Analysis of physical traits, clinical parameters, and energy metabolism of in vivo- and in vitro-derived Flemish newborn calves during the first day of life.

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.

Inter-software reliability and agreement for follicular and luteal morphometric and echotextural ultrasonographic parameters in beef cattle.

This study aimed to compare the inter-software and inter-observer reliability and agreement for the assessment of follicular and luteal morphometry and echotexture parameters in beef crossbreed females (3/8 Bos taurus indicus and 5/8 Bos taurus taurus). B-mode and color Doppler ultrasonographic ovarian images were obtained at specific time points of estradiol-progesterone-based protocols for timed artificial insemination (TAI). Sonograms were analyzed by two observers using a licensed (IASP1) and an open access (IASP2) software package. A total of 292 snap-shot sonograms were analyzed for morphometric parameters and 504 for echotexture parameters. inter-software reliability was judged moderate to excellent (ICC or CCC=0.73-0.98), whereas inter-observer reliability for morphometric parameters was deemed good to excellent (ICC or CCC=0.82-0.98). A small percentage (up to 10.95 %) of measured parameters fell outside the limits of inter-software and inter-observer agreement. For echotexture parameters, inter-software reliability varied widely (ICC or CCC=0.16-0.95) based on the size of regions of interest (ROI), while inter-observer reliability ranged from moderate to excellent (ICC or CCC= 0.71-0.97). The highest inter-software reliability for pixel value and heterogeneity value was observed for the corpus luteum (ICCs=0.81-0.95; P>0.05), followed by the peripheral follicular antrum (ICCs=0.75-0.78; P<0.05). However, lower reliability was determined for the follicular wall (ICCs=0.08-0.33; P<0.0001) and perifollicular stroma (ICCs=0.16-0.46; P<0.05). In conclusion, both software packages showed high reproducibility for morphometric measurements, while echotexture measurements were more challenging to replicate based on ROI sizes. Caution is advised when selecting ROI sizes for echotexture measurements in bovine ovaries.

In vitro and in vivo embryo production efficiency in Flemish and Holstein donor females.

The aim of this study was to compare embryo production efficiency in Flemish and Holstein donor females using ovum pick-up and in vitro fertilization (OPU-IVF) or in vivo production (superovulation; SOV) procedures. The study was conducted using a split-plot design, with eight Flemish and eight Holstein non-lactating cycling females. Females were subjected to ten weekly OPU/IVF sessions and/or two SOV/embryo collections sessions at a 63-day interval, for a total of 160 OPU-IVF and 32 SOV sessions. Mean numbers of follicles and corpora lutea, and cumulus-oocyte complex (COC) recovery rates were similar between breeds after the OPU and SOV sessions. However, Flemish donors yielded better quality grade II COCs (301, 41.9%) than Holstein females (609, and 202, 33.1%). Also, cleavage and blastocyst rates, and the total number and the mean number of viable embryos obtained after OPU-IVF were higher in Flemish (49.6% and 11.8%, and 63 and 11.8 per donor, respectively) than in Holstein (32.8% and 7.2%, and 34 and 7.2 per donor, respectively) females. Flemish females were also more efficient in yielding viable embryos after SOV (111, 7.3 per donor) than Holstein (48, 3.3 per donor) females. Overall, Flemish donor females had better responses to OPU-IVF or SOV procedures than Holstein counterparts. Irrespective of the breeds, SOV procedures were more efficient than OPU-IVF in yielding more viable embryos, under the conditions of this study. Both reproductive procedures were useful tools for the genetic conservation of the Flemish cattle breed in Southern Brazil.

ß-Mercaptoethanol in culture medium improves cryotolerance of in vitro-produced bovine embryos.

