h. Aliquots of fresh ejaculates and of each cooled diluted subgroup, according to extender, dilution, and aerobiosis, were collected at times T0 and T72 for determination of acrosome status and membrane integrity by the chlortetracycline (CTC) and trypan blue-Giemsa stainings, respectively. No differences were detected in sperm cell motility (M) and motility vigor (V) between fresh and diluted semen. After cooling, a significant decrease in M was observed after 48 h in CY and SM compared with fresh semen and 0 h of cooling, while V started to decrease after 24 h in CY compared with TY. Likewise, M/V from different dilutions and aerobic conditions decreased more significantly after 48 and 24 h of cooling, respectively. The sperm capacitation status did not show differences in the proportion of non-capacitated (NCA), CA and AR sperm cells between TY, CY, and SM extenders (NCA: 75.0%, 71.3%, 74.0%; CA: 15.7%, 17.2%, 15.9%; AR: 9.3%, 11.5%, 10.2%) or between Bi and Mi dilutions (NCA: 74.0%, 72.9%; CA: 15.9%, 16.6%; AR: 10.1%, 10.5%), respectively. However, differences (P < 0.05) were observed between A and SA aerobic conditions, with CA (17.0% vs. 15.5%) and AR (11.9% vs. 8.7%) rates being higher in A than SA, respectively, with no differences in NCA (71.1% vs. 75.8%), irrespective of the storage time. Sperm cell viability decreased after 48 h, especially in CY (P < 0.05). Discussion: Ram sperm cells can suffer irreversible damage due to thermal shock during cooling. Egg yolk-based extenders provide phospholipids and cholesterol to protect the sperm cell membrane during the thermal shock caused by the change in temperature. In this study, sperm cells had irreversible decreases in M/V, with increase in acrosome and plasma membrane damage after cooling to 5ºC. The largest and smallest decreases in M and V over time were observed in the CY and TY extenders, respectively. In addition to the extender type, the semen preservation method and storage time promoted changes in the capacitation status, AR and in sperm cell viability, which per se were associated with a decrease in semen fertility. In fact, the proportions of CA and/or AR sperm cells gradually increased over time after dilution and storage at 5ºC, with a negative correlation between sperm cell viability and M/V over time. In summary, extender and cooling time affected mostly M/V, while aerobiosis condition and dilution factor were more associated with acrosome status and sperm survival, with the extender having less impact on the acrosome status as a function of time. [ABSTRACT FROM AUTHOR]

Contents: The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft. [ABSTRACT FROM AUTHOR]