RESUMEN
BACKGROUND: Sperm miRNAs were reported to regulate spermatogenesis and early embryonic development in some mammals including bovine. The dairy cattle breeding industry now tends to collect semen from younger bulls under high selection pressure at a time when semen quality may be suboptimal compared to adult bulls. Whether the patterns of spermatic miRNAs are affected by paternal age and/or impact early embryogenesis is not clear. Hence, we generated small non-coding RNA libraries of sperm collected from same bulls at 10, 12, and 16 months of age, using 16 months as control for differential expression and functional analysis. RESULTS: We firstly excluded all miRNAs present in measurable quantity in oocytes according to the literature. Of the remaining miRNAs, ten sperm-borne miRNAs were significantly differentially expressed in younger bulls (four in the 10 vs 16 months contrast and six in the 12 vs 16 months contrast). Targets of miRNAs were identified and compared to the transcriptomic database of two-cell embryos, to genes related to two-cell competence, and to the transcriptomic database of blastocysts. Ingenuity pathway analysis of the targets of these miRNAs suggested potential influence on the developmental competence of two-cell embryos and on metabolism and protein synthesis in blastocysts. CONCLUSIONS: The results showed that miRNA patterns in sperm are affected by the age of the bull and may mediate the effects of paternal age on early embryonic development.
Asunto(s)
Desarrollo Embrionario , MicroARNs , Análisis de Semen , Animales , Blastocisto , Bovinos , Desarrollo Embrionario/genética , Femenino , Masculino , MicroARNs/genética , Embarazo , EspermatozoidesRESUMEN
Two experiments were performed to determine effects of follicular ablation (FA) and GnRH treatment on conception rate and synchronization in timing of ovulation among Holstein heifers. In Experiment 1, heifers were randomly allocated to four groups: Control (n = 84): prostaglandin F2α (PGF) IM on Day 0; FA-5/GnRH (n = 43): FA 5 days before PGF and GnRH on Day 2; FA-4/GnRH (n = 48):FA 4 days before PGF and GnRH on Day 2; andFA-3/GnRH (n = 21): FA 3 days before PGF and GnRH on Day 2. Ultrasonography was performed to determine follicular size, ovulation occurrence, and size of CL. In Experiment 2, heifers were assigned to three groups: Control (n = 264), FA-5/GnRH, and FA-4/GnRH. Pregnancy diagnosis was performed at Days 30 and 60. In Experiment 1, size of largest follicle at time of PGF was less variable (P ≤ 0.05) in all FA groups compared to the Control group. With the FA-5/GnRH and FA-4/GnRH treatments, there were greater (P ≤ 0.05) proportions of timing of ovulation synchronization (86 % and 85 %, respectively) compared to the Control (61 %) and FA-3/GnRH (62 %) groups. In Experiment 2, conception rates did not differ among groups, however, there were more pregnancies per cow when timing-of-ovulation treatments were imposed. In conclusion, follicular ablation combined with GnRH treatment resulted in an increased proportion of heifers having synchronized ovulation and, therefore, number of recipient heifers available for embryo transfer. Additionally, there was no effect on conception rate when there was greater synchronization in timing of ovulation among heifers.
Asunto(s)
Bovinos/fisiología , Transferencia de Embrión/veterinaria , Fertilización/efectos de los fármacos , Hormona Liberadora de Gonadotropina/farmacología , Folículo Ovárico , Ovulación/efectos de los fármacos , Animales , Cloprostenol/administración & dosificación , Cloprostenol/farmacología , Dinoprost/administración & dosificación , Dinoprost/farmacología , Sincronización del Estro , Femenino , Luteolíticos/administración & dosificación , Luteolíticos/farmacología , Ovulación/fisiología , Oxitócicos/administración & dosificación , Oxitócicos/farmacologíaRESUMEN
In the dairy industry, the high selection pressure combined with the increased efficiency of assisted reproduction technologies (ART) are leading toward the use of younger females for reproduction purposes, with the aim to reduce the interval between generations. This situation could impair embryo quality, decreasing the success rate of the ART procedures and the values of resulting offspring. Young Holstein heifers (n = 10) were subjected to ovarian stimulation and oocyte collection at 8, 11, and 14 months of age. All the oocytes were fertilized in vitro with semen from one adult bull, generating three pools of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used to compare the DNA methylation status of blastocysts obtained from oocytes collected at 8 versus 14 and 11 versus 14 months of age. Age-related contrast analysis identified 5,787 and 3,658 differentially methylated regions (DMRs) in blastocysts from heifers at 8 versus 14 and 11 versus 14 months of age, respectively. For both contrasts, the DMRs were distributed nonrandomly in the different DNA regions. The DNA from embryos from 8-month-old donors was more hypermethylated, while the DNA from embryos from 11-month-old donors displayed an intermediate phenotype. According to Ingenuity Pathway Analysis, the upstream regulator genes cellular tumor antigen p53, transforming growth factor ß1, tumor necrosis factor, and hepatocyte nuclear factor 4α are particularly associated with methylation sensitive targets, which were more hypermethylated in embryos from younger donors.
