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The objectives of this study were (1) to investigate the effects of the preovulatory follicle (POF) size on the accuracy of Doppler-based early pregnancy detection, and (2) to determine whether the removal of PGF2α (PGF) treatment during the resynchronisation protocol would affect fertility in beef cows. In Experiment 1, Nelore suckling cows (n = 224) were enrolled in an estradiol-progesterone-based timed artificial insemination (TAI) protocol. At TAI, cows were separated based on the range of POF diameters, as follows: ≤11.0 mm (n = 50), 11.1-12.9 mm (n = 64), 13.0-14.4 mm (n = 62) and ≥14.5 mm (n = 48). On day 22 after TAI, the corpus luteum (CL) blood flow (CLBF) of all cows was examined by colour Doppler ultrasonography to diagnose nonpregnant cows. The cows with the largest POF had the greatest positive predictive value (88.6%; 31 of 35) and diagnostic accuracy (91.7%; 44 of 48). In Experiment 2, Nelore cows (n = 233) were subjected to the same TAI protocol. Fourteen days after TAI, all cows were started on a resynchronisation protocol. Cows diagnosed as nonpregnant based on CLBF, on day 22, received 0.5 mg estradiol cypionate intramuscular (im) and were assigned to receive either 150 µg of PGF (PGF; n = 50) or 2 mL of saline (control; n = 47). Cows treated with PGF had a P/AI of 30.0% compared with a 48.9% P/AI in controls (p = 0.06). Our findings demonstrate that the POF size affects the accuracy of a CLBF-based early pregnancy diagnosis and that the removal of PGF treatment from the resynchronisation protocol tended to increase P/AI of the second TAI.
Assuntos
Corpo Lúteo , Dinoprosta , Estradiol , Sincronização do Estro , Inseminação Artificial , Folículo Ovariano , Progesterona , Animais , Feminino , Bovinos , Gravidez , Inseminação Artificial/veterinária , Sincronização do Estro/métodos , Progesterona/administração & dosagem , Progesterona/sangue , Progesterona/farmacologia , Estradiol/análogos & derivados , Estradiol/administração & dosagem , Estradiol/farmacologia , Corpo Lúteo/efeitos dos fármacos , Dinoprosta/administração & dosagem , Dinoprosta/farmacologia , Dinoprosta/análogos & derivados , FertilidadeRESUMO
This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus-oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro-fertilized or activated with ionomycin and cultured in vitro for 192 hr post-in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat-shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down-regulated the expression of AQP3 (p < .01) and up-regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down-regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat-shocked oocytes.
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Blastocisto/fisiologia , Bovinos/embriologia , Resposta ao Choque Térmico/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Partenogênese/fisiologia , Animais , Células do Cúmulo , Desenvolvimento Embrionário , Feminino , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Masculino , OócitosRESUMO
Brucellosis is an infectious and contagious disease that profoundly impacts public health. However, in many countries, disease prevention is restricted to the vaccination of calves, and there is no prophylactic strategy for pregnant heifers and cows. The aim of this study was to evaluate the safety of the rough strain vaccine against brucellosis in pregnant cattle. Crossbred cows (N = 96) at three gestational periods (early, mid, or late pregnancy) were randomly allocated into the vaccine treatment group or to the control group. We then compared the percentage of pregnancies reaching full term, live calves 60 days after delivery, and seropositive calves. There was no effect of vaccination in any of the gestational periods on the evaluation endpoints. In conclusion, vaccination against brucellosis with the rough strain is safe for pregnant cattle at all gestational periods.
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Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Brucelose Bovina/prevenção & controle , Vacinação/veterinária , Animais , Vacina contra Brucelose/efeitos adversos , Bovinos , Feminino , Parto , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controle , Complicações Infecciosas na Gravidez/veterinária , Vacinação/efeitos adversosRESUMO
The aim of the present study was to evaluate the effect of differences in body weight gain after embryo transfer on the pregnancy rates of crossbred heifers used as recipients and raised under a grazing system. The study was performed during the dry (April to September) and the rainy (October to March) seasons. The embryos transferred were produced by in vitro fertilization. The body weight of each recipient was measured immediately before the embryo transfer and 23 to 25 days later, when the diagnosis of pregnancy was performed by ultrasonography. The associations among initial body weight (IBW), daily body weight gain (DWG), season, and pregnancy rate were evaluated using a logistic procedure that included the effect of the IBW, season, and linear and quadratic effects of the DWG. Altogether, there was no effect of season and pregnancy rates did not change between the dry and rainy seasons (42.3 vs. 45.8%, respectively; P > 0.05). However, the pregnancy rate was greater in the recipients with daily body weight gains over 250 g/day, regardless of the season. In addition, the pregnancy rate of the recipients was better (P < 0.04) explained by a logistic regression model that included the linear and quadratic effects of the DWG. The probability of each heifer to become pregnant according to DWG is explained by the follow equation: P(y = 1) = (Exp((-1.06703 + 0.0108 * DWG - 0.00002 * DWG ^ 2)))/(1 + Exp((-1.6703 + 0.0108 * DWG - 0.00002 * DWG ^ 2))). In conclusion, body weight gain potential is a critical factor for the pregnancy rates of in vitro embryo recipients managed under grazing systems.
