Use of thermography in the long-term evaluation of scrotal surface temperature and its impact on seminal quality in stallions.

Scrotal surface thermography is a non-invasive method for assessing testicular thermoregulation in stallions; however, few studies have explored the application of this technique concerning the thermal physiology of equine reproductive systems. This study aimed to evaluate the consistency of testicular thermoregulation in stallions over a year using thermography to measure the scrotal surface temperature (SST). Moreover, we assessed the best region for measuring the surface body temperature compared with the SST. Ten light-breed stallions were used in the experiment. Thermographic images of the scrotal and body surfaces (neck and abdomen) were captured. Fresh, cooled and frozen-thawed semen samples were evaluated to verify the impact of thermoregulation on semen quality. Testicular thermoregulation was maintained throughout the year in stallions amidst changes in the external temperature, as evidenced by the weak correlation between the SST and ambient temperature. A lower correlation was observed between the environmental temperature and body surface temperature (BTS) obtained from the abdomen (BTS-A; R = .4772; p < .0001) than with that obtained from the neck (BTS-N; R = .7259; p < .0001). Moreover, both BTS-A and SST were simultaneously captured in a single image. The consistent quality of the fresh, cooled and frozen semen suggests efficient thermoregulation in stallions throughout the year.

Genome transfer technique for bovine embryo production using the metaphase plate and polar body.

Despite many studies in humans and mice using genome transfer (GT), there are few reports using this technique in oocytes of wild or domestic animals. Therefore, we aimed to establish a GT technique in bovine oocytes using the metaphase plate (MP) and polar body (PB) as the sources of genetic material. In the first experiment, GT was established using MP (GT-MP), and a sperm concentration of 1 × 106 or 0.5 × 106 spermatozoa/ml gave similar fertilization rates. The cleavage rate (50%) and blastocyst rate (13.6%) in the GT-MP group was lower than that of the in vitro production control group (80.2% and 32.6%, respectively). The second experiment evaluated the same parameters using PB instead of MP; the GT-PB group had lower fertilization (82.3% vs. 96.2%) and blastocyst (7.7% vs. 36.8%) rates than the control group. No differences in the amount of mitochondrial DNA (mtDNA) were observed between groups. Finally, GT-MP was performed using vitrified oocytes (GT-MPV) as a source of genetic material. The cleavage rate of the GT-MPV group (68.4%) was similar to that of the vitrified oocytes (VIT) control group (70.0%) and to that of the control IVP group (81.25%, P < 0.05). The blastocyst rate of GT-MPV (15.7) did not differ neither from the VIT control group (5.0%) nor from the IVP control group (35.7%). The results suggested that the structures reconstructed by the GT-MPV and GT-PB technique develop in embryos even if vitrified oocytes are used.

Maturation system affects lipid accumulation in bovine oocytes.

This study evaluated the effects of three maturation systems, namely invitro (MatV) and invivo (MatS) systems, as well as intrafollicular transfer of immature oocytes (IFIOT; MatT), on the accumulation of lipid droplets in bovine oocytes. Lipids were evaluated using confocal microscopy and transmission electron microscopy. The expression of genes related to lipid metabolism, namely acyl-CoA synthetase short chain family member 2 (ACSS2), ELOVL fatty acid elongase 1 (ELOVL1) and fatty acid binding protein 3 (FABP3), was quantified by quantitative polymerase chain reaction. The mean (±s.d.) area occupied by lipids in immature oocytes (13±2%) was similar to those matured invivo (MatS, 16±2%; MatT, 12±2%). However, there was a significant increase in lipids in oocytes in the MatV group (24±2%) compared with all other groups (P<0.001). In the ultrastructural evaluations, MatV oocytes also showed the highest lipid content. The expression of ELOVL1 and FABP3 was similar in the MatS and IFIOT groups. However, transcript levels of ACSS2 were lower in IFIOT than MatV oocytes. These results indicate, for the first time, that oocytes matured by IFIOT are similar to those matured invivo with regard to lipid accumulation, which indicates better quality than those matured invitro.

Effect of sex on cryotolerance of bovine embryos produced in vitro.

