RESUMEN

Superovulation is a technique used to increase the number of oocytes released for fertilization. This study investigated the effects of short-term differences in concentrate feed intake on in vivo embryo production through superovulation in indigenous Korean (Hanwoo) cows. The cows were given fresh water and hay ad libitum and randomly divided into three groups (control (CON, n = 9): 2.0 kg/day (unchanged diet); low concentrate (LC, n = 10): 0 kg/day; and high concentrate (HC, n = 8): 4.0 kg/day) according to the amount of formula they were fed. This feeding treatment began seven days before the start of the hormonal treatment for superovulation. From the results, the LC group had the greatest weight change and the lowest body condition score at harvest, followed by the CON and HC groups (p < 0.05). The LC group had the highest number of harvesting embryos, followed by the HC and CON groups (p < 0.05). Estradiol, progesterone, glucose, total cholesterol, high-density lipoprotein, low-density lipoprotein, total protein, and blood urea nitrogen concentrations did not differ between the groups, except for a temporary increase in the HC group on day 0. These findings suggest that more embryos may be harvested when short-term changes in concentrate intake are made during superovulatory responses in Hanwoo cows.

RESUMEN

We evaluated the growth performance, serum testosterone, carcass traits, histological characteristics, and economic efficacy of castrated and hemi-castrated Korean beef cattle. Thirty-two Hanwoo calves (Initial body weight: 148.4 ± 19.8 kg) were randomly assigned into the castrated Hanwoo (CH) and hemi-castrated Hanwoo (HH) group. The experiment lasted 18 months; the animals were all slaughtered on the same day. Final body weight and average daily gain (ADG) tended to increase in the HH group compared to the CH group. Testosterone concentration was higher in HH group (5.27-14.27 ng/dL) than in the CH group (0.47-0.70 ng/dL) during the whole experimental period after castration (p < 0.05). Rib eye area was 17.08 cm2 wider in HH group than in CH group, but marbling score was improved by 3.33 in CH group compared to HH group (p < 0.01). Deposition area of adipocytes in Longissimus dorsi were higher in CH group than in HH group (p < 0.001). Net income per head was 1760 US dollar higher in the CH group than in the HH group (p < 0.04). Thus, our findings suggest that hemi-castration had positive effects on the increase in ADG and meat yield traits, with negative effects on marbling and profitability.

RESUMEN

Hematological reference intervals must consider several parameters, including genotype, age, sex, management, and analytic process. Work is needed to evaluate hematological changes specifically in Hanwoo calves and according to calf sex. Therefore, in this study, we sought to confirm the complete blood cell count (CBC) reference intervals in Hanwoo calves, to monitor changes in hematologic values in Hanwoo calves from birth until 28 weeks of life, and to compare the hematologic values of male and female calves. A total of 35 male calves and 35 female calves was studied. Calf blood was sampled at multiple intervals from the time of birth until 28 weeks of age (including within 6 h of birth and at 2 days, 7 days, and 4 weeks and then at 4-week intervals through 28 weeks). In addition, blood samples were collected from 210 clinically healthy pregnant Hanwoo cows to establish CBC reference intervals for adult cattle. There were significant differences in the results of the cows and calves in all 14 parameters considered. The CBC reference intervals of the calves were wider than those of the cows in all parameters except mean corpuscular volume, mean corpuscular hemoglobin concentration, and mean platelet volume. We also identified differences from birth through 28 weeks between male and female calves at only some ages and some parameters. These results suggest that CBC reference intervals specific to Hanwoo calves are necessary for accurate diagnosis of calf diseases.