Assuntos
Bovinos/crescimento & desenvolvimento , Transferência Embrionária/veterinária , Taxa de Gravidez , Aumento de Peso , Aborto Animal , Ração Animal/análise , Criação de Animais Domésticos , Fenômenos Fisiológicos da Nutrição Animal , Animais , Peso Corporal , Bovinos/fisiologia , Dieta/veterinária , Feminino , Gravidez , Estações do AnoRESUMO
The aim of this study was to evaluate the superestimulatory and superovulatory responses of cattle treated with corifollitropin-alpha, a long-acting human recombinant FSH (rhFSH). In the first and second experiments, we used Nelore (Bos indicus) heifers previously submitted to follicular wave suppression by active immunization against GnRH. In Experiment 1 (a dose-response study), heifers (n = 20) were randomly allocated into five groups, which received placebo (saline) or a single sc dose of 7.5, 15.0, 22.5 or 30.0 µg rhFSH. The heifers were subjected to daily ovarian scan and blood sampling during 11 days. We observed group, time, and group x time effects (P<0.0001) for both average follicle size and circulating FSH concentrations, with a strong correlation (R = 0.82, P<0.0001) between the area under curve (AUC) for both parameters. The peak concentration of FSH 24h after treatment and average follicle size at all timepoints, however, were similar (P>0.05) between groups 22.5 and 30.0 µg. In Experiment 2, heifers (n = 18) were allocated into three groups, which received (0h) either placebo (control), 25 µg rhFSH or 130 mg pFSH (Folltropin). There was no difference (P>0.05) in average follicle size at any moment, as well as in intrafollicular E2 at 120h or in plasma P4 seven days later between groups rhFSH and pFSH. In Experiment 3, cycling Nelore heifers (n = 20) were subjected to a wave synchronization protocol and superovulated (day 0) using a standard pFSH protocol (120 mg split in eight decreasing im doses) or with a single sc injection of 20 µg rhFSH. The number of follicles >7 mm on day 4 did not differ (P=0.4370). Heifers receiving rhFSH had greater average follicle size on day 4 (P=0.0005), ovulation rate (P<0.0001), and number of CL (P=0.0155), as well as a trend towards a greater number of ova (P=0.07) and viable embryos (P=0.0590). In Experiment 4, superovulation was induced with a single sc injection of 25 µg rhFSH in Girolando and Nelore cows and heifers (n = 20). None of the embryo yield endpoints differed between the two breeds (P>0.05). In conclusion, cattle superstimulation and superovulation can be successfully induced with a single dose of a long-acting rhFSH (corifollitropin-alpha).
Assuntos
Relação Dose-Resposta a Droga , Hormônio Foliculoestimulante Humano , Ovário , Superovulação , Bovinos , Animais , Feminino , Superovulação/efeitos dos fármacos , Hormônio Foliculoestimulante Humano/administração & dosagem , Hormônio Foliculoestimulante Humano/farmacologia , Ovário/efeitos dos fármacos , Meia-Vida , Gravidez , Hormônio Foliculoestimulante/farmacologia , Hormônio Foliculoestimulante/administração & dosagem , Folículo Ovariano/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/administração & dosagem , HumanosRESUMO
With the development of in vitro technologies, embryos can be produced using oocytes retrieved directly from the ovaries, i.e., regardless of ovulation. This has allowed the use of different animal categories as oocyte donors, including prepubertal cattle. The advantages of using this strategy to shorten the generation interval and accelerate genetic gain over time were soon recognized, and the first offspring generated using oocytes collected from calves were born in the early 1990s. Nevertheless, embryo production from calves and prepubertal heifers remains a challenge. The oocytes collected before puberty present low in vitro developmental potential, and the subsequent blastocyst rates are consistently lower than those from pubertal females. The acquisition of developmental competence by the oocytes occurs progressively throughout the prepubertal period, which can be subdivided into an early, intermediate, and late prepubertal (or peripubertal) phases, each characterized by different physiological and endocrine features. Therefore, embryo yield increases with age but will only achieve its maximum after puberty. The most common strategy to improve oocyte developmental potential before puberty is the use of gonadotrophic stimulation prior to oocyte retrieval. The results with superstimulation, however, vary among studies, depending on the source, dose, and length of FSH treatment, as well as the age and breed of the donors. The use of calves and prepubertal heifers as oocyte donors should also consider the possible impacts of the oocyte retrieval technique (LOPU or OPU) and the use of exogenous hormones on their subsequent fertility and productive life.