Male and female embryos are known to be different in developmental kinetics, metabolism, gene expression, and epigenetic patterns. Therefore, the objective of this study was to clarify whether the morphological criteria used to select embryos for cryopreservation lead to a deviation in the male:female ratio, and whether vitrification effects vary according to embryo sex. Initially, five sires were tested to evaluate the effect of the bull on embryo development, sex ratio, speed of development, and response to cryopreservation. Results showed that bulls affected (P < 0.05) embryo production, response to cryopreservation, and sex ratio. Then, one bull was selected, and used to produce embryos in vitro to characterize the responses of male and female embryos to vitrification. Results suggested that male and female embryos have the same morphological responses to vitrification, as no differences (P > 0.05) were observed between the two sexes in post-warming survival and re-expansion rates. However, their molecular responses as evaluated by gene expression (FOSL1, HSPB1, CASP3, CASP8, HSPA5, HSPA1A, G6PD, and PGK1) analysis indicated an effect of sex on vitrification; vitrified female embryos exhibited higher mRNA levels of HSPA1A, CASP3, and G6PD compared to their male counterparts. In conclusion, bulls affected embryo production, speed of development, sex ratio, and response to cryopreservation. Male and female embryos differed in their molecular responses to vitrification; and also, deviations in the male:female ratio when selecting embryos for cryopreservation were confirmed.

Phospholipid composition and resistance to vitrification of in vivo blastocyst of a Brazilian naturalized porcine breed / Composição de fosfolipídios e resistência à vitrificação de blastocistos produzidos in vivo de uma raça de suíno naturalizada brasileira

Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)

Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)

Phospholipid composition and resistance to vitrification of in vivo blastocyst of a Brazilian naturalized porcine breed / Composição de fosfolipídios e resistência à vitrificação de blastocistos produzidos in vivo de uma raça de suíno naturalizada brasileira

Piau porcine blastocysts were submitted to MALDI-TOF to identify the main phospholipids (PL). After that, in vivo blastocysts (D6) were vitrified (n=52), non-vitrified were used as control (n=42). After warming, blastocysts were in vitro cultured to assess re-expansion and hatching at 24 and 48 hours. Finally, at 48 hours, hatched blastocysts were submitted to RT-qPCR searching for BCL2A1, BAK, BAX and CASP3 genes. For MALDI-TOF, the ion intensity was expressed in arbitrary units. Blastocyst development was compared by Qui-square (P< 0.05). Among the most representative PL was the phosphatidylcholine [PC (32:0) + H]+; [PC (34:1) + H]+ and [PC (36:4) + H]+. Beyond the PL, MALDI revealed some triglycerides (TG), including PPL (50:2) + Na+, PPO (50:1) + Na+, PLO (52:3) + Na+ and POO (52:2) + Na. Re-expansion did not differ (P> 0.05) between fresh or vitrified blastocysts at 24 (33.3%; 32.7%) or 48 hours (2.4%; 13.5%). Hatching rates were higher (P< 0.05) for fresh compared to vitrified at 24 (66.7%; 15.4%) and 48 hours (97.6%; 36.0%). BAX was overexpressed (P< 0.05) after vitrification. In conclusion, Piau blastocysts can be cryopreserved by Cryotop. This study also demonstrated that the apoptotic pathway may be responsible for the low efficiency of porcine embryo cryopreservation.(AU)

Blastocistos de suínos foram submetidos ao MALDI-TOF para se identificarem os principais fosfolipídios (PL). Depois, parte destes embriões (D6) foram vitrificados (n=52), ou permaneceram frescos (grupo controle, n=42). Após o aquecimento, os blastocistos foram cultivados in vitro para se avaliar a reexpansão e a eclosão (BE) às 24 e 48 horas. Finalmente, às 48 horas, os BE foram submetidos ao RT-qPCR em busca dos genes BCL2A1, BAK, BAX e CASP3. No MALDI-TOF, a intensidade do íon foi expressa em unidades arbitrárias. O desenvolvimento embrionário foi comparado por qui-quadrado (P<0,05). Entre os PL mais representativos estavam as fosfatidilcolinas [PC (32: 0) + H] +; [PC (34: 1) + H] + e [PC (36: 4) + H] +. Além do PL, o MALDI revelou alguns triglicerídeos (TG), incluindo PPL (50: 2) + Na +, PPO (50: 1) + Na +, PLO (52: 3) + Na + e POO (52: 2) + Na. A reexpansão não diferiu (P>0,05) entre blastocistos frescos ou vitrificados às 24 (33,3%, 32,7%) e 48 horas (2,4%, 13,5%). As taxas de eclosão foram maiores (P<0,05) para o grupo fresco comparado ao vitrificado às 24 (66,7% x 15,4%) e 48 horas (97,6% x 36,0%). O BAX estava mais expresso (P<0,05) após a vitrificação. Concluindo, os blastocistos Piau podem ser criopreservados por Cryotop. Este estudo também demonstrou que a via apoptótica pode ser responsável pela baixa eficiência da criopreservação de embriões suínos.(AU)

Identification of molecular markers for oocyte competence in bovine cumulus cells.