RESUMEN

Here, we investigated the effects of different nutrient requirements (NR) on blood metabolites, transferable embryo number after multiple superovulations with artificial insemination (AI), body condition score (BCS), and estrus cycle in Hanwoo cow. Nineteen Hanwoo cows were randomly divided into three groups (80%, 100%, and 120% NR, containing 6, 8, and 5 individuals, respectively) and fed based on the NR. In experiment 1, glucose, total cholesterol, triglyceride, blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), non-esterified fatty acids (NEFA), albumin (ALB), and total protein (TP) were analyzed. In experiment 2, total number of recovered embryos and transferable embryos was examined after embryo recovery and multiple superovulations with AI. In experiment 3, body weight, BCS, and estrus cycle were examined. In experiment 1, total cholesterol was significantly different among the 80%, 100%, and 120% NR groups (126.5 ± 3.3, 152.6 ± 2.4, and 177.4 ± 1.8 mg/dL, respectively, p < 0.05). The triglyceride and BUN levels in the 120% NR group were significantly higher than those in the 80% and 100% groups (p < 0.05). The NEFA levels were significantly different among the 80%, 100%, and 120% NR groups (440.5 ± 18.2, 318.5 ± 23.1, and 195.1 ± 8.5 ЧEq/L, respectively, p < 0.05). The AST and TP levels in the 80% NR group were significantly lower than those in the 100% and 120% NR groups (p < 0.05). In experiment 2, the 120% NR group showed a higher percentage of transferable embryos than the 80% and 100% groups (p < 0.01). The mean body weight and BCS among the 80%, 100%, and 120% NR groups were significantly different (p < 0.05). The estrus cycle in the 80% NR group was delayed compared with the 100% and 120% NR groups (20.8 ± 0.2 and 21.2 ± 0.5 days, respectively). In conclusion, the blood metabolic tests proved that Hanwoo cows with 120% NR can produce a large number of transferable embryos. Thus, 120% NR is the appropriate feeding level for this type of cows as it results in the production of a large number of transferable embryos by multiple superovulations with AI.

RESUMEN

Until recently, genome-scale phasing was limited due to the short read sizes of sequence data. Though the use of long-read sequencing can overcome this limitation, they require extensive error correction. The emergence of technologies such as 10X genomics linked read sequencing and Hi-C which uses short-read sequencers along with library preparation protocols that facilitates long-read assemblies have greatly reduced the complexities of genome scale phasing. Moreover, it is possible to accurately assemble phased genome of individual samples using these methods. Therefore, in this study, we compared three phasing strategies which included two sample preparation methods along with the Long Ranger pipeline of 10X genomics and HapCut2 software, namely 10X-LG, 10X-HapCut2, and HiC-HapCut2 and assessed their performance and accuracy. We found that the 10X-LG had the best phasing performance amongst the method analyzed. They had the highest phasing rate (89.6%), longest adjusted N50 (1.24 Mb), and lowest switch error rate (0.07%). Moreover, the phasing accuracy and yield of the 10X-LG stayed over 90% for distances up to 4 Mb and 550 Kb respectively, which were considerably higher than 10X-HapCut2 and Hi-C Hapcut2. The results of this study will serve as a good reference for future benchmarking studies and also for reference-based imputation in Hanwoo.

Asunto(s)

Bovinos/genética , Genómica/métodos , Secuenciación Completa del Genoma/métodos , Animales , Genómica/normas , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Programas Informáticos/normas , Secuenciación Completa del Genoma/normas

RESUMEN

[This corrects the article DOI: 10.5187/jast.2020.62.4.449.].

RESUMEN

This study aimed to investigate the effects of temperature and retention time of the pressurized steam chamber on the ruminal fermentation characteristics and nutrient degradability of corn flakes in three Korean native Hanwoo cows and three Holstein cows implanted with a ruminal fistula. Corn kernels were categorized into 13 groups based on the chamber temperature (range, 100°C-116°C) and retention time (range, 700-950 s). The pH value was lowest in T1 regardless of breed. Propionate concentration was the highest in T2 (p < 0.05). Total-volatile fatty acid (VFA) concentration was slightly but not significantly greater in T2 than in other conditions. Dry matter (p < 0.05), starch, and crude protein (p < 0.05) degradability were the highest in T1. At different incubation times and with different breeds, dry matter, starch, and crude protein degradability of corn flakes were the highest in T1. Thus, the present results indicate that the optimal temperature and retention time of the pressurized steam chamber should be 100°C-105°C and 700-720 s.

