Morphometric developmental pattern of bovine handmade cloned concepti in late pregnancy.

Cloning procedures often interfere with conceptus growth and life ex utero, in a set of symptoms known as abnormal offspring syndrome (AOS). The aim of the present study was to compare the developmental pattern of in vivo-derived (IVD), IVF-derived and handmade cloning-derived (NT-HMC) Day 225 bovine concepti using established procedures. Pregnancy diagnosis was performed on Day 30 following blastocyst transfer on Day 7. Conceptus morphometry was assessed by ultrasonography on Day 51, and on Day 225 pregnant cows were killed for morphological examination of concepti. Pregnancy outcome was similar between groups, with greater pregnancy losses in the first trimester (70.6%) and smaller fetuses on Day 51 in the NT-HMC group than in the IVD (14.3%) and IVF (20.0%) groups. However, NT-HMC-derived concepti were twofold larger on Day 225 of gestation than controls. A higher frequency (63.5%) of placentomes larger than the largest in the IVD group was observed in the NT-HMC group, which may be relevant to placental function. Conceptus traits in the IVF group were similar to the IVD controls, with only slight changes in placentome types. Morphological changes in cloned concepti likely affected placental function and metabolism, disrupting the placental constraining mechanism on fetal growth in mid- to late pregnancy.

A fast and simple method for the polymerase chain reaction-based sexing of livestock embryos.

Embryo sexing is a powerful tool for livestock producers because it allows them to manage their breeding stocks more effectively. However, the cost of supplies and reagents, and the need for trained professionals to biopsy embryos by micromanipulation restrict the worldwide use of the technology to a limited number of specialized groups. The aim of this study was to couple a fast and inexpensive DNA extraction protocol with a practical biopsy approach to create a simple, quick, effective, and dependable embryo sexing procedure. From a total of 1847 sheep and cattle whole embryos or embryo biopsies, the sexing efficiency was 100% for embryo biopsies, 98% for sheep embryos, and 90.2% for cattle embryos. We used a primer pair that was common to both species and only 10% of the total extracted DNA. The whole protocol takes only 2 h to perform, which suggests that the proposed procedure can be readily applied to field conditions. Moreover, in addition to embryo sexing, the procedure can be used for further analyses, such as genotyping and molecular diagnosis in preimplantation embryos.

The transgenic animal platform for biopharmaceutical production.

The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.

Systemic immunosuppression by methylprednisolone and pregnancy rates in goats undergoing the transfer of cloned embryos.

The presence of the zona pellucida has been perceived as a requirement for the oviductal transfer of cloned embryos at early stages of development while protecting the embryo from an immune system response. We hypothesized that steroid hormone therapy could reduce a potential cellular immune response after the transfer of zona-free cloned embryos into the oviduct of recipient female goats. In Experiment 1, seven does were used to study the systemic immunosuppressant effect of the methylprednisolone administration (for 3 days) on blood cell counts. Whole blood was collected prior to treatment with methyprednisolone and then on Days 1, 2, 3, 4, 7, 14, 21 and 28 after the first dose of methylprednisolone for the analysis of haematological parameters. Methylprednisolone treatment significantly reduced circulating white blood cells and neutrophils in comparison with pre-treatment levels, demonstrating a systemic immunosuppressant effect. In Experiment 2, a group of 58 does were used as recipient females to study the effect of administration of methylprednisolone for 3 days on the establishment of pregnancies after the transfer of zona-free cloned embryos into the oviducts. No effects on pregnancy rates on Day 30 were observed regarding the distinct treatment groups (control vs. methylprednisolone), the source of oocytes (in vivo- vs in vitro-matured) or the presence or absence of the zona pellucida in embryos. In summary, methylprednisolone was effective at inducing a systemic immunosuppressed state in goats, but the treatment prior to embryo transfer did not affect pregnancy rates. Moreover, pregnancy rates were similar between zona-free and zona-intact goat cloned embryos.

Comparative expression profiles of genes related to oocyte development in goats after long-term feeding with biodiesel castor industry residues.