Asunto(s)
Blastocisto/metabolismo , Metilación de ADN/fisiología , Donación de Oocito/veterinaria , Factores de Edad , Animales , Estudios de Casos y Controles , Bovinos , Células Cultivadas , Embrión de Mamíferos , Desarrollo Embrionario , Femenino , Fertilización In Vitro/veterinaria , Regulación del Desarrollo de la Expresión Génica , Masculino , Oocitos/metabolismo , Maduración Sexual/fisiologíaRESUMEN
Genetic selection for the best suited offspring drives the dairy industry to use young genitors and assisted reproductive technologies (ART) to reduce generation intervals. However, sperm samples collected from peri-pubertal bulls have lower counts and quality compared to samples from adult bulls. Moreover, our previous study identified differentially methylated regions (DMRs) in sperms from early-, peri- and post-pubertal bulls. The aim of this study was to further investigate the impacts of paternal age on early embryos. To achieve this, we evaluated the transcriptome and the epigenome of bovine blastocysts generated from spermatozoa of bulls at 10, 12, and 16 months of age and used in vitro fertilization (IVF) of oocytes recovered from the same adult cows. A total of 259 probes were differentially expressed and 6953 probes were differentially methylated in the 10- vs 16-month and the 12- vs 16-month groups. Ingenuity Pathway Analysis (IPA) of transcriptomic data demonstrated that energy-related pathways such as oxidative phosphorylation, EIF2 signaling, and mitochondrial dysfunction were affected the most by the age of the bull. Meanwhile, IPA analysis of the epigenome revealed that protein kinase A signaling, RAR activation, and other pathways were influenced by paternal age. Overall, we showed that the bull's age mainly influenced metabolism-related pathways in blastocysts, and this could therefore impact subsequent development.
Asunto(s)
Envejecimiento , Blastocisto/fisiología , Bovinos/fisiología , Epigenoma , Fertilización In Vitro/veterinaria , Transcriptoma , Animales , Regulación del Desarrollo de la Expresión Génica , MasculinoRESUMEN
One of the major challenges of artificial reproductive technologies is to develop new methods for producing greater numbers of embryos. An oocyte fosters the ability to develop into an embryo before oocyte meiotic resumption. The aim of the present study was to assess the effect of adenosine (ADO), a purine nucleoside found in follicular fluid, on the inhibition of oocyte meiotic resumption and the production of blastocysts. The results showed the efficacy of ADO to inhibit oocyte meiotic resumption. The use of ADO (3â¯mM) during a pre-in vitro maturation (pre-IVM) culture period of 6â¯h resulted in a significant increase (pâ¯<â¯0.05) of blastocysts compared to control conditions with no pre-IVM culture period. No effect on the percentage of cleavage was observed. The effect of adenosine on blastocyst yield was time- and concentration-dependent with an optimum effect at 3â¯mM for 6â¯h. Supplementing the ADO pre-IVM culture medium with estradiol, follicle-stimulating hormone, progesterone, epidermal growth factor, insulin-like growth factor-2 or reelin did not improve the blastocyst yield. Transcriptional analyses of ADO-treated cumulus cells revealed that NRP1, RELN, MAN1A1, THRA and GATM were up-regulated. Finally, bioinformatic analysis identified mitochondrial function as the top canonical pathway affected by ADO. This opens up new opportunities for further investigations.