RESUMEN

To make an optimal treatment decision for early stage breast cancer, it is important to identify risk of recurrence. Here, we developed and validated a new prognostic model for predicting the risk of distant metastasis in patients with pN0-N1, hormone receptor-positive, HER2-negative (HR+/HER2-) breast cancer treated with hormone therapy alone. RNA was extracted from formalin-fixed, paraffin-embedded tumor tissues and gene expression was measured by quantitative real-time reverse transcription-PCR. The relative expression of six novel prognostic genes was combined with two clinical variables (nodal status and tumor size) to calculate a risk score (BCT score). In the validation cohort treated with hormone therapy alone, the 10 year rate of distant metastasis in the high-risk group (26.3%) according to BCT score was significantly higher than that in the low-risk group (3.8%) (P < 0.001). Multivariate analysis adjusted for clinical variables revealed that BCT score is an independent predictor of distant metastasis. Moreover, the C-index estimate revealed that BCT score has a prognostic power superior to that of prognostic models based on clinicopathological parameters. The BCT score outperforms prognostic models based on traditional clinicopathological factors and predicts the risk of distant metastasis in patients with HR+/HER2- early breast cancer.

Asunto(s)

Neoplasias de la Mama/patología , Neoplasias de la Mama/secundario , Técnicas de Apoyo para la Decisión , Metástasis de la Neoplasia/diagnóstico por imagen , Patología Molecular/métodos , Receptor ErbB-2/análisis , Factores de Transcripción/análisis , Femenino , Perfilación de la Expresión Génica , Humanos , Persona de Mediana Edad , Pronóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

BACKGROUND: Meningocele and meningoencephalocele of the skull are congenital deformities. Various species, such as pigs, dogs, and cats, are susceptible to congenital meningocele and meningoencephalocele and the incidence is higher in large white and landrace pigs. CASE PRESENTATION: In this study, swelling was observed in the fontanel areas of the median planes of the skull cap in two female piglets of the same litter. Gross clinical examination, neurological examination, computed tomography (CT), and magnetic resonance imaging (MRI) were conducted on the symptomatic piglets. The gross clinical and neurological examinations revealed no specific findings, except for the swellings. According to the CT results, the length of the defect on the sagittal section of the skull was 4.7 mm in case 1 and 20.62 mm in case 2. Connected flow between the skull swellings and the cerebrospinal fluid (CSF) of the lateral ventricles was observed, and partial herniation was identified in case 2. On MRI, CSF with high T2 signals was identified in the arachnoid spaces between the cerebrum and the cerebellum in the two cases, which is consistent with intracranial hypertension. The size of the swelling formed in the parietal bones was 1.6 × 1.1 × 1.8 cm(3) (case 1) and 1.2 × 1.38 × 1.7 cm(3) (case 2). The increase in intracranial pressure was more obvious in case 2 than in case 1, and was accompanied by posterior displacements of the mesencephalon and cerebellum. CONCLUSIONS: Case 1 was diagnosed as meningocele resulting from meningeal herniation and case 2 was diagnosed as meningoencephalocele caused by brain tissue herniation.

Asunto(s)

Encefalocele/veterinaria , Meningocele/patología , Enfermedades de los Porcinos/congénito , Animales , Encefalocele/patología , Femenino , República de Corea , Porcinos , Enfermedades de los Porcinos/patología

RESUMEN

Various methods have been used to remove reactive oxygen species (ROS) generated from in vitro culture (IVC) conditions that can cause cell injury or death, including the application of low oxygen (O(2)) tension and the addition of antioxidants. The beneficial effects of antioxidants and O(2) tension on IVC of porcine embryos, however, are controversial among researchers. In this study, we sought to determine the effects and optimal concentrations of antioxidants for the development of porcine embryos in an IVC system. Specifically, we examined the synergistic effects of antioxidants on development to the blastocyst stage in a culture system supplemented with L-cysteine during IVM. Of the antioxidants tested (melatonin, glutathione (GSH), ß-mercaptoethanol (ß-ME), N-acetylcysteine (NAC) and dithiothreitol (DTT)), addition of GSH (1 mM) or ß-ME (25 µM) significantly increased development to the blastocyst stage compared with the controls without antioxidant treatment (22.2 ± 4.2% for 1 mM GSH, 25.9 ± 2.2% for 25 µM ß-ME and 12-13% for the control, P<0.05). In addition, the mean cell number per blastocyst was increased by approximately 1.7-fold in the presence of GSH or ß -ME. These GSH- and ß-ME-induced increases in development to the blastocyst stage and total cell number, however, were not mimicked by melatonin, NAC or DTT, all of which are ROS scavengers. The combination of GSH or ß-ME with L-cysteine significantly reduced high O(2) tension-induced ROS production (P<0.05). These results suggest that a combination of 1 mM GSH or 25 µM ß-ME with 1 mM L-cysteine could be used for production of high quality porcine blastocysts in IVC systems.