Cumulus cells (CCs) have an important role during oocyte growth, competence acquisition, maturation, ovulation and fertilization. In an attempt to isolate potential biomarkers for bovine in vitro fertilization, we identified genes differentially expressed in bovine CCs from oocytes with different competence statuses, through microarray analysis. The model of follicle size, in which competent cumulus-oocyte complexes (COCs) were recovered from bigger follicles (≥8.0 mm in diameter) and less competent ones from smaller follicles (1-3 mm), was used. We identified 4178 genes that were differentially expressed (P < 0.05) in the two categories of CCs. The list was further enriched, through the use of a 2.5-fold change in gene expression as a cutoff value, to include 143 up-regulated and 80 down-regulated genes in CCs of competent COCs compared to incompetent COCs. These genes were screened according to their cellular roles, most of which were related to cell cycle, DNA repair, energy metabolism, metabolism of amino acids, cell signaling, meiosis, ovulation and inflammation. Three candidate genes up-regulated (FGF11, IGFBP4, SPRY1) and three down-regulated (ARHGAP22, COL18A1 and GPC4) in CCs from COCs of big follicles (≥8.1 mm) were selected for qPCR analysis. The selected genes showed the same expression patterns by qPCR and microarray analysis. These genes may be potential genetic markers that predict oocyte competence in in vitro fertilization routines.

Meiotic arrest as an alternative to increase the production of bovine embryos by somatic cell nuclear transfer.

This study aimed to evaluate the effect of meiotic arrest using phosphodiesterase type 3A (PDE 3A) inhibitors, cilostamide and C-type natriuretic peptide (NPPC), on pre-maturation (PM) of oocytes to be used in the production of cloned embryos. Nuclear maturation, in vitro embryo production (IVP), somatic cell nuclear transfer (SCNT) and parthenogenetic activation (PA), and total cells number of cloned embryos were evaluated. The results were analysed by chi-squared and Kruskal-Wallis test with a P-value 0.05) between control and PM, both for cleavage (78.2% and 76.9%) and blastocyst (35.5% and 29.3%) rates. After SCNT, cleavage rate was also similar (P > 0.05) between control and PM (66% and 51.9%) however, blastocyst rate was lower (P < 0.05) in the PM group than in the control group (7.4% and 30.2%). After 6 h of PM with 100 nM of NPPC, approximately 84.9% of the oocytes remained at GV. No difference was found between control and PM in cleavage (69.2% and 76.1%) and blastocyst rates (37,4% and 35%) after IVP. Similarly, no differences between PM and control groups were observed for cleavage (69.2% and 68.4%) and blastocyst (24.4% and 21.5%) rates. SCNT and PA embryos from control or PM oocytes had similar total cell number. It can be concluded that PM for 6 h with 100 nM NPPC is feasible for cloned embryo production without affecting embryo outcome.

Effect of insulin-transferrin-selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation on in vitro bovine embryo development.

The aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal-Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1-3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

Update and overview on assisted reproductive technologies (ARTs) in Brazil

The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.

Update and overview on assisted reproductive technologies (ARTs) in Brazil

The impressive increase in the use of assisted reproductive technologies (ARTs), especially in cattle, during the last few years in Brazil is well known worldwide. In 2015, there were over 13.7 million artificial inseminations (AI), of which, about 77% were carried out using fixed-time AI (FTAI). This technology has helped to substantially improve reproductive efficiency in beef and dairy cattle. In relation to embryo transfer, production of in vivo derived (IVD) embryos remained relatively stable, with average production of 30-40,000 embryos per year, whereas in vitro production (IVP) of embryos had a substantial increase, from about 12,500 IVP embryos in 2000 to more than 300,000 IVP embryos after 2010. The increasing availability and use of sex-sorted sperm was one of the factors responsible for a recent shift from the predominance of IVP embryos from beef breeds to dairy breeds in Brazil. Moreover, there was also an increase from 13% in 2014 to 29% in 2015 in the percentage of vitrified/frozen embryos. Moreover, the successful use of protocols for fixed-time ET (FTET) due to their high efficiency and ease of implementation, has facilitated the dissemination of ET programs all over Brazil. However, there is room for improvement, since there are several reports of high pregnancy loss and high peripartum loss, when IVP embryos are used. The production of healthy cattle by somatic cell nuclear transfer has also increased in the last few years in Brazil, but despite substantial progress in reducing postnatal losses, no drastic increase in cloning efficiency up to parturition has occurred.(AU)

Molecular markers for oocyte competence in bovine cumulus cells.