The objective of this study was to investigate the effects of adding ß-mercaptoethanol (ßME) to culture medium of bovine in vitro-produced (IVP) embryos prior to or after vitrification on embryo development and cryotolerance. In Experiment I, Day-7 IVP blastocysts were vitrified and, after warming, cultured in medium containing 0, 50 or 100 µM ßME for 72 h. Embryos cultured in 100 µM ßME attained higher hatching rates (66.7%) than those culture in 0 (47.7%) and 50 (52.4%) µM ßME. In Experiment II, IVP embryos were in vitro-cultured (IVC) to the blastocyst stage in 0 (control) or 100 µM ßME, followed by vitrification. After warming, embryos were cultured for 72 h (post-warming culture, PWC) in 0 (control) or 100 µM ßME, in a 2 × 2 factorial design: (i) CTRL-CTRL, control IVC and control PWC; (ii) CTRL-ßME, control IVC and ßME-supplemented PWC; (iii) ßME-CTRL, ßME-supplemented IVC and control PWC; or (iv) ßME-ßME, ßME-supplemented IVC and ßME-supplemented PWC. ßME during IVC reduced embryo development (28.0% vs. 43.8%) but, following vitrification, higher re-expansion rates were seen in ßME-CTRL (84.0%) and ßME-ßME (87.5%) than in CTRL-CTRL (71.0%) and CTRL-ßME (73.1%). Hatching rates were higher in CTRL-ßME (58.1%) and ßME-ßME (63.8%) than in CTRL-CTRL (36.6%) and ßME-CTRL (42.0%). Total cell number in hatched blastocysts was higher in ßME-ßME (181.2 ± 7.4 cells) than CTRL-CTRL (139.0 ± 9.9 cells). Adding ßME to the IVC medium reduced development but increased cryotolerance, whereas adding ßME to the PWC medium improved embryo survival, hatching rates, and total cell numbers.

Impact of donor nutritional balance on the growth and development of mesenchymal stem cells from caprine umbilical cord Wharton´s jelly.

Mesenchymal stem cells (MSCs) from the umbilical cord (UC) have aroused considerable interest. However, little is known about the maternal effect on these cells. The aim of this study was to verify the impact of the nutritional status of donor goats on the growth and differentiation of MSCs from the UC. At parturition, 19 goats were grouped based on their low or high body mass index (low BMI, LBMI, n = 9; and high BMI, HBMI, n = 10). UCs were collected during delivery and Wharton's jelly (WJ) fragments cultured. WJ-MSCs were differentiated into osteocytes, adipocytes, chondrocytes, and the population doubling time (PDT) was determined. Samples of WJ-MSCs were also used to verify the expression of the CD90, CD73, CD34, CD45, and CD105 genes. Media used for WJ-MSC primary cultures were analyzed using near-infrared spectroscopy. The lag phase was 7.5 ± 0.6 days and the entire culture took 26.7 ± 1.3 days, with a cell proliferation rate of 8.500 cells/day. The mean PDT from subculture was 30.0 ± 0.7 h. The CD105 gene was sub-expressed in LBMI, and the spectra of the spent media from the second to fourth day of WJ-MSC primary culture were segregated into negative scores by multivariate analysis. We conclude that, in goats, the nutritional balance of the donor did not affect the in vitro growth of MSCs derived from the UC. However, the molecular profile observed in the low BMI group suggests that the use of MSCs for therapeutic purposes should be considered more carefully.

Components of the insulin-like growth factor system in in vivo - and in vitro-derived fetuses of cattle, and the association with growth and development.

This experiment was designed to study mechanisms affecting growth of in vivo-derived (IVD) and in vitro-produced (IVP) fetuses of cattle. Day-7 IVD or IVP cattle blastocysts were transferred to recipients, with pregnant females being slaughtered on Days 90 or 180 of gestation or allowed to undergo parturition. Uteri and contents were dissected and physically measured, and maternal and fetal plasma and amniotic and allantoic fluids were collected for IGF-1 and IGF-2 determinations, and IGFBP profile characterization. Transcripts for IGF-1 and IGF-2 mRNA in placental and fetal tissues, and IGF-1r and IGF-2r in placentomes were determined. There was a greater fetal weight in the IVP group, which was associated with greater IGF-1 and IGF-2 concentrations in maternal circulation, and changes in IGFBP profiles within fetal fluids. Day-90 IVP-derived fetuses were longer, had greater organ weights, larger placentomes, less placentome IGF-2r mRNA transcript, and greater maternal IGF-1 and IGF-2 concentrations than controls. On Day 180 and at parturition tissues from IVP-derived fetuses/calves were from larger uteri, with larger placentomes/fetal membranes, fetuses/calves weighed more, had greater fetal hepatic IGF-2 mRNA transcript, had less fetal plasma IGF-1 and greater allantoic IGF-2 concentrations, greater and lesser IGFBP activities in the allantoic and amniotic fluids, respectively, and greater glucose and fructose accumulation in fetal fluids. Components of the IGF system were differentially regulated not only according to the gestation period (Days 90 or 180) and fluid type (maternal or fetal plasma, amniotic or allantoic fluids), but also based on conceptus origin (IVP or IVD) in cattle.