Asunto(s)

Antioxidantes/farmacología , Blastocisto/efectos de los fármacos , Cisteína/metabolismo , Ectogénesis/efectos de los fármacos , Técnicas de Cultivo de Embriones/veterinaria , Oocitos/efectos de los fármacos , Sus scrofa/embriología , Crianza de Animales Domésticos , Animales , Blastocisto/citología , Blastocisto/metabolismo , Recuento de Células , Sinergismo Farmacológico , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/veterinaria , Glutatión/farmacología , Masculino , Mercaptoetanol/farmacología , Oocitos/citología , Oocitos/metabolismo , Concentración Osmolar , Estrés Oxidativo/efectos de los fármacos , Oxígeno/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sus scrofa/metabolismo

RESUMEN

This study evaluated the pregnancy rates following either a controlled internal drug release (CIDR)-based timed artificial insemination (TAI) or an embryo transfer (TET) protocol compared with that following a single PGF(2alpha) injection and AI after estrus (AIE) in lactating repeat breeder dairy cows. Fifty-three lactating dairy cows diagnosed as repeat breeders were used in this study and were randomly assigned to the following three treatments. (1) Cows, at random stages of the estrous cycle, received a CIDR device and 2 mg estradiol benzoate (EB; Day 0), a 25 mg PGF(2) (alpha) injection at the time of CIDR removal on Day 7 and a 1 mg EB injection on Day 8. The cows then received TAI 30 h (Day 9) after the second EB injection using dairy semen (TAI group, n=13). (2) Cows, at random stages of the estrous cycle, received the same hormonal treatments as in the TAI group. The cows then received TET on Day 16 using frozen-thawed blastocysts or morula embryos collected from Korean native cattle donors (TET group, n=13). (3) Cows, at the luteal phase, received a 25 mg injection of PGF(2alpha) and AIE using dairy semen (control group, n=27). The ovaries of the cows in the TET group were examined by transrectal ultrasonography to determine ovulation of the preovulatory follicles, and blood samples were collected for serum progesterone (P4) analysis. The pregnancy rate was significantly higher in the TET group (53.8%) than in the control (18.5%) or TAI (7.7%) groups (P<0.05). The ultrasonographic observations demonstrated that all the cows in the TET group ovulated the preovulatory follicles and concomitantly formed new corpora lutea. Accordingly, the mean serum P4 concentration remained constant between Day 0 and Day 7 of the luteal phase, decreased dramatically on Day 8 (P<0.01) and subsequently increased by Day 16 (P<0.01). These data suggest that the CIDR-based TET protocol can be used to effectively increase the pregnancy rate in lactating repeat breeder dairy cows.

Asunto(s)

Cruzamiento , Bovinos , Transferencia de Embrión/métodos , Transferencia de Embrión/veterinaria , Lactancia , Animales , Industria Lechera , Implantes de Medicamentos , Sincronización del Estro , Femenino , Inseminación Artificial/métodos , Inseminación Artificial/veterinaria , Embarazo , Índice de Embarazo , Progesterona/sangre

