Claudia Barbosa Fernandes

Reproductive Stem Cell Differentiation Extracellular Matrix, Tissue Microenvironment, and Growth Factors Direct the Mesenchymal Stem Cell Lineage Commitment

The mesenchymal stem cells (MSCs) have awakened interest in regenerative medicine due to its high capability to proliferate and differentiate in multiple specialized lineages under defined conditions. The reproductive system is considered a valuable source of MSCs, which needs further investigations. Many factors have been reported as critical for these cell lineage specification and determination. In this review, we discuss the main effects of extracellular matrix or tissue environment and growth factors in the cell lineage commitment, including the reproductive stem cells. The MSCs responses to culture medium stimuli or to soluble factors probably occur through several intracellular activation pathways. However, the molecular mechanisms in which the cells respond to these mechanical or chemical perturbations remain elusive. Recent findings suggest a synergic effect of microenvironment and soluble cell culture factors affecting cell differentiation. For future applications in cell therapy, protocols of reproductive MSCs differentiation must be established.

Use of Bayesian Inference to Correlate In Vitro Embryo Production and In Vivo Fertility in Zebu Bulls

The objective of this experiment was to test in vitro embryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilized in vitro using sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimate in vivo bull fertility using data from IVP.

Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Influence of Maternal Age and Parity on Placental Structure and Foal Characteristics From Birth up to 2 Years of Age

ABSTRACT The foals weight and height at birth are regarded as important parameters for the equestrian sports industry. Moreover, scientific studies indicate that maternal environment can influence postnatal genetic potential of foals. The purpose of this study was to evaluate the influence of maternal age and parity on placental efficiency and size of the newborn foal using stereological analysis tools of the terms allantochorion. Furthermore, the influence of maternal age and parity on weight, height, and girth circumference of the offspring from birth until 2 years of age was investigated. Forty pregnant mares were categorized according to age: 4–8 years (n = 15); 9–12 years (n = 15); ≥13 years (n = 10) and parity: nulliparous (n = 12), 1–3 parities (n = 18); ≥4 parities (n = 10). Parturition was monitored and the allantochorion membranes were collected after delivery. The stereological analysis determined the volumetric composition of the different compartments of the placenta along with the area of fetomaternal contact surface. It was observed that maternal age and parity significantly increased the development of the microcotyledons and total villi surface density, as well as the vascularization and tissue composition of different regions of the placenta, which contributes to fetomaternal contact. We also found a significant relationship between maternal age and parity on the size of the neonate foals but not after the first year of age, possibly because environmental and genetic factors contribute to postnatal growth. HIGHLIGHTSMaternal age and parity influence placental characteristics and fetomaternal contact.Mares age and parity influence neonatal size at birth.After the first year, differences in height, weight, and girth circumference disappear.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

ABSTRACT.- Martins L.R., Fernandes , C.B., Villaverde A.I.S.B., Landim-Alvarenga F.C. & Lo-pes M.D.2014. The seasonal and ovarian status effects on in vitro production of do-mestic cat embryos in a region between Equator and Tropic of Capricorn. Pesquisa Veterinaria Brasileira 34(3):277-280 . Laboratorio de Reproducao Animal, Curso de Medici-na Veterinaria, Universidade Federal de Mato Grosso, Av. Alexandre Ferronato 1200, Sinop, MT 78557-267, Brazil. E-mail: lirigatto@yahoo.com.brFrom the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo produc-tion. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53’09” S and 48°26’42” W) – non breeding season (NBS), comprehending January to March; and breeding season (BS), August to Oc-tober. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were wa-shed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mine-ral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham’s F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100 mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of moru-lae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries,

Factors affecting gestational length in the Mangalarga Paulista breed

The Mangalarga Paulista is a Brazilian horse breed that originated from a cross between Andalusian and Alter Real horses, and despite its national importance, no data exist regarding their gestational length. This study was conducted to determine the variation in gestation length and the influence of factors such as the sex of the foal, maternal age and parity, stallion and mating period. Retrospectively, data from 208 gestations of Mangalarga Paulista foals located in Piracaia, SP, Brazil, during the period from 1991 to 2012 were analyzed. The mean gestational length was 343.08 ± 0.66 days, with a range between 317 and 404 days. Gestation of mares carrying colts had a tendency to be longer than mares carrying fillies. Maternal parity significantly affected gestational length, with longer gestation for primiparous mares until the third gestation, shorter pregnancies between 4th and 8th gestation, and longer thereafter. Regarding mating period, mares that were mated at the end of the breeding season (January and February) had shorter pregnancies (P < 0.05). These results are of significant clinical and economic relevance for the equine industry, so that the monitoring of gestation and parturition can be performed effectively.

Transplacental transmission of Theileria equi in mules: Should we worry?

Equine piroplasmosis, disease globally responsible for significant economic losses, is caused by the transmission of the hemoprotozoa Theileria equi and Babesia caballi by ticks. Transplacental transmission has been reported previously in horses, which is not the case for mules´ foals. The present study investigated, by Real Time-PCR (RT-PCR), the presence of both Theileria equi and Babesia caballi in newborn mules before the ingestion of colostrum. Blood samples of 18 mares and their respective mules´ foals were collected and analyzed by RT-PCR. Only one mare was positive for B. caballi. In contrast, 13 mares were positive for T. equi, with two foals (11.1%) positive for the same protozoon, both of affected mares. We may conclude that transplacental transmission of Theileria equi also occurs in mules which may be an important concern while breeding such animals.