Asunto(s)
Adenosina/farmacología , Bovinos , Técnicas de Cultivo de Célula/métodos , Desarrollo Embrionario/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos , Meiosis/efectos de los fármacos , Oocitos/efectos de los fármacos , Animales , Bovinos/embriología , Bovinos/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Técnicas de Cultivo de Embriones/métodos , Técnicas de Cultivo de Embriones/veterinaria , Desarrollo Embrionario/genética , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/veterinaria , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos/métodos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Análisis por Micromatrices , Oocitos/citología , Oocitos/fisiologíaRESUMEN
Genomic selection is accelerating genetic gain in dairy cattle. Decreasing generation time by using younger gamete donors would further accelerate breed improvement programs. Although ovarian stimulation of peripubertal animals is possible and embryos produced in vitro from the resulting oocytes are viable, developmental competence is lower than when sexually mature cows are used. The aim of the present study was to shed light on how oocyte developmental competence is acquired as a heifer ages. Ten peripubertal Bos taurus Holstein heifers underwent ovarian stimulation cycles at the ages of 8, 11 (mean 10.8) and 14 (mean 13.7) months. Collected oocytes were fertilised in vitro with spermatozoa from the same adult male. Each heifer served as its own control. The transcriptomes of granulosa cells recovered with the oocytes were analysed using microarrays. Differential expression of certain genes was measured using polymerase chain reaction. Principal component analysis of microarray data revealed that the younger the animal, the more distinctive the gene expression pattern. Using ingenuity pathway analysis (IPA) and NetworkAnalyst (www.networkanalyst.ca), the main biological functions affected in younger donors were identified. The results suggest that cell differentiation, inflammation and apoptosis signalling are less apparent in peripubertal donors. Such physiological traits have been associated with a lower basal concentration of LH.
Asunto(s)
Transferencia de Embrión/veterinaria , Células de la Granulosa/metabolismo , Inducción de la Ovulación , Transcriptoma , Factores de Edad , Animales , Bovinos , Técnicas de Cultivo de Embriones/veterinaria , Femenino , Recuperación del Oocito/veterinaria , Oocitos/metabolismoRESUMEN
Oocytes collected from prepubertal animals are known to be less developmentally competent than those from adult animals. There is evidence suggesting that acquisition of developmental competence in bovine oocytes may be linked to the expression profile of genes in the granulosa cells (GCs). Cumulus-oocyte complexes (COC) and GCs were collected from 12 Holstein heifers between 2 and 6 months of age (nine follicle-stimulating hormone [FSH] treated and three untreated) and eight FSH-treated cows. The COCs from prepubertal animals were matured, fertilized, and cultured in vitro to assess development to the blastocyst stage. The relative messenger RNA (mRNA) abundance of FSHR, StAR, CYP19A1, HSD3B1, CX43, FOXO1, and XIAP in GCs were quantified by real-time quantitative polymerase chain reaction. Results from this study revealed that GCs of prepubertal animals respond to FSH treatment by increasing mRNA levels of genes promoting estradiol synthesis and follicular growth ( FSHR and CYP19A1), and preventing cell apoptosis ( XIAP), and by decreasing mRNA levels of genes promoting progesterone production ( StAR and HSD3B1). This study also revealed that the relative mRNA abundance of FOXO1 in GCs is associated with oocyte competence to support embryo development to the blastocyst stage in prepubertal Holstein heifers.
Asunto(s)
Apoptosis/efectos de los fármacos , Hormona Folículo Estimulante/farmacología , Células de la Granulosa/metabolismo , Oocitos/metabolismo , Maduración Sexual/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Bovinos , Femenino , Células de la Granulosa/citología , Oocitos/citologíaRESUMEN