RESUMEN

The objective of this study was to investigate the effect of dosage and number of days of follicle stimulating hormone (FSH) treatment on superovulatory response in controlled internal drug release (CIDR)-treated Korean native cows. Forty cows underwent two superovulatory treatments with a crossover design. Cows, at random stages of the estrous cycle, received a CIDR together with injections of 1 mg estradiol benzoate and 50 mg progesterone, and gonadotropin treatment began 4 days later. The cows were divided into 2 groups based on the dosage and number of days of treatment with porcine FSH; a total of 28 mg FSH was given in twice daily intramascular injections in decreasing doses over 4 days (5, 5, 4, 4, 3, 3, 2 and 2 mg; T1 group, n=20) or a total of 24 mg FSH was given in twice daily decreasing doses over 3 days (5, 5, 4, 4, 3 and 3 mg; T2 group, n=20). This was followed by the alternate treatment in the subsequent superovulation. The cows were treated identically in all other respects. PGF(2alpha) (25 mg and 15 mg) was given with the 5th and 6th injections of FSH, CIDR were withdrawn at the 6th FSH injection and the cows received 200 microg GnRH 36 h after CIDR withdrawal. The cows were artificially inseminated twice, at 48 and 60 h after CIDR withdrawal, using commercial semen from four Korean native bulls, and embryos were recovered 6 or 7 days after the 2nd insemination. The numbers of corpora lutea (CL; 7.9+/-1.0 vs. 8.3+/-1.1) and large follicles (1.2+/-0.2 vs. 1.3+/-0.3) present at the time embryo recovery, as detected by ultrasonography, did not differ between the T1 and T2 groups (P>0.05). Similarly, the numbers of total ova/embryos (6.2+/-0.9 vs. 6.4+/-1.1), transferable embryos (3.4+/-0.8 vs. 3.2+/-0.7), degenerate embryos (0.8+/-0.2 vs. 1.0+/-0.3) and unfertilized ova (2.1+/-0.5 vs. 2.2+/-0.5) did not differ between the groups (P>0.05). These data indicate that a reduced dose (24 vs. 28 mg) and number of treatments (6 vs. 8) of FSH for superovulation of CIDR-treated Korean native cows does not affect the embryo yield.

Asunto(s)

Transferencia de Embrión/veterinaria , Sincronización del Estro/efectos de los fármacos , Hormona Folículo Estimulante/farmacología , Superovulación/efectos de los fármacos , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Implantes de Medicamentos , Ciclo Estral/efectos de los fármacos , Femenino , Corea (Geográfico)

RESUMEN

This study evaluated the effects of two different oxygen (O2) concentrations on in vitro embryo development, embryo quality, and gene expression and the in vivo development following embryos transfer to recipients of natural and synchronized estrus in bovines. Cumulus oocyte complexes were in vitro matured in TCM199 supplemented with FSH (10 microg/ml), LH (10 microg/ml), and 10% (v/v) FBS. Presumptive zygotes were cultured in SOF medium either under 5% (low) or 20% (high) O2 in air. Cleavage rates did not differ between groups. Blastocyst and hatched blastocyst development in 5% O2 were significantly (P < 0.05) higher than in 20% O2. Total cell number of in vivo blastocyst was significantly (P < 0.05) higher than that of in vitro blastocyst. ICM ratio and apoptosis of in vivo blastocyst were significantly (P < 0.05) lower than that of in vitro blastocyst. Using real time PCR, we have found that for the set of genes (GLUT-1, MnSOD, VEGF, Bax, and Bcl-2) analyzed, there were differences in mRNA expression between in vitro produced (IVP) and in vivo produced embryos. Interestingly, the abundance of transcript for IFN-tau in IVP embryos produced under 5% O2 concentration was similar to in vivo counterparts. The pregnancy and twin rates of natural recipients were significantly (P < 0.05) higher than those of synchronized counterparts. No significant difference in the offspring sex was observed. In conclusion, low (5%) O2 concentration during IVC was beneficial for enhancing the embryo quality and recipients of natural estrus were more suitable than synchronized estrus for stable production of Hanwoo calves.