A rare case of heteropaternal twin calves after natural mating in Brazil.

Twin birth is a complex condition observed in most livestock animals, when the female gives birth to two or more offspring, generally out of the same mating. In cattle, it is a rare condition (3 to 5%) and depends on the genetic background and environmental factors. Twin birth is a result of multiple ovulations, being more common in dairy rather than in beef cattle. Calves could be monozygous or dizygous, with the same or of different sexes. When twins are born with different sexes, a sexual condition called Freemartinism occurs in between 90 to 97% of pregnancies, causing infertility in the female calf. Knowing that the twin rate is rare in commercial beef cattle, here we present an even rarer case of twin birth from two different sires after natural mating, also called heteropaternal superfecundation.

Promoter-specific expression of the imprinted IGF2 gene in bovine oocytes and pre-implantation embryos.

The bovine IGF2 locus is a genomic region with alternative transcripts controlled by five promoters (P0, P1, P2, P3 and P4). As transcriptional regulation can affect messenger RNA (mRNA) stability and translation, and thus, subsequent biological effects, this study evaluated the bovine IGF2 promoter-specific expression patterns in oocytes and pre-implantation embryos produced in vitro by our standard IVP procedures. Immature and matured oocytes, and pre-implantation embryos at the 1-, 2-, 4-, 8- and 16-cell, and at early morula, compact morula, blastocyst and expanded blastocyst stages were collected in three pools of five structures per stage, in four replicates. Total RNA was extracted and subjected to RT-qPCR, using four sets of IGF2 promoter-specific primers covering transcripts driven by promoters P0/P1, P2, P3 and P4, with fragments sequenced for confirmation. Expression of P2- and P4-derived transcripts showed an initial peak between immature (P4) or matured (P2/P4) oocytes and 2-cell embryos, gradually falling until embryo genome activation (EGA), rising again at compaction and cavitation. P0/P1-derived transcripts were identified after EGA, during compaction, whereas P3 activity was not detected at any stage. Our findings suggest that P0/P1 and P2 likely have secondary roles during early stages, whereas P3 may be more relevant later in development. P4 seems to be the main pathway for bovine IGF2 expression during oocyte maturation and embryo development and, therefore, the main target to influence IVP in modulation of embryo growth and in studies in developmental biology.

Proteomic profile of pre-implantational ovine embryos produced in vivo.

The present study was conducted to decipher the proteome of in vivo-produced pre-implantation ovine embryos. Ten locally adapted Morana Nova ewes received hormonal treatment and were inseminated 12 hr after ovulation. Six days later, 54 embryos (morula and blastocyst developmental state) were recovered from eight ewes and pooled to obtain sufficient protein for proteomic analysis. Extracted embryo proteins were analysed by LC-MS/MS, followed by identification based on four database searches (PEAKS, Proteome Discoverer software, SearchGUI software, PepExplorer). Identified proteins were analysed for gene ontology terms, protein clusters and interactions. Genes associated with the ovine embryo proteome were screened for miRNA targets using data sets of TargetScan (http://www.targetscan.org) and mIRBase (http://www.mirbase.org) servers. There were 667 proteins identified in the ovine embryos. Biological processes of such proteins were mainly related to cellular process and regulation, and molecular functions, to binding and catalytic activity. Analysis of the embryo proteins revealed 49 enriched functional clusters, linked to energy metabolism (TCA cycle, pyruvate and glycolysis metabolism), zona pellucida (ZP), MAPK signalling pathway, tight junction, binding of sperm to ZP, translation, proteasome, cell cycle and calcium/phospholipid binding. Sixteen miRNAs were related to 25 pre-implantation ovine embryo genes, all conserved in human, bovine and ovine species. The interaction network generated by miRNet showed four key miRNAs (hsa-mir-106b-5p; hsa-mir-30-5p; hsa-mir-103a-5p and hsa-mir-106a-5p) with potential interactions with embryo-expressed genes. Functional analysis of the network indicated that miRNAs modulate genes related to cell cycle, regulation of stem cell and embryonic cell differentiation, among others. Retrieved miRNAs also modulate the expression of genes involved in cell signalling pathways, such as MAPK, Wnt, TGF-beta, p53 and Toll-like receptor. The current study describes the first major proteomic profile of 6-day-old ovine embryos produced in vivo, setting a comprehensive foundation for our understanding of embryo physiology in the ovine species.