Laparoscopic Ovum Pick-Up (LOPU) in calves followed by in vitro embryo production (IVEP) and transfer (ET) into adult recipients has great potential for accelerated genetic gain through shortening of the generation interval. In this study, 11 Holstein calves were subjected to up to six LOPU procedures between the ages of 2-6â¯monthsâ¯at 2-3 weeks interval. In all cases, the animals received a CIDR 5 days prior to LOPU and were gonadotropin-stimulated starting at 72â¯h before LOPU using one of three protocols that were rotated twice among the animals during the study. Calves were injected with FSH every 12â¯h (FSH12h), or every 8â¯h (FSH8h) or every 8â¯h until -36â¯h from LOPU at which point the FSH was replaced with a single dose of 400 IU eCG (FSH8h-eCG). No statistical differences were observed among the 3 treatments in terms of mean follicles available for aspiration (35.7⯱â¯16 vs. 38.5⯱â¯25 vs. 31.1⯱â¯22), mean oocytes recovered (26.5⯱â¯14 vs. 21.6⯱â¯10 vs. 19.4⯱â¯14) and cleavage rate (66.0⯱â¯14 vs. 61.1⯱â¯11 vs. 72.2⯱â¯8), for FSH12h, FSH8h and FSH8h-eCG, respectively. However, FSH8h-eCG resulted in a significantly higher rate of transferable embryos (17.5⯱â¯8%) compared with FSH12h (8.9⯱â¯5%, Pâ¯<â¯0.05). Oocytes from follicles of ≥5â¯mm in diameter yielded a higher rate (Pâ¯<â¯0.05) of development to the blastocyst stage (13.8%) than those collected from <5â¯mm follicles (6.8%). Animal age, by comparing animals at <100, 101 to 130 andâ¯>â¯130 days of age, did not affect the mean number of follicles (34.2⯱â¯15 vs. 39.3⯱â¯26 vs. 31.6⯱â¯25), the mean number of oocytes recovered (21.2⯱â¯10 vs. 24.5⯱â¯15 vs. 22.6⯱â¯17), and the cleavage rate (68.6⯱â¯11 vs. 61.7⯱â¯12 vs. 70.7⯱â¯10%), respectively. However, animals in the older age range had significantly higher development to the blastocyst stage (19.9⯱â¯6 vs. 9.5⯱â¯8%, Pâ¯<â¯0.01) and better embryo quality, as evidenced by higher average cell numbers (119.1⯱â¯47 vs. 91.5⯱â¯25, Pâ¯<â¯0.05) compared with those in the lower age. Finally, we tested the benefits of relieving endoplasmic reticulum stress by supplementing the culture medium with 50⯵M tauroursodeoxycholic acid (TUDCA) and found a numerically higher rate of development to the blastocyst stage (21.1⯱â¯8 vs. 18.6⯱â¯4%), but not statistically different, compared with control culture. Overall, our findings indicate that a significant number of transferable embryos (range 10-30) can be produced from Holstein calves before they reach 6 months of age.
Asunto(s)
Bovinos/fisiología , Fertilización In Vitro/veterinaria , Gonadotropinas/uso terapéutico , Oocitos/efectos de los fármacos , Animales , Transferencia de Embrión/veterinaria , Femenino , Fertilización In Vitro/métodos , Hormona Folículo Estimulante/administración & dosificación , Hormona Folículo Estimulante/uso terapéutico , Laparoscopía/veterinaria , Oocitos/crecimiento & desarrolloRESUMEN
The selection of the best dairy heifers is mainly driven by the genetic value of their parents. The phenotype analysis of cows and of the daughters of bulls has been used to identify the best genetic value for decades before being replaced by genomic selection of individuals that are not yet parents. Because it is possible to predict the future value of an individual by its genetic makeup, it becomes feasible to do it as early as the blastocyst stage and to decide which should be transferred or not. Because we know the genotype of an animal at birth, or even before, it is becoming desirable to reproduce this animal as soon as possible to reduce generation interval and improve selection speed. Nature provides constraints that can be overcome: a single oocyte per cycle and age at puberty. Indeed, it is now possible to super-stimulate the ovary at any age and to start collecting oocytes at 6 mo by trans-vaginal ultrasonography. The challenge becomes the production of good eggs and embryos capable of implanting and developing into healthy calves. Our understanding of ovarian follicular physiology has been instrumental in designing stimulation protocols that may be adjusted to any physiological context including age, and even the individual animal, to obtain a good response. Therefore, the combination of procedures developed in cows to optimize oocyte quality, for example, FSH coasting, in association with in vitro fertilization and optimal culture conditions can now result in the production of several female embryos twice a month from animals 6 to12 mo of age. The transcriptomic and epigenetic analyses of embryos produced from the same females at different ages were compared and few differences were noted in particular in relation to embryo metabolism. These embryos are as good as the ones obtained from adult animals and can be produced with sexed sperm of bulls 12 mo of age. This combination of these technical optimizations with blastocyst genotyping allows the selection of a second generation within a year.