The aim of this study was to determine whether the consumption of detoxified castor meal (DCM) by goats over a long period of time affects mRNA levels in oocytes, and in mural granulosa and cumulus cells. A total of 41 adult does were supplemented (DCM group, n=21) or not (control group, n=20) with detoxified castor meal (DCM) for a period of 500 days. Then, 13 and 12 does were randomly selected for slaughter from the DCM and control treatments groups, respectively, for the determination of the number of visible ovarian follicles, retrieved cumulus-oocyte complexes (COCs), and viable and non-viable oocytes. The relative expression levels for distinct genes were determined by quantitative PCR in viable immature oocytes prior to in vitro maturation (IVM), in oocytes attaining or not the metaphase stage after IVM, as well as in granulosa cells obtained upon oocyte collection, and in cumulus cells obtained after IVM. The number of follicles ≥4 mm did not differ between treatments (overall mean 23.3 ± 2.0) and no significant differences were observed in the recovery of viable, non-viable, or total mean numbers of oocytes (control group: 44.7 ± 4.6, DCM group: 54.9 ± 5.9, respectively) between control and DCM fed goats. The maturation rate was significantly higher for control than DCM oocytes (58.0% vs. 45.3%; P<0.05). The mRNA levels in immature COC for controls were significantly higher for GLUT1 and lower for HSP70 (P<0.05) than for DCM. Following maturation, MII oocytes from both treatments had mRNA levels that were significantly higher for GDF9 and lower for BMP15 than for NC oocytes (P<0.05). In cumulus cells, the mRNA levels were significantly higher for LHR, FSHR, LeptinR, and IGF1, and lower for MnSOD in the control group compared with the DCM group (P<0.05). In conclusion, the inclusion of DCM in goat feed for long periods of time changed gene expression in immature oocytes and in cumulus cells. This was reflected by a decrease in the in vitro oocyte maturation rate.

Goat oocyte quality and competence to undergo IVM and embryo development after parthenogenetic activation from goats fed with different levels of cashew nut bran as source of dietary lipids.

Lipid-rich and energy-dense diets can have significant effects on the reproductive physiology, including the ovarian function and fertility. The aim of this study was to assess the effect of cashew nut bran supplementation as a lipid source on follicle development, plasma and intrafollicular concentrations of cholesterol, and developmental competence of in vitro-matured goat oocytes. The inclusion of cashew nut bran as 24% of the goats' diet for 28 days increased the percentage and number of degenerated oocytes compared with the control (P < 0.05), and also the plasma cholesterol levels and the proportion of grade IV oocytes compared with all other treatments (P < 0.05). Moreover, a significant reduction was observed in the proportion of viable oocytes compared with the control and in the percentage of grade II oocytes compared with all other treatments (P < 0.05). Oocyte maturation, cleavage, and blastocyst rates after parthenogenetic activation of viable oocytes were not affected by the type of diet. In conclusion, the inclusion of cashew nut bran as 24% of the diet of adult goats for 28 days changed plasma cholesterol levels and reduced the proportion of viable immature oocytes; however, the 12% and 24% diet supplementations with cashew nut bran did not interfere with competence of resulting viable oocytes to reach the metaphase II stage after IVM, and to develop after parthenogenetic activation.

Effect of supplementing fat to pregnant nonlactating cows on colostral fatty acid profile and passive immunity of the newborn calf.

The objectives were to evaluate the effect of supplementing saturated or unsaturated long-chain fatty acids (FA) to nulliparous and parous Holstein animals (n=78) during late gestation on FA profile of colostrum and plasma of newborn calves and on production and absorption of IgG. The saturated FA supplement (SAT) was enriched in C18:0 and the unsaturated FA supplement (ESS) was enriched in the essential FA C18:2n-6. Fatty acids were supplemented at 1.7% of dietary dry matter to low-FA diets (1.9% of dietary dry matter) during the last 8 wk of gestation. Calves were fed 4 L of colostrum within 2h of birth from their own dam or from a dam fed the same treatment. Feeding fat did not affect prepartum dry matter intake, body weight change, or gestation length. Parous but not nulliparous dams tended to give birth to heavier calves if fed fat prepartum. Parous dams were less able to synthesize essential FA derivatives, as evidenced by lower desaturase indices, compared with nulliparous dams, suggesting a greater need for essential FA supplementation. The FA profile of colostrum was modified to a greater degree by prepartum fat feeding than was that of neonatal calf plasma. The placental transfer and synthesis of elongated n-3 FA (C20:5, C22:5, and C22:6) were reduced, whereas the n-6 FA (C18:2, C18:3, and C20:3) were increased in plasma of calves born from dams fed ESS rather than SAT. Supplementing unsaturated FA prepartum resulted in elevated concentrations of trans isomers of unsaturated monoene and diene FA, as well as C18:2n-6 in colostrum. Serum concentrations of IgG tended to be increased in calves born from dams fed fat compared with those not fed fat, and prepartum feeding of SAT tended to improve circulating concentrations of IgG in newborn calves above the feeding of ESS. Apparent efficiency of absorption of IgG was improved in calves born from dams fed fat, and SAT supplementation appeared more effective than supplementation with ESS. Feeding SAT prepartum may be of greater benefit based upon greater circulating IgG concentrations of calves after colostrum feeding. Feeding moderate amounts of saturated or unsaturated long-chain FA during the last 8 wk of gestation changed the FA profile of colostrum and plasma of neonates to reflect that of the supplements.