Asunto(s)

Transferencia de Embrión , Desarrollo Embrionario/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Oxígeno/farmacología , Preñez , Animales , Blastocisto/efectos de los fármacos , Peso Corporal , Bovinos , Femenino , Fertilización In Vitro , Freemartinismo , Masculino , Embarazo , Gemelos

RESUMEN

This study was conducted to evaluate the effects of exposing porcine ovaries to 30-33 C during transportation for 4 h and subsequently room temperature (25 C) for 6 h of storage on in vitro maturation (IVM) and subsequent parthenogenetic development of oocytes collected from the ovaries. After IVM, oocytes having a tight oopalsm membrane and no signs of degeneration were exposed to Dulbecco's phosphate-buffered saline (DPBS) with 7% ethanol (v/v) for 7 min to induce parthenogenetic activation. Moreover, we also determined whether exposure of the collected oocytes to room temperature for 1, 2 and 4 h in DPBS or porcine follicular fluid (pFF) affected parthenogenetic development. When porcine ovaries were stored after transportation, oocytes collected from the stored ovaries showed a significantly higher rate of degeneration after 65 h of IVM (58.4%) and a significantly lower rate of cleavage after parthenogenetic activation (40.1%) than oocytes collected from ovaries immediately after transportation (38.9% and 47.4%, respectively). However, there was no significant difference in developmental rates to the morula and blastocyst stages between these two groups (14.4% and 14.3%, respectively). The duration of preservation, 1, 2, and 4 h, of oocytes in DPBS did not affect parthenogenetic development. In contrast, when preserved for 4 h in pFF, the developmental rates of the oocytes were significantly decreased. This suggested that some factor(s) in follicular fluid affects the developmental rate of oocytes with the passage of time in ambient conditions. These results suggest that even after 6 h storage of ovaries, oocytes having normal morphology after IVM have the same rate of parthenogenetic development as oocytes collected from ovaries just after 4 h of transportation, except for a lower cleavage rate, and that exposure of oocytes to room temperature for 4 h in DPBS does not affect their parthenogenetic developmental competence.

Asunto(s)

Oocitos/crecimiento & desarrollo , Ovario/fisiología , Partenogénesis/fisiología , Temperatura , Animales , Femenino , Líquido Folicular , Porcinos , Factores de Tiempo

RESUMEN

The success of AI technology is based on both semen quality and freezing process. In order to establish the semen freezing techniques in Korean native bucks, factors affecting the success were evaluated in the present study. Semen collected by electro-ejaculation from bucks during four distinct seasons was evaluated for semen volume and pH, sperm motility and survivability. The semen volume, concentration and total cell were higher in spring, summer and less in winter. Yet, there were no seasonal differences in the proportional data of live sperm, motility score and pH of semen among seasons. The percentage of live sperm after thawing was found to increase with increased concentration of lactose in Tris-Egg yolk-glycerol (TY-G), being highest in TY-G supplemented with 180 mm lactose (TYL180-G), but did not differ between TY-G and TYL120-G. Sperm motility was enhanced by employing 2.0 h equilibration time with rapid freezing method. In conclusion, semen could be frozen with high success rates for further use of AI in breeding techniques and to preserve the Korean native bucks.

Asunto(s)

Criopreservación/veterinaria , Crioprotectores/farmacología , Cabras/fisiología , Preservación de Semen/veterinaria , Recuento de Espermatozoides/veterinaria , Motilidad Espermática/fisiología , Animales , Supervivencia Celular , Criopreservación/métodos , Criopreservación/normas , Concentración de Iones de Hidrógeno , Masculino , Estaciones del Año , Preservación de Semen/métodos , Preservación de Semen/normas , Factores de Tiempo