Asunto(s)
Bovinos/fisiología , Oocitos/fisiología , Reproducción , Espermatozoides/fisiología , Animales , Blastocisto/fisiología , Bovinos/genética , Femenino , Fertilización In Vitro/veterinaria , Masculino , Recuperación del Oocito/veterinaria , Folículo Ovárico/fisiología , Ovulación , Maduración SexualRESUMEN
In cows, the use of follicle-stimulating hormone (FSH) to stimulate follicular growth followed by a short period of FSH withdrawal has been shown to be beneficial for oocyte developmental competence. Although this treatment represents a useful optimization to generate highly competent oocytes, the underlying physiological process is not completely understood. The goal of this study was to investigate the role of luteinizing hormone (LH) action during FSH withdrawal before ovulation. To accomplish this, LH release was pharmacologically inhibited during the coasting period with gonadotropin-releasing hormone (GnRH) antagonists. Granulosa cells samples were obtained from cows stimulated with FSH during 3 days followed by a coasting period of 68 h and treated with a GnRH antagonist (cetrorelix group) or not (control). A significant reduction in the number of follicles at >10 mm diameter was observed with the cetrorelix group and gene expression of granulosa cells reveals that 747 transcripts are potentially regulated by LH. Further analysis indicates how the absence of LH may trigger early atresia, the upregulation of atretic agent as tumor protein P53 and transforming growth factor ß1 and the inhibition of growth support. This work allows identification of genes that are associated with maintained follicular growth and conversely the ones leading to atresia in dominant pre-ovulatory follicles.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Células de la Granulosa , Hormona Luteinizante/fisiología , Folículo Ovárico/crecimiento & desarrollo , Transcriptoma/genética , Animales , Bovinos , Femenino , Hormona Folículo Estimulante/fisiología , Atresia Folicular/genética , Hormona Liberadora de Gonadotropina/análogos & derivados , Hormona Liberadora de Gonadotropina/antagonistas & inhibidores , Hormona Liberadora de Gonadotropina/farmacología , Hormona Luteinizante/metabolismo , Ovulación , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia ArribaRESUMEN
In the dairy industry, using semen as soon as the bull is mature enough to produce it is advantageous for breeding purposes. Mammalian spermatogenesis is a hormone-dependent developmental program in which a complex cascade of events must take place to ensure that germ cells reach the proper stage of development at the proper time. Conventional indicators of semen quality such as sperm cell motility and viability usually improve as bulls mature, meeting quality criteria satisfactorily at around 16 months. Using semen before that age may affect embryo viability, but other changes occurring during the peripubertal period should be considered. Although it is known that establishment of these patterns begins during foetal life, the extent to which sperm cell DNA methylation changes during puberty has not been studied. The aim of this study is to correlate the age of a young bull with the overall DNA methylation pattern of its spermatozoa. Spermatozoa were collected from bulls at the ages of 10 months (early pubertal), 12 months (late pubertal) and 16 months (pubertal). Each animal (n = 4) was compared to itself with 16 months as control. Genome-wide DNA methylation was analyzed by microarray using the EmbryoGENE DNA Methylation Analysis platform. Using a fold change over 1.5 and a 5% FDR p-value correction, a total of 2602 differently methylated regions were found in common between 10 months of age and 16 months of age. No differently methylated regions between 12 months and 16 months of age were found at the same level of statistical significance. We conclude that spermatozoa from bulls aged 10 months have a different epigenetic profile, which could compromise their value.
Asunto(s)
Bovinos/fisiología , Maduración Sexual/fisiología , Espermatozoides/metabolismo , Animales , Metilación de ADN , Masculino , Análisis de Semen , Espermatogénesis/fisiologíaRESUMEN
The use of oocytes recovered from prepubertal donors for in vitro embryo production has great potential for accelerating the rate of genetic gain in the dairy industry. However, these oocytes are known to be less developmentally competent than those from adult donors. In this study, we investigated the effect of age and gonadotropin stimulation in Holstein heifers subjected to oocyte collection every two weeks between 2 and 6 months of age. In order to assess the effect of gonadotropin stimulation, animals were subjected to one of three treatments, namely Short (ST; 36-42 h), Long (LT; ≥72 h) and No Treatment (NT) prior to laparoscopic ovum pick-up (LOPU). Our results show that the LT significantly improved the proportion of large follicles (>5 mm diameter) present in the ovary (LT 34.0% vs. ST 11.2% vs. NT 2.4%, P < 0.05), as well as the percentage of good-quality cumulus oocyte complexes (COCs) recovered (LT 95.3 ± 18% vs. ST 85.4 ± 22% vs. NT 82.2 ± 14%, P < 0.05) and blastocyst rate (LT 36.7 ± 26% vs. ST 18.3 ± 15% vs. NT 16.7 ± 9%, P < 0.05). Recovery rate was affected by treatment (LT 70.4 ± 25 vs. ST 85.4 ± 29 vs. NT 72.7 ± 23, P < 0.05). To assess the impact of age, data was grouped into <100 days (A), 100-130 days (B) and >130 days (C) of age at LOPU. We found that as animals got older, although the average number of COCs per donor per LOPU declined (A: 17.5 ± 11 vs. B: 14.7 ± 7 vs. C: 11.9 ± 8), the blastocyst rate increased (A: 12.8 ± 20% vs. B: 17.1 ± 21% vs. C: 21.8 ± 25%, P < 0.05). We also evaluated the incidence of polyspermy and confirmed it is a critical limitation for IVF in calf oocytes. The incidence of polyspermy was unaffected by gonadotropin treatment, but significantly decreased with age. The capacity for full development to term of in vitro produced embryos from calf oocytes was tested by embryo transfer into 21 synchronized adult recipients, which resulted in 13 pregnancies (62%), full development to term and healthy calves born. Finally, the study allowed evaluating the safety of the procedure since, on average, each animal was subjected to 8 LOPU procedures over a period of 4 months. Our results showed that the procedure is safe (no incidents during laparoscopy), and was not harmful for the reproductive future of the animals, as those that were bred became pregnant after reaching sexual maturity.