This study aimed to quantify the expression of candidate genes in cumulus cells (CCs) from cumulus-oocyte complexes (COCs) with high and low potential for in vitro development up to the blastocyst stage. First, the effects of individual culture and biopsy on embryo development were evaluated. Individuals cultured using the well of the well system were compared with individuals cultured in 20 µL droplets (microdroplets) and those cultured in groups (control). Blastocyst rates were lower for the individual culture systems (P < 0.05; well of the well = 17.9%, n = 95; microdrop = 26.3%, n = 95) than for the control group (45.0%, n = 209). Second, the effects of biopsy on embryo production were compared between the control and microdroplet cultures, and no effects (P > 0.05) were observed for either group. Finally, the expression profiles of glypican 4 (GPC4), IGF4-binding protein, follicle-stimulating hormonereceptor, growth hormone receptor, epidermal growth factor receptor, fibroblast growth factor 11, solute carrier family 2 member 1, solute carrier family 2 member 3,sprouty homolog 1, versican, and keratin protein 8 in CCs obtained by biopsy were quantified by real-time polymerase chain reaction. Cumulus cells were categorized on the basis of the fates of the COCs: expanded blastocyst, cleaved and arrested, and uncleaved. The GPC4 gene was overexpressed (P = 0.007) in CCs from oocytes that formed embryos compared with those that produced cleaved and arrested embryos. We concluded that individual culture reduced blastocyst production; however, biopsy did not affect embryo development. The profile of GPC4 expression can be used as a marker to distinguish COCs with potential for embryo development from those with limited developmental potential.

The effect of pre-maturation culture using phosphodiesterase type 3 inhibitor and insulin, transferrin and selenium on nuclear and cytoplasmic maturation of bovine oocytes.

This study aims to evaluate if a pre-maturation culture (PMC) using cilostamide as a meiotic inhibitor in combination with insulin, transferrin and selenium (ITS) for 8 or 24 h increases in vitro embryo production. To evaluate the effects of PMC on embryo development, cleavage rate, blastocyst rate, embryo size and total cell number were determined. When cilostamide (20 µM) was used in PMC for 8 or 24 h, 98% of oocytes were maintained in germinal vesicles. Although the majority of oocytes resumed meiosis after meiotic arrest, the cleavage and blastocyst rates were lower than the control (P 0.05) to the control. The deleterious effect of 20 µM cilostamide treatment for 24 h on a PMC was confirmed by lower cumulus cell viability, determined by trypan blue staining, in that group compared with the other groups. A lower concentration (10 µM) and shorter exposure time (8 h) minimized that effect but did not improve embryo production. More studies should be performed to determine the best concentration and the arresting period to increase oocyte competence and embryo development.

Vitrification of bovine oocytes at different meiotic stages using the Cryotop method: assessment of morphological, molecular and functional patterns.

This study aimed to investigate the functional, morphological and molecular patterns of bovine oocytes vitrified at different times during in vitro maturation (IVM). Four groups of oocytes were used: non-vitrified control oocytes (CG), oocytes vitrified at 0 h (V0), oocytes vitrified after 8 h of IVM (V8) and oocytes vitrified after 22 h of IVM (V22). After vitrification, the oocytes were warmed and then returned to the incubator to complete a total of 24h of IVM. To evaluate the effect of vitrification, the nuclear maturation and fertilization rates were assessed by lacmoid staining and ultrastructural electron microscopy. The cleavage and blastocyst rates were evaluated at D2, D7 and D8. The expression levels of CASP3, TP53, HDAC2, SUV39H1 and DNMT1 were investigated by RT-qPCR. The nuclear maturation, oocyte fertilization, cleavage and blastocyst rates were higher (P < 0.05) in the CG group (80%; 81.3%; 88.5%; and 35.8%) than in the V0 (44%; 44.6%; 22.7%; and 2.6%), V8 (50%; 63%; 21.5%; and 2.2%) and V22 (55.5%; 66.9%; 24.1%; and 4.6%) groups. Ultrastructural analysis revealed significant damage within the cytoplasm of all vitrified groups, but more severe degeneration was observed in the V22 group. The gene expression profiles were not affected by vitrification (P > 0.05). In conclusion, cytoplasm degeneration seems to be the most severe form of damage caused by vitrification. The use of the Cryotop method for vitrification severely reduces bovine oocyte viability regardless of whether it is performed at GV, GVBD or MII stage.

RNAs de espermatozoide: qual a sua função fisiológica? / Spermatozoal RNA: what is its physiological function?