RESUMEN

The timing between round spermatid(s) (RS) injection and oocyte activation are critical for spermatid remodeling and embryo development in intracytoplasmic injection of round spermatid (ROSI) procedure. The objective of the present study was to develop an appropriate oocyte activation method for producing developmentally competent bovine embryos reconstructed with RS. Embryos reconstructed by ROSI were compared with three activation treatments for the rates of pronuclear formation, development and ploidy. RS were isolated from bull testes by Percoll density gradients. Matured oocytes were divided into three activation groups. In Group 1, oocytes were activated with ionomycin (5 microM, 5 min) before ROSI. In Group 2, oocytes were activated with ionomycin after ROSI. In Group 3, oocytes were activated twice with ionomycin before and after ROSI. All the eggs were then incubated in cycloheximide (CHX, 10 microg/mL) for 5 h and cultured in CR1aa medium for up to 8 days. Three methods of oocyte activation were also compared for the activation and development of parthenotes. Activation rates among the groups were 70-79% and did not differ. Cleavage rates in parthenotes were significantly (P < 0.05) higher in Group 3 than in Groups 1 and 2, but blastocyst rates did not differ among the groups. In ROSI embryos, the rates of cleavage and development into blastocysts were significantly (P < 0.05) greater in Group 3 (82.3% and 13.1%) than in Groups 1 and 2 (53.7, 5.8% and 64.2, 1.7%, respectively). Ploidy analysis by examining the metaphase spreads of ROSI blastocysts displayed greater numbers of diploid chromosomal complements. These results suggest that intracytoplasmic RS injection combined with repeated ionomycin activation followed by CHX treatment is more efficient for producing developmentally competent embryos.

Asunto(s)

Bovinos , Oocitos/fisiología , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Espermátides/fisiología , Animales , Blastocisto/fisiología , Células Cultivadas , Cicloheximida/farmacología , Desarrollo Embrionario , Femenino , Ionomicina/farmacología , Masculino , Oocitos/efectos de los fármacos , Partenogénesis , Inyecciones de Esperma Intracitoplasmáticas/métodos , Testículo/citología , Recolección de Tejidos y Órganos/veterinaria

RESUMEN

The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 microm nylon mesh, the cells were centrifuged at 650 x g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that approximately 40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen-thawed cells to those of untreated cells. The percentage (mean+/-S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P<0.05) than in untreated and frozen-thawed cells from 35% Percoll gradient (11.7+/-3.1% compared with 26.3+/-2.0% and 53.5+/-1.3%, respectively), but the rate of apoptosis did not differ (1.2+/-0.49% compared with 2.5+/-0.8% and 0.9+/-0.04%, respectively). The proportional data (mean+/-S.E.M.) of live cells in Percoll treated group were greater (P<0.05) than in untreated and frozen-thawed cells from the 35% Percoll gradient (86.7+/-3.26% compared with 70.8+/-2.73% and 41.9+/-1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen-thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P>0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P<0.05) cleavage and blastocyst rates in frozen-thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.

Asunto(s)

Centrifugación por Gradiente de Densidad/veterinaria , Recuento de Espermatozoides/veterinaria , Espermátides/fisiología , Testículo/citología , Animales , Apoptosis , Blastocisto/citología , Blastocisto/fisiología , Bovinos , Células Cultivadas , Centrifugación por Gradiente de Densidad/métodos , ADN/análisis , Desarrollo Embrionario , Femenino , Citometría de Flujo/veterinaria , Masculino , Povidona , Dióxido de Silicio , Recuento de Espermatozoides/métodos

RESUMEN

The levels of glutamic acid decarboxylase (GAD) were strongly increased in the cortex and the striatum in dopamine D2 receptors null (D2R-/-) mice, which show a significant locomotor impairment. In this study, the effects of different GABAergic drugs on locomotor activity were analyzed in D2R-/- mice. After administering muscimol (1 mg/kg), a GABA(A) receptor agonist, the D2R-/- mice showed increased locomotor activity up to 200%. When the muscimol dose was increased (4-6 mg/kg), the D2R-/- mice exhibited seizure-like behavior, and the electroencephalographic (EEG) recordings during these behaviors showed a high amplitude rhythmic epileptiform activity in these mice. In situ hybridization showed that after injecting muscimol in the D2R-/- mice, the expression of enkephalin and immediate early gene, NGFI-A, was closely regulated with the locomotor activity regulated by GABAergic stimulation. These results suggest that the absence of D2R alters the GABAergic neurotransmission, specifically on GABA(A)-receptor mediated signaling, and stimulating the GABA(A) receptor can reverse the dysfunction of GABAergic inhibition in the motor circuits in the basal ganglia.