Assessment of binder of sperm protein 1 (BSP1) and heparin effects on in vitro capacitation and fertilization of bovine ejaculated and epididymal sperm.

The present study evaluated the effect of binder of sperm protein 1 (BSP1) and/or heparin on in vitro bovine capacitation and fertilization rates using epididymal and ejaculated bovine sperm. Frozen-thawed sperm were selected and used in the following treatments. Control group: Fert-TALP medium without heparin; heparin (HEP) group: Fert-TALP with heparin (10 UI/ml); BSP1 group: Fert-TALP medium with BSP1 (10 µg/ml for ejaculated sperm; 40 µg/ml for epididymal sperm); HEP + BSP1 group: Fert-TALP medium with heparin (5 UI/ml) and BSP1 (5 µg/ml for ejaculated sperm; 20 µg/ml for epididymal sperm) and determined in vitro capacitation rates in different interval times (0, 15, 30 and 60 min) using the chlortetracycline fluorescence (CTC) method. Also, we evaluated the development rates of oocytes fertilized with ejaculated or epididymal sperm into the same treatments. Capacitation was greater and faster when ejaculated sperm were treated for 60 min with heparin compared with other treatments. However, developmental rates were similar in all treatments. For epididymal sperm, the treatments with BSP1 presented higher capacitation and fertilization rates compared with heparin (P < 0.05). The effects of heparin + BSP1 on capacitation and developmental rates did not cause any increase in capacitation or blastocyst rates compared with other groups for ejaculated or epididymal sperm. In conclusion, this study confirmed that either BSP1 and heparin can be used as capacitator agents for bovine ejaculated sperm during IVF. However, BSP1 seems to be more efficient compared with heparin for epididymal sperm. Furthermore, BSP1 and heparin have no synergic effects on sperm capacitation.

Influence of oocyte selection, activation with a zinc chelator and inhibition of histone deacetylases on cloned porcine embryo and chemically activated oocytes development.

The aim of this study was to evaluate the effects of alternative protocols to improve oocyte selection, embryo activation and genomic reprogramming on in vitro development of porcine embryos cloned by somatic cell nuclear transfer (SCNT). In Experiment 1, in vitro-matured oocytes were selected by exposure to a hyperosmotic sucrose solution prior to micromanipulation. In Experiment 2, an alternative chemical activation protocol using a zinc chelator as an adjuvant (ionomycin + N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) + N-6-dimethylaminopurine (6-DMAP)) was compared with a standard protocol (ionomycin + 6-DMAP) for the activation of porcine oocytes or SCNT embryos. In Experiment 3, presumptive cloned zygotes were incubated after chemical activation in a histone deacetylase inhibitor (Scriptaid) for 15 h, with the evaluation of embryo yield and total cell number in day 7 blastocysts. In Experiment 1, cleavage rates tended to be higher in sucrose-treated oocytes than controls (123/199, 61.8% vs. 119/222, 53.6%, respectively); however, blastocyst rates were similar between groups. In Experiment 2, cleavage rates were higher in zygotes treated with TPEN than controls but no difference in blastocyst rates between groups occurred. For Experiment 3, the exposure to Scriptaid did not improve embryo development after cloning. Nevertheless, the total number of cells was higher in cloned zygotes treated with Scriptaid than SCNT controls. In conclusion, oocyte selection by sucrose as well as treatments with zinc chelator and an inhibitor of histone deacetylases did not significantly improve blastocyst yield in cloned and parthenotes. However, the histone deacetylases inhibitor produced a significant improvement in the blastocyst quality.