Asunto(s)
Envejecimiento/fisiología , Técnicas de Cultivo de Embriones/veterinaria , Desarrollo Embrionario/efectos de los fármacos , Gonadotropinas/farmacología , Oocitos/efectos de los fármacos , Animales , Bovinos , Transferencia de Embrión , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Masculino , EmbarazoRESUMEN
Ovarian stimulation with exogenous FSH followed by FSH withdrawal or 'coasting' is an effective means of increasing the number of oocytes obtainable for the in vitro production of cattle embryos. However, the quality of the oocytes thus obtained varies considerably from one cow to the next. The aim of the present study was to gain a better understanding of the follicular conditions associated with low oocyte developmental competence. Granulosa cells from 94 Holstein cows in a commercial embryo production facility were collected following ovarian stimulation and coasting. Microarray analysis showed 120 genes expressed with a differential of at least 1.5 when comparing donors of mostly competent with donors of mostly incompetent oocytes. Using ingenuity pathway analysis, we revealed the main biological functions and potential upstream regulators that distinguish donors of mostly incompetent oocytes. These are involved in cell proliferation, apoptosis, lipid metabolism, retinol availability and insulin signalling. In summary, we demonstrated that differences in follicle maturity at collection could explain differences in oocyte competence associated with individual animals. We also revealed deficiencies in lipid metabolism and retinol signalling in granulosa cells from donors of mostly incompetent oocytes.
Asunto(s)
Hormona Folículo Estimulante/administración & dosificación , Expresión Génica/efectos de los fármacos , Células de la Granulosa/metabolismo , Oocitos/metabolismo , Inducción de la Ovulación/veterinaria , Animales , Bovinos , Femenino , Fertilización In Vitro/métodos , Fertilización In Vitro/veterinaria , Células de la Granulosa/efectos de los fármacos , Oocitos/efectos de los fármacos , Inducción de la Ovulación/métodosRESUMEN
Assisted reproduction technologies (ART) and high selection pressure in the dairy industry are leading towards the use of younger females for reproduction, thereby reducing the interval between generations. This situation may have a negative impact on embryo quality, thus reducing the success rate of the procedures. This study aimed to document the effects of oocyte donor age on embryo quality, at the transcriptomic level, in order to characterize the effects of using young females for reproduction purpose. Young Holstein heifers (n = 10) were used at three different ages for ovarian stimulation protocols and oocyte collections (at 8, 11 and 14 months). All of the oocytes were fertilized in vitro with the semen of one adult bull, generating three lots of embryos per animal. Each animal was its own control for the evaluation of the effects of age. The EmbryoGENE platform was used for the assessment of gene expression patterns at the blastocyst stage. Embryos from animals at 8 vs 14 months and at 11 vs 14 months were used for microarray hybridization. Validation was done by performing RT-qPCR on seven candidate genes. Age-related contrast analysis (8 vs 14 mo and 11 vs 14 mo) identified 242 differentially expressed genes (DEGs) for the first contrast, and 296 for the second. The analysis of the molecular and biological functions of the DEGs suggests a metabolic cause to explain the differences that are observed between embryos from immature and adult subjects. The mTOR and PPAR signaling pathways, as well as the NRF2-mediated oxidative stress response pathways were among the gene expression pathways affected by donor age. In conclusion, the main differences between embryos produced at peri-pubertal ages are related to metabolic conditions resulting in a higher impact of in vitro conditions on blastocyts from younger heifers.