A constatação da presença de RNAs em espermatozoides trouxe diversos questionamentos a respeito dacontribuição dessa célula para o desenvolvimento embrionário. O gameta masculino, que até então eraconsiderado apenas um transportador do genoma paterno, começou a ser estudado na busca de se descobrir seessa célula tão especializada possuiria outros atributos. Ainda existem muitas dúvidas quanto às funções dosRNAs presentes nos espermatozoides. Acredita-se que essas moléculas possam ter alguma função no início dodesenvolvimento embrionário, ou ainda que possam indicar a qualidade da espermatogênese e estar envolvidascom a fertilidade masculina, no entanto mais estudos são necessários para defini-las.

The findings of spermatozoa RNAs brought several questions regarding potential contributions of thiscell to embryo development. The male gamete, considered until recently as a simple transporter of male genome,began to be studied to discover if such a specialized cell would also have other functions. There are still manydoubts related to the functions of RNAs found on spermatozoa, it is believed that these molecules may have somerole in early embryonic development, or may indicate the quality of spermatogenesis and be related to malefertility, but further studies are necessary to better define them.

RNAs de espermatozoide: qual a sua função fisiológica? / Spermatozoal RNA: what is its physiological function?

A constatação da presença de RNAs em espermatozoides trouxe diversos questionamentos a respeito dacontribuição dessa célula para o desenvolvimento embrionário. O gameta masculino, que até então eraconsiderado apenas um transportador do genoma paterno, começou a ser estudado na busca de se descobrir seessa célula tão especializada possuiria outros atributos. Ainda existem muitas dúvidas quanto às funções dosRNAs presentes nos espermatozoides. Acredita-se que essas moléculas possam ter alguma função no início dodesenvolvimento embrionário, ou ainda que possam indicar a qualidade da espermatogênese e estar envolvidascom a fertilidade masculina, no entanto mais estudos são necessários para defini-las.(AU)

The findings of spermatozoa RNAs brought several questions regarding potential contributions of thiscell to embryo development. The male gamete, considered until recently as a simple transporter of male genome,began to be studied to discover if such a specialized cell would also have other functions. There are still manydoubts related to the functions of RNAs found on spermatozoa, it is believed that these molecules may have somerole in early embryonic development, or may indicate the quality of spermatogenesis and be related to malefertility, but further studies are necessary to better define them.(AU)

Different ways to evaluate bovine sexed sperm in vitro

Over the years, many techniques for in vitro evaluation of sperm have been developed. Those assessments allow to perform structural, functional and molecular evaluations of the sperm cell. A combination of laboratory tests used simultaneously can provide more accurate information on sperm function and quality because sperm have multiple compartments with different functions. Many of those analyses have been used to assess the effect of sexing by flow cytometry on sperm cellular and molecular levels such as DNA methylation pattern, sperm shape, sperm morphology and capacity to remain viable after thawing. Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, evaluation and identification of the possible damages caused by the sexing process are needed. It is expected that those information will help to develop procedures to improve results when sexed sperm is used. This review is focused on the recent results using structural, functional and molecular tests to evaluate sperm viability after sexing by flow cytometry. (AU)

Different ways to evaluate bovine sexed sperm in vitro

Over the years, many techniques for in vitro evaluation of sperm have been developed. Those assessments allow to perform structural, functional and molecular evaluations of the sperm cell. A combination of laboratory tests used simultaneously can provide more accurate information on sperm function and quality because sperm have multiple compartments with different functions. Many of those analyses have been used to assess the effect of sexing by flow cytometry on sperm cellular and molecular levels such as DNA methylation pattern, sperm shape, sperm morphology and capacity to remain viable after thawing. Considering that sexed sperm are submitted to a variety of adverse conditions during sorting, evaluation and identification of the possible damages caused by the sexing process are needed. It is expected that those information will help to develop procedures to improve results when sexed sperm is used. This review is focused on the recent results using structural, functional and molecular tests to evaluate sperm viability after sexing by flow cytometry.

Ovogênese e modificações epigenéticas / fications

A reprogramação epigenética se refere a modificações programadas do genoma, que ocorrem nos períodos da gametogênese e embriogênese e que regulam a atividade dos genes sem alteração da sequência primária de DNA, sendo herdáveis ao longo das divisões celulares. Entender como ocorrem esses mecanismos proporcionará maior compreensão acerca das modificações necessárias para a melhoria do sistema de cultivo in vitro, viabilizando ainda mais as biotécnicas de reprodução assistida, em especial a produção in vitro de embriões (AU)

The epigenetic reprogramming refers to programmed genome modifications that occur during gametogenesis and embryogenesis and regulate gene activity without altering the primary sequence of DNA, heritable during cell divisions. Understanding how these mechanisms occur, would give us a clearer indication of the which modifications are needed to improve the in vitro systems in, enabling further assisted reproductive biotechnologies, in particular, in vitro embryo production. (AU)