Fertility in dairy cows following presynchronization and administering twice the luteolytic dose of prostaglandin F2α as one or two injections in the 5-day timed artificial insemination protocol.

The objectives were to evaluate pregnancy per AI (P/AI) of dairy cows subjected to the 5-day timed AI protocol under various synchronization and luteolytic treatments. Cows were either presynchronized or received supplemental progesterone during the synchronization protocol, and received a double luteolytic dose of PGF2α, either as one or two injections. In Experiment 1, dairy cows (n=737; Holstein=250, Jersey=80, and crossbred=407) in two seasonal grazing dairy farms were randomly assigned to one of four treatments in a 2×2 factorial arrangement. The day of AI was considered study Day 0. Half of the cows were presynchronized (G6G: PGF2α on Day -16 and GnRH on Day -14) and received the 5-day timed AI protocol using 1 mg of cloprostenol, either as a single injection (G6G-S: GnRH on Day -8, PGF2α on Day -3, and GnRH+AI on Day 0) or divided into two injections of 0.5 mg each (G6G-T: GnRH on Day -8, PGF2α on Day -3 and -2, and GnRH+AI on Day 0). The remaining cows were not presynchronized and received a controlled internal drug-release (CIDR) insert containing progesterone from GnRH to the first PGF2α injection of the 5-day timed AI protocol, and 1 mg of cloprostenol either as a single injection on Day -3 (CIDR-S) or divided into two injections of 0.5 mg each on Days -3 and -2 (CIDR-T). Ovaries were examined by ultrasonography on Days -8 and -3 and plasma progesterone concentrations were determined on Days -3 and 0. In Experiment 2, 655 high-producing Holstein cows had their estrous cycle presynchronized with PGF2α at 46±3 and 60±3 days postpartum and were randomly assigned to receive 50 mg of dinoprost during the 5-day timed AI protocol, either as a single injection or divided into two injections of 25 mg each. Pregnancies per AI were determined on Days 35 and 64 after AI in both experiments. In Experiment 1, presynchronization with G6G increased the proportion of cows with a CL on Day -8 (80.6 vs. 58.8%), ovulation to the first GnRH of the protocol (64.2 vs. 50.2%), and the presence (95.6 vs. 88.4%) and number (1.79 vs. 1.30) of CL at PGF(2α) compared with CIDR cows. Luteolysis was greater for two injections compared to a single PGF2α injection (two PGF2α=95.9 vs. single PGF2α=72.2%), especially in presynchronized cows (G6G-T=96.2 vs. G6G-S=61.7%). For cows not presynchronized, two PGF2α injections had no effect on P/AI (CIDR-S=30.2 vs. CIDR-T=34.3%), whereas for presynchronized cows, it improved P/AI (G6G-S=28.7 vs. G6G-T=45.4%). In Experiment 2, the two-PGF2α injection increased P/AI on Days 35 (two PGF2α=44.5 vs. single PGF2α=36.4%) and 64 (two PGF2α=40.3% vs. single PGF2α=32.6%) after AI. Presynchronization and dividing the dose of PGF2α (either cloprostenol or dinoprost) into two injections increased P/AI in lactating dairy cows subjected to the 5-day timed AI protocol.

Internal artificial vagina (IAV) to assess breeding behavior of young Bos taurus and Bos indicus bulls.