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene.

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus-oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80 ng/µl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day 7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/µl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0 ± 3.0) than non-CMI controls (23.3%, 75.0% and 75.0 ± 6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/µl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.

Impact of short nutrient stimuli with different energy source on follicle dynamics and quality of oocyte from hormonally stimulated goats.

The aim of this study was to verify the effect of the energy source for a short-term diet supplementation on follicular dynamics, ovarian response and oocyte recovery in goats. Thirty Anglo Nubian crossbred does received a diet for 4 weeks to satisfy the nutritional requirements of breeding for adult non-dairy goats. Seven days prior to oocyte recovery (OR), a group of does (n = 10) was supplemented with ground full-fat linseed in the diet (Diet A), whereas a second group of does (n = 10) received crude glycerine in the diet (Diet B). The total mixed ration (TMR) diet was maintained as the Control Diet (n = 10). All animals were oestrous-synchronized by the use of a progesterone insert for 12 days prior to OR. Follicles were stimulated by using pFSH (five 40-mg/ml doses) during the supplementation time. At OR, follicles were counted and recovered oocytes were classified as viable or degenerated. Follicular dynamics was monitored by ultrasonography, and plasma glucose, cholesterol and triglyceride levels were measured during supplementation. Glucose was higher in Diet B and cholesterol in Diet A. Diet B had a lower proportion of small (<3 mm) and large follicles (≥3 mm; p = 0.01). The follicular growth rate was higher in Diet A (p < 0.01), with follicles emerging in the 5th day of supplementation. No differences were observed for follicles counted and oocytes recovered. Thus, the type of energy source supplemented for a short term was capable to alter the follicular dynamics, without affecting the proportion of morphologically viable oocytes upon recovery.

Etiology and severity of diarrheal diseases in infants at the semiarid region of Brazil: A case-control study.

BACKGROUND: Diarrheal diseases are an important cause of morbidity and mortality among children in developing countries. We aimed to study the etiology and severity of diarrhea in children living in the low-income semiarid region of Brazil. METHODOLOGY: This is a cross-sectional, age-matched case-control study of diarrhea in children aged 2-36 months from six cities in Brazil's semiarid region. Clinical, epidemiological, and anthropometric data were matched with fecal samples collected for the identification of enteropathogens. RESULTS: We enrolled 1,200 children, 596 cases and 604 controls. By univariate analysis, eight enteropathogens were associated with diarrhea: Norovirus GII (OR 5.08, 95% CI 2.10, 12.30), Adenovirus (OR 3.79, 95% CI 1.41, 10.23), typical enteropathogenic Escherichia coli (tEPEC), (OR 3.28, 95% CI 1.39, 7.73), enterotoxigenic E. coli (ETEC LT and ST producing toxins), (OR 2.58, 95% CI 0.99, 6.69), rotavirus (OR 1.91, 95% CI 1.20, 3.02), shiga toxin-producing E. coli (STEC; OR 1.77, 95% CI 1.16, 2.69), enteroaggregative E. coli (EAEC), (OR 1.45, 95% CI 1.16, 1.83) and Giardia spp. (OR 1.39, 95% CI 1.05, 1.84). By logistic regression of all enteropathogens, the best predictors of diarrhea were norovirus, adenovirus, rotavirus, STEC, Giardia spp. and EAEC. A high diarrhea severity score was associated with EAEC. CONCLUSIONS: Six enteropathogens: Norovirus, Adenovirus, Rotavirus, STEC, Giardia spp., and EAEC were associated with diarrhea in children from Brazil's semiarid region. EAEC was associated with increased diarrhea severity.

Effects of fusion-activation interval and embryo aggregation on in vitro and in vivo development of bovine cloned embryos.