Asunto(s)
Bovinos/embriología , Fertilización In Vitro/veterinaria , Donación de Oocito/veterinaria , Oocitos/fisiología , Transcriptoma/fisiología , Factores de Edad , Envejecimiento , Animales , Blastocisto/fisiología , Femenino , Masculino , Análisis por Micromatrices , Factor 2 Relacionado con NF-E2/genética , Recuperación del Oocito/veterinaria , Estrés Oxidativo/genética , Receptores Activados del Proliferador del Peroxisoma/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Maduración Sexual , Transducción de Señal/genética , Serina-Treonina Quinasas TOR/genéticaRESUMEN
Monozygotic (MZ) twins are of great interest to elucidate the contributions of pre- and postnatal environmental factors on epigenetics in the expression of complex traits and diseases. Progeny testing recently revealed that MZ twin bulls do not necessarily lead to identical genetic merit estimates (i.e. breeding values). Therefore, to explain differences in offspring productivity of MZ twin bulls despite their identical genetic backgrounds, we hypothesised that paternal sperm epigenomes vary between MZ twin bulls. In the present study, semen characteristics and global sperm DNA methylome were profiled for four pairs of MZ twin bulls. Some MZ twin pairs had divergent semen quality (sperm morphology, motility and viability). Comparative genome-wide DNA methylome surveys were performed using methyl-sensitive enrichment and microarray identification. Between 2% and 10% of all probes (400000) were differentially methylated between MZ twin pairs. In addition, there were 580 loci differentially methylated across all pairs of MZ twins. Furthermore, enrichment analysis indicated a significant enrichment for fertility associated quantitative trait loci (P=0.033). In conclusion, differences in the sperm epigenome may contribute to incongruous diverging performances of daughters sired by bulls that are MZ twins.
Asunto(s)
Metilación de ADN , Genoma , Espermatozoides/citología , Espermatozoides/metabolismo , Animales , Bovinos , Forma de la Célula/fisiología , Supervivencia Celular/fisiología , Masculino , Análisis de Semen , Motilidad Espermática/fisiologíaRESUMEN
Oocyte developmental competence in superstimulated cows is dependent in part on the duration of the FSH coasting. FSH coasting refers to superstimulation with FSH (2 days of endogenous FSH following follicle ablation and 3 days of FSH injections) followed by no FSH for a specific duration. The optimal duration varies among individuals. FSH coasting appears to modulate the transcriptome of different follicular compartments, which cooperate as a single functional unit. However, the integrative effects of FSH coasting on different follicular compartments remain ambiguous. Meta-analysis of three independent transcriptome studies, each focused on a single cell type (granulosa, cumulus, and oocyte) during FSH coasting, allowed the identification of 12 gene clusters with similar time-course expression patterns in all three compartments. Network analysis identified HNF4A (involved in metabolic functions) and ELAVL1 (an RNA-binding protein) as hub genes regulated respectively upward and downward in the clusters enriched at the optimal coasting time, and APP (involved in mitochondrial functions) and COPS5 (a member of the COP9 signalosome) as hub genes regulated respectively upwards and downwards in the clusters enriched progressively throughout the coasting period. We confirmed the effects on HNF4A downstream targets (TTR, PPL) and other hub genes (ELAVL1, APP, MYC, and PGR) in 30 cows with RT-quantitative PCR. The correlation of hub gene expression levels with FSH coasting indicated that a combination of these genes could predict oocyte competence with 83% sensitivity, suggesting that they are potential biomarkers of follicle differentiation. These findings could be used to optimize FSH coasting on an individual basis.
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Oocitos/crecimiento & desarrollo , Oocitos/metabolismo , Folículo Ovárico/metabolismo , Transcriptoma/fisiología , Animales , Bovinos , Femenino , Hormona Folículo Estimulante/metabolismo , Perfilación de la Expresión Génica/métodosRESUMEN
The use of oocytes obtained from younger donors for IVF followed by embryo transfer represents an opportunity to accelerate genetic gain by reducing generation time. In this study, we investigated the relationship between donor age and the in vitro developmental competence of oocytes obtained from Holstein females (aged 5-18 months) after FSH stimulation and coasting. The follicle size patterns showed a significantly higher total number of small follicles (5-6 mm) from donors aged 5 to 10 months and a higher total number of medium-sized follicles (7-10 mm) in donors aged 6 to 7 months. Our analysis also revealed that the total number of follicles was significantly higher (P < 0.05) in donors aged 5 to 8 months and tended to be higher (P = 0.053) in nine-month-old donors. However, oocytes obtained from donors aged 5 to 10 months yielded fewer embryos reaching the morula and blastocyst stages. In summary, our results demonstrate that a higher number of oocytes can be obtained from younger animals but lower developmental competence negates this gain.