Bull breeding soundness evaluation (BBSE) usually neglects the libido and mating ability evaluation. The internal artificial vagina (IAV) permits semen sampling, as well as mating ability evaluation. Few studies have been performed using IAV with young bulls and there are none with Bos indicus bulls. The present study evaluated sexual behavior, mating ability and semen quality in young Bos taurus (Devon) and B. indicus (Nellore) bulls using the IAV device. In the first experiment, 52 Devon bulls, 18-25 months old were observed, and the behavior and mating ability recorded over a 10-min period within a restrained mount-cow with an IAV inserted. In the second experiment, 20 Nellore bulls, 20-30 months old were evaluated over a 20 min period. Of the 52 Devon bulls, 45 (86.5%) had semen recovered with the IAV, 31 (69.0%) were considered satisfactory. Nellore bulls exhibited a different sexual behavior, with 10 bulls not showing any interest in the females. Four bulls demonstrated sexual interest only once, e.g., sniffing, two showed interest on more than one occasion, and four had more than two mounts or mounting attempts. None out of the Nellore bulls was collected with IAV. The IAV was an effective and welfare-promoting animal technology for the evaluation of semen quality and mating ability of B. taurus bulls. However, the IAV was not adequate for young Nellore bulls, probably due to their quiescent sexual behavior and delayed sexual maturity. Further studies are needed to evaluate the performance of the IAV for older Nellore bulls.

Effect of interval between induction of ovulation and artificial insemination (AI) and supplemental progesterone for resynchronization on fertility of dairy cows subjected to a 5-d timed AI program.

Objectives were to investigate 2 intervals from induction of ovulation to artificial insemination (AI) and the effect of supplemental progesterone for resynchronization on fertility of lactating dairy cows subjected to a 5-d timed AI program. In experiment 1, 1,227 Holstein cows had their estrous cycles presynchronized with 2 injections of PGF(2α) at 46 and 60 d in milk (DIM). The timed AI protocols were initiated with GnRH at 72 DIM, followed by 2 injections of PGF(2α) at 77 and 78 DIM and a second injection of GnRH at either 56 (OVS56) or 72h (COS72) after the first PGF(2α) of the timed AI protocols. All cows were time-inseminated at 72h after the first PGF(2α) injection. Pregnancy was diagnosed on d 32 and 60 after AI. In experiment 2, 675 nonpregnant Holstein cows had their estrous cycles resynchronized starting at 34 d after the first AI. Cows received the OVS56 with (RCIDR) or without (RCON) supplemental progesterone, as an intravaginal insert, from the first GnRH to the first PGF(2α). Pregnancy diagnoses were performed on d 32 and 60 after AI. During experiment 2, subsets of cows had their ovaries scanned by ultrasonography at the first GnRH, the first PGF(2α), and second GnRH injections of the protocol. Blood was sampled on the day of AI and 7 d later, and concentrations of progesterone were determined in plasma. Cows were considered to have a synchronized ovulation if they had progesterone <1 and >2.26 ng/mL on the day of AI and 7 d later, respectively, and if no ovulation was detected between the first PGF(2α) and second GnRH injections during resynchronization. In experiment 1, the proportion of cows detected in estrus at AI was greater for COS72 than OVS56 (40.6 vs. 32.4%). Pregnancy per AI (P/AI) did not differ between OVS56 (46.4%) and COS72 (45.5%). In experiment 2, cows supplemented with progesterone had greater P/AI compared with unsupplemented cows (51.3 vs. 43.1%). Premature ovulation tended to be greater for RCON than RCIDR cows (7.5 vs. 3.6%), although synchronization of the estrous cycle after timed AI was similar between treatments. Timing of induction of ovulation with GnRH relative to insemination did not affect P/AI of dairy cows enrolled in a 5-d timed AI program. Furthermore, during resynchronization starting on d 34 after the first AI, supplementation with progesterone improved P/AI in cows subjected to the 5-d timed AI protocol.

Acute hemiplegia associated with cat-scratch disease.

Cat scratch disease (CSD) is an infectious illness caused by a Gram-negative rod named Bartonella henselae. Typical CSD is characterized by a small skin lesion at the site of a scratch or a bite, followed by regional lymphadenopathy, one to two weeks later. Atypical forms may present as ocular manifestations, neurological manifestations, hepatosplenic involvement and vertebral osteomyelitis. Among neurological complications, encephalopathy is by far the most common. Other neurological manifestations are very rare. We report a case of an 11-year-old boy, with a posterior cervical lymphadenopathy and fever. Cat scratch disease was diagnosed and treated after a positive "Whartin-Starry" stain on lymph node biopsy. Two weeks after treatment, the patient was readmitted presenting an acute episode of left hemiplegia. A brain MRI demonstrated a right subcortical fronto-parietal lesion with no contrast enhancement. Complete recovery was observed after corticosteroid treatment.