Nuclear reprogramming in somatic cell cloning is one of the key factors for proper development, with variations in the protocol appearing to improve cloning efficiency. This study aimed to determine the effects of two fusion-activation intervals and the aggregation of bovine cloned embryos on subsequent in vitro and in vivo development. Zygotes produced by handmade cloning were exposed to two fusion-activation intervals (2 h or 4 h), and then cultured in microwells either individually (1 × 100%) or after aggregation of two structures (2 × 100%). Zona-intact oocytes and zona-free oocytes and hemi-oocytes were used as parthenote controls under the same fusion-activation intervals. Day-7 cloned blastocysts were transferred to synchronous recipients. Cleavage (Day 2), blastocyst (Day 7) and pregnancy (Day 30) rates were compared by the χ2 test (P < .05). Extending fusion-activation interval from 2 to 4 h reduced cleavage (91.0 vs. 74.4%) but not blastocyst (34.8 vs. 42.0%) rates. On a microwell basis, cloned embryo aggregation (2 × 100%) increased cleavage (91.5% vs. 74.4%) and blastocyst (46.0% vs. 31.3%) rates compared to controls (1 × 100%), but did not improve the overall embryo production efficiency on Day 7 (23.0% vs. 31.3%), on a per reconstructed embryo basis, respectively. Treatments had no effects on in vitro developmental kinetics, embryo quality, and in vivo development. In summary, the fusion-activation interval and/or the aggregation of cloned bovine embryos did not affect cloning efficiency based on the in vitro development to the blastocyst stage and on pregnancy outcome.

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification.

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.

Stroma cell-derived factor 1 and connexins (37 and 43) are preserved after vitrification and in vitro culture of goat ovarian cortex.

This study aimed to evaluate the follicular morphology and development (follicular activation, cell proliferation, and hormone production), as well as the distribution pattern of Connexins 37 and 43 and SDF-1α after vitrification and in vitro culture of goat ovarian tissue. The study involved four experimental groups: fresh control, vitrified control, fresh culture and vitrified culture. The ovarian fragments were vitrified by a solid surface technique using the Ovarian Tissue Cryosystem and subsequently in vitro cultured for 7 days. The percentage of normal preantral follicles was similar between vitrified control and vitrified culture. However, both vitrified control and vitrified culture treatments showed a significant reduction of morphologically normal follicles in comparison to fresh control. A higher percentage of developing follicles (transition, primary and secondary) was observed in both fresh culture and vitrified culture treatments. Progesterone and estradiol production decreased (P < 0.05) during in vitro culture. SDF-1α and Cx37 proteins were detected in oocytes and granulosa cells from all the treatments. However, in vitrified cultured tissue, only granulosa cells were labeled with Cx37. Connexin 43 was detected in the granulosa, theca cells and zona pellucida in all the treatments. In conclusion, in vitro culture of vitrified goat ovarian cortex was able to promote follicle survival and did not alter the expression of SDF-1α and 43. However, the expression of Cx 37 was modified after in vitro culture of vitrified tissue.

Mid-pregnancy ewe shearing and the effects on fetus liver and muscle glycoprotein deposits.

The reason why shearing ewes in mid-pregnancy does increase the lamb birth weight is not completely clears. Therefore, we focused on the analyses of the deposition of glycogen in different fetal tissues to investigate this issue. Thirteen pregnant Australian Merino ewes, raised in native pasture, were separated in two groups. One group (n = 7) was shorn (SE) at 70 days of pregnancy, whereas another group (n = 6) remained unshorn (NSE). Cesarean section was conducted in all the ewes at near parturition, when placenta and fetuses sampling were collected. Placenta, liver and muscle samples were fixed and stained with glycoprotein-reactive acid-Schiff acid for analysis under light microscopy. The quantification of these glycoproteins was performed with the support of a program that analyzes the measurement of the intensity of staining by field. Five random fields from each sample were used, where statistical analyzes was used as normal test T. Among the analyzed regions, the deposition of glycoprotein between SE and NSE groups was statistically different in the hepatic portal vein (54,499.23 µm 2 in SE and 34,830.73 µm2 in NSE) and in the total muscle area of the sample fragment (41,128, 7 µm2 and 31,942.7 µm2 , respectively; P < 0.05). We conclude that shearing ewes at the 70th day of gestation lead to accumulation of glycoproteins in the liver and muscle of fetuses, which may be responsible for the increase in birth weights in that group.