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Envejecimiento/fisiología , Bovinos/fisiología , Hormona Folículo Estimulante/farmacología , Oocitos/fisiología , Animales , Técnicas de Cultivo de Embriones , Femenino , Estudios RetrospectivosRESUMEN
Some embryos exhibit better survival potential to cryopreservation than others. The cause of such a phenotype is still unclear and may be due to cell damage during cryopreservation, resulting from overaccumulation and composition of lipids. In cattle embryos, in vitro culture conditions have been shown to impact the number of lipid droplets within blastomeres. Thus far, the impact of breed on embryonic lipid content has not been studied. In the present study were compared the colour, lipid droplet abundance, lipid composition, mitochondrial activity and gene expression of in vivo-collected Jersey breed embryos, which are known to display poor performance post-freezing, with those of in vivo Holstein embryos, which have good cryotolerance. Even when housed and fed under the same conditions, Jersey embryos were found to be darker and contain more lipid droplets than Holstein embryos, and this was correlated with lower mitochondrial activity. Differential expression of genes associated with lipid metabolism and differences in lipid composition were found. These results show genetic background can impact embryonic lipid metabolism and storage.
RESUMEN
BACKGROUND: Genome-wide profiling of single-nucleotide polymorphisms is receiving increasing attention as a method of pre-implantation genetic diagnosis in humans and of commercial genotyping of pre-transfer embryos in cattle. However, the very small quantity of genomic DNA in biopsy material from early embryos poses daunting technical challenges. A reliable whole-genome amplification (WGA) procedure would greatly facilitate the procedure. RESULTS: Several PCR-based and non-PCR based WGA technologies, namely multiple displacement amplification, quasi-random primed library synthesis followed by PCR, ligation-mediated PCR, and single-primer isothermal amplification were tested in combination with different DNA extractions protocols for various quantities of genomic DNA inputs. The efficiency of each method was evaluated by comparing the genotypes obtained from 15 cultured cells (representative of an embryonic biopsy) to unamplified reference gDNA. The gDNA input, gDNA extraction method and amplification technology were all found to be critical for successful genome-wide genotyping. The selected WGA platform was then tested on embryo biopsies (n = 226), comparing their results to that of biopsies collected after birth. Although WGA inevitably leads to a random loss of information and to the introduction of erroneous genotypes, following genomic imputation the resulting genetic index of both sources of DNA were highly correlated (r = 0.99, P<0.001). CONCLUSION: It is possible to generate high-quality DNA in sufficient quantities for successful genome-wide genotyping starting from an early embryo biopsy. However, imputation from parental and population genotypes is a requirement for completing and correcting genotypic data. Judicious selection of the WGA platform, careful handling of the samples and genomic imputation together, make it possible to perform extremely reliable genomic evaluations for pre-transfer embryos.
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Bovinos/genética , ADN/análisis , Embrión de Mamíferos/citología , Técnicas de Genotipaje/métodos , Animales , Cruzamiento , Bovinos/embriología , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Genoma , Genómica/métodos , Polimorfismo de Nucleótido Simple , Reproducibilidad de los ResultadosRESUMEN
Mitochondria play an important role during early development in mammalian embryos. It has been shown that properly controlled follicular preparation increases the likelihood of in-vitro-produced bovine embryos reaching the blastocyst stage and that competent embryos exhibit heightened expression of genes associated with mitochondrial function. We hypothesized that apparently incompetent embryos could be rescued by restoring mitochondrial function. It has been shown that vitamin K2 (a membrane-bound electron carrier similar to ubiquinone) can restore mitochondrial dysfunction in eukaryotic cells. The aim of this study was therefore to investigate the effects of vitamin K2 on bovine embryonic development in vitro. The vitamin was found most effective when added 72âh after fertilization. It produced a significant (P<0.05) increase in the percentage of blastocysts (+8.6%), more expanded blastocysts (+7.8%), and embryos of better morphological quality. It improved the mitochondrial activity significantly and had a measurable impact on gene expression. This is the first demonstration that current standard conditions of in vitro production of bovine embryos may be inadequate due to the lack of support for mitochondrial function and may be improved significantly by supplementing the culture medium with vitamin K2.