Acute hemiplegia associated with cat-scratch disease

Cat scratch disease (CSD) is an infectious illness caused by a Gram-negative rod named Bartonella henselae. Typical CSD is characterized by a small skin lesion at the site of a scratch or a bite, followed by regional lymphadenopathy, one to two weeks later. Atypical forms may present as ocular manifestations, neurological manifestations, hepatosplenic involvement and vertebral osteomyelitis. Among neurological complications, encephalopathy is by far the most common. Other neurological manifestations are very rare. We report a case of an 11-year-old boy, with a posterior cervical lymphadenopathy and fever. Cat scratch disease was diagnosed and treated after a positive Whartin-Starry stain on lymph node biopsy. Two weeks after treatment, the patient was readmitted presenting an acute episode of left hemiplegia. A brain MRI demonstrated a right subcortical fronto-parietal lesion with no contrast enhancement. Complete recovery was observed after corticosteroid treatment.

Developmental milestones of vertically HIV infected and seroreverters children: follow up of 83 children.

The aim of the study was to detect neurological abnormalities in human immunodeficiency virus (HIV) infected children. This was achieved by a prospective evaluation, from November/1995 to April/2000, of 43 HIV infected children (group I) and 40 HIV seroreverters children (group II) through neurological exam and neurodevelopmental tests: Denver Developmental Screening Test (DDST) and Clinical Adaptive Test/Clinical Linguistic and Auditory Milestone Scale (CAT/CLAMS). A control group (III), of 67 children, were evaluated by CAT/CLAMS. Hyperactivity, irritability and hypotonia were the findings on neurological examination, without statistical differences between group I and II. On CAT/CLAMS, the group I developmental quotient (DQ) was significantly lower than the other groups. The same occurred in DDST, with group I presenting significantly more failures than group II. Nineteen HIV children of group I had brain computed tomographic scan, with abnormalities in three of them (basal ganglia calcification, white matter hypodensity and asymmetry of lateral ventricles). We conclude that in HIV infected children a neurodevelopment delay occur early in the disease, and it can be detected by screening tests.

Multiple brain abscesses caused by Salmonella typhi: case report.

BACKGROUND: Focal intracranial infections caused by Salmonella species are uncommon. The authors report a case of multiple brain abscesses caused by Salmonella typhi. CASE DESCRIPTION: A 2-month-old girl was admitted to the hospital because of diarrhea, vomiting, fever, and poor feeding. Neurological examination revealed cervical hyperextension and absence of sucking and Moro reflexes. During the next 20 hours she developed complex partial seizures with secondary generalization and alternated irritability with drowsiness. Investigation showed hemoglobin 6.3 g/dl; white blood cell count of 19500/mm3 with a marked shift to the left. The analysis of the cerebrospinal fluid revealed white cell count of 1695/mm3, lymphocytes 61%, protein 300 mg/dl and glucose 6 mg/dl. The patient was treated for acute gastroenterocolitis, sepsis, and meningitis. Blood culture taken on the day of admission showed gram-negative bacilli, later identified as S. typhi. Computed tomography scan demonstrated a lesion in the right parietal lobe compatible with a brain abscess. Follow-up computed tomography after 7 days showed several other lesions with the same features. Surgical drainage of the right parietal lesion was performed on the 13th day, through a burr hole. The patient was discharged 5 weeks after admission without neurological deficit. CONCLUSION: Bacteremia, sepsis, and meningitis are relatively common in children with Salmonella infection but intracranial abscesses are very rare. Surgical drainage combined with prolonged antibiotic therapy (drug of choice: chloramphenicol) is the best treatment for Salmonella brain abscesses. The possibility of intracranial infection should be considered in patients with Salmonellosis and neurological dysfunction.

[Acute hepatic failure associated with valproic acid in children. Report of 3 cases]. / Insuficiência aguda hepática associada ao ácido valpróico na infância. Relato de três casos.

We report the cases of three epileptic children who developed hepatotoxicity induced by valproic acid. Two patients had developmental delay. Including the one who died, all patients were receiving polytherapy (carbamazepine in two and phenobarbital in one). The patients age ranged from 2 years and 8 months to 5 years and 1 month. The onset of hepatic complications occurred within 6 months of valproate therapy in two patients and 12 months in one. All patients developed the classical clinical signs of hepatotoxicity. Vomiting, edema and jaundice were the initial symptoms. Fever occurred in two patients. The serum levels of glutamic oxaloacetic transaminase were mildly elevated with a maximum of 194 IU. The bilirubin levels ranged from 5.5 to 19.8 mg%. Two patients recovered clinically and showed normalization of the laboratory abnormalities and one had fatal course. The hepatotoxicity must be considered as a side effect of valproic acid mainly in children under two years age, with polytherapy regimen and neurologic damage. The hepatic insufficiency can be reversible.