Asunto(s)

Ganglios Basales/metabolismo , Química Encefálica/genética , Receptores de Dopamina D2/genética , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Ganglios Basales/efectos de los fármacos , Ganglios Basales/fisiopatología , Dopamina/metabolismo , Electroencefalografía/efectos de los fármacos , Encefalinas/metabolismo , Agonistas del GABA/farmacología , Antagonistas del GABA/farmacología , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Glutamato Descarboxilasa/metabolismo , Masculino , Ratones , Ratones Noqueados , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Receptores de Dopamina D2/deficiencia , Receptores de GABA-A/metabolismo , Convulsiones/inducido químicamente , Convulsiones/enzimología , Transmisión Sináptica/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología

RESUMEN

Stable rat pituitary tumor cell lines expressing two isoforms of the dopamine D2 receptor, D2L (long) and D2S (short) (the GH3D2L and GH3D2S cell lines, respectively), were established, and the signaling pathway underlying the anti-proliferative and cell death effects of dopaminergic agonists was examined in these cells. After either dopamine or quinpirole treatment, the cell viability decreased significantly only in GH3D2L cells and GH3D2S cells, but not in GH3 cells where D2 receptors are absent. Treatment with haloperidol, a specific D2 receptor antagonist, rescued the dopamine-mediated decreased cell viability in both the GH3D2L and GH3D2S cells. Treatment of these cells with dopamine decreased the DNA synthesis rate, as demonstrated by the incorporation of 5-bromo-2'-deoxyuridine (BrdU). Dopamine-induced cell death was observed in the GH3D2L and GH3D2S cells, and was accompanied by DNA laddering and caspase-3 activation, which were blunted by haloperidol, indicating that dopamine-induced cell death in these cells is mediated by the dopamine D2 receptors. D2 receptor-mediated cell death in these cells correlated with the sustained and enhanced activation of p38 mitogen-activated protein kinase (MAPK) and the extracellular-signal regulated kinase (ERK)1/2 pathways. Treatment with SB203580, which is a specific p38 MAPK inhibitor and PD98059, which is an inhibitor of MEK1/ERK signaling, selectively abrogates dopamine-induced cell death. It was further shown that p38 MAPK and ERK activation was inhibited by the antioxidant, N-acetylcysteine (NAC), and that a treatment with haloperidol completely blocked the p38 and ERK activation induced by dopamine. These results suggest that dopamine induces an anti-proliferative effect and cell death via the dopamine D2 receptors, by means of the p38 MAPK and ERK pathways involving oxidative stress, in the pituitary tumor cells.

Asunto(s)

Apoptosis/efectos de los fármacos , Neoplasias Hipofisarias/patología , Receptores de Dopamina D2/fisiología , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Dopamina/farmacología , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/farmacología , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Isoformas de Proteínas/fisiología , Ratas , Receptores de Dopamina D2/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos

RESUMEN

PURPOSE: Culture systems affect the development of IVP embryos and consequently their cryosurvival potential. The viability of postthawed bovine IVP embryos developed from IVM/IVC medium in the presence or absence of serum was compared. METHODS: Cumulus-oocyte complexes were matured in IVM medium supplemented with or without serum. Some oocytes were evaluated for nuclear maturation status and others were inseminated with semen. Presumptive zygotes were cultured in IVC medium supplemented with or without serum for 9 days. Blastocysts were cryopreserved with 1.5 M ethylene glycol in PBS. RESULTS: No difference was observed in the nuclear maturation status and cleavage rates in both groups, but significantly (P < 0.05) higher in blastocyst rates in the serum-supplemented group. After freezing, survival of blastocysts was higher in the serum-free group. At 36 h culture after thawing, blastocysts developed without serum had significantly (P < 0.05) higher cell number than those cultured with serum. CONCLUSIONS: We conclude that serum-free culture system enhances the viability of frozen-thawed bovine embryos.

Asunto(s)

Blastocisto/fisiología , Fase de Segmentación del Huevo/fisiología , Criopreservación , Medio de Cultivo Libre de Suero , Fertilización In Vitro/veterinaria , Animales , Bovinos , Técnicas de Cultivo de Célula/métodos , Supervivencia Celular/fisiología , Transferencia de Embrión/veterinaria