Milk from transgenic goat expressing human lysozyme for recovery and treatment of gastrointestinal pathogens.

Lysozyme is an important non-specific immune protein in human milk, modulating the immune response against bacterial infections. The aim of this study was to characterize the milk of a transgenic goat expressing a recombinant human lysozyme (rhLZ) in the milk, also testing the in vitro antibacterial activity of the rhLZ milk against pathogens of the gastrointestinal tract. Milk samples collected from Tg and non-transgenic goats (nTg) from the 3rd to the 11th week of lactation were submitted to physicochemical analyses, rhLZ semi-quantification, and to rhLZ antimicrobial activity against Micrococcus luteus, Shiguella sonnei and Enterococcus faecalis. Viability and cell migration were studied in ileum epithelial cells (IEC-18) in absence or presence of E. faecalis, Staphylococcus aureus, Escherichia coli (EPEC) and S. sonnei. The expression of ZO-1 and IL-6 genes was evaluated in IEC-18 to evaluate the effect of rhLZ milk on intestinal barrier function and intestinal inflammation. Physicochemical parameters between goat Tg and nTg milk were similar and within normal values for human consumption, with hLZ concentrations being similar between Tg (224µg/mL) and human (226µg/mL) milk. The Tg milk had bactericidal activity against M. luteus, no bactericidal effect on S. sonnei, and relative to discrete sensitivity against E. feacalis than controls. Better migrating parameters were observed in cells in culture with nTg and Tg than controls. In the presence of pathogens, the Tg milk promoted improved migrating parameters than controls, except for S. sonnei, with lower cell numbers in the presence of nTg samples and E. faecalis and S. sonnei. No differences in ZO-1 relative expression patterns were observed in cultured cells, with increased expression in IL-6 in cells exposed to nTg milk than controls, with the Tg group being similar to all groups. In conclusion, goat milk containing rhLZ demonstrated valid evidence for its potential use as a nutraceutical for improvement of health and nutrition quality in humans.

Impact of cumulative gain in expertise on the efficiency of handmade cloning in cattle.

The aim of this study was to determine the effects of the cumulative gain in expertise in carrying out handmade cloning (HMC) procedures on embryo yield and pregnancy outcome in cattle. Results from in vitro and in vivo embryo development after HMC during three periods of 7 months, separated by 3-month intervals, were compiled and designated as P1, P2 and P3. Blastocyst yield, morphological quality and stage of development, and pregnancy per embryo transfer (ET) on Day 30 of gestation were compared. Zona-intact oocytes were activated chemically in each experiment replicate, and development of parthenogenetic blastocysts was used as a control measurement of oocyte quality and in vitro culture conditions. A total of 21,231 cumulus-oocyte complexes (COCs) were in vitro-matured, with 5,432, 10,721 and 5078 COCs used in 16, 18 and 10 replicates for P1, P2 and P3, respectively. Cloned blastocyst yields on Day 7 increased from 15.5% (124/798) in P1 to 21.6% (309/1428) and 36.6% (280/764) in P2 and P3, respectively. No differences were observed in blastocyst development of parthenogenetic embryos, which average 30.0, 37.6, and 36.4% in P1, P2, and P3, respectively. A 10-fold higher probability of obtaining cloned blastocysts at more advanced stages of development and of higher morphological grade was seen during P3 compared with P1. Pregnancy per ET on Day 30 also increased with gain in expertise, being 6.7% (2/30), 20.8% (10/48) and 40.0% (24/60) for P1, P2 and P3, respectively. The relative efficiency for the establishment of pregnancies (per total COC) increased from 0.04% (1:2716) in P1 to 0.22% (1:460) in P2, reaching 0.47% (1:212) in P3. Results demonstrated a gradual improvement in in vitro and in vivo embryo development over time after establishment of HMC procedures in the laboratory, highlighting the importance of gaining experience and technical skills on the overall cloning efficiency.