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The aims of this study were to determine anti-müllerian hormone (AMH) cutoff values for selecting Gir (Bos taurus indicus) oocyte donors and estimate the impact of using AMH concentrations as a selection criterion. In Exp. 1, Gir heifers (n=120) were sampled for AMH analysis and submitted to ovum pick-up and in vitro embryo production (OPU-IVEP). AMH cutoff values were calculated using ROC analysis or, alternatively, by the successive exclusion of heifers with the lowest AMH values. The correlations between AMH and OPU-IVEP outcomes were significant (P<0.001), though low or moderate (r= 0.34-0.52). We estimated an improvement (P<0.05) after the use of AMH cutoff values to select donors of +15.3% for total oocyes, +19.4% for viable COC, and +23.4% for blastocysts. This selection pressure, however, led to the exclusion of 32.8%, 37.9%, and 50.0% of the initial potential donors, respectively. In Exp. 2, we analyzed data from OPU-IVEP sessions of 658 Gir donors with known genomic values for predicted transmitting ability for milk (GPTAm) and age at first calving (GPTAafc). The selection based on the number of oocytes recovered had no effect (P>0.05) on the average GPTAm nor GPTAafc values of the remaining donors. In summary, plasma AMH ≥700â¯pg/mL is a cutoff value that can be used to select Gir heifers with a greater potential as oocyte donors. Nevertheless, this selection leads to the exclusion of up to 50% of potential donors. Finally, exclusion of poor responders had no effect on mean genomic estimates for milk production or age at first calving in the selected subset of donors.
Assuntos
Hormônio Antimülleriano , Fertilização in vitro , Animais , Hormônio Antimülleriano/sangue , Bovinos/sangue , Bovinos/fisiologia , Feminino , Fertilização in vitro/veterinária , Técnicas de Cultura Embrionária/veterinária , Oócitos/fisiologia , Doação de Oócitos/veterinária , Recuperação de Oócitos/veterinária , Recuperação de Oócitos/métodos , Transferência Embrionária/veterináriaRESUMO
A number of potentials uses of Doppler ultrasonography have been explored in the last decades, both as research tools in reproductive physiology investigations and for the reproductive management of farm animals. The objective of this review was to address some of the recent strategies developed in fixed-time reproductive programs and resynchronization of ovulation in cattle, based on the evaluation of corpus luteum function by color-Doppler ultrasound imaging. Recent studies in dairy and beef cattle pointed out to a high accuracy when Doppler ultrasonography is used to assess the functionality of the corpus luteum and identify non-pregnant females at 20-24 days after breeding. Therefore, super-early resynchronization programs starting in the second week after timed-artificial insemination or embryo transfer have been developed and are being implemented in commercial assisted reproduction programs; thus, anticipating conception with proven semen or genetically superior embryos. In addition, assessment of corpus luteum blood perfusion can be used for identifying high fertility embryo recipients in fixed-time embryo transfer programs.
Assuntos
Corpo Lúteo , Progesterona , Feminino , Bovinos , Animais , Corpo Lúteo/diagnóstico por imagem , Ovulação , Reprodução , Ultrassonografia/veterinária , Ultrassonografia Doppler/veterinária , Inseminação Artificial/veterinária , Sincronização do EstroRESUMO
The effects of seasonality on the reproduction of stallions vary based on the latitude. Although previous studies have shown the influence of seasonality in raw semen quality in south-eastern Brazil, data regarding the influence of seasonality in cooled and frozen stored semen in Brazil is limited. Therefore, in this study, we have analysed if seasonality influences the hormone production (i.e., cortisol and testosterone), spermatogenesis, and quality of fresh, cooled, and frozen semen of stallions in central Brazil, and established the season most suitable for semen cryopreservation in a latitude of 15°S. Ten stallions were followed-up for one year, which was divided into two seasons, namely, drought, and rainy. Fresh, cooled, and frozen-thawed semen samples were assessed using CASA and flow cytometry. Additionally, the temperature and humidity index (THI) was calculated to determine the thermal stress. Although the THI varied between the two seasons, no thermal stress was observed throughout the year, nor were there differences in the physiological parameters of the stallions or plasma cortisol or testosterone levels. Furthermore, differences were not detected in total and progressive motility, sperm capacitation, and sperm membrane integrity, as well as in the number of live sperm with intact acrosomes and high mitochondrial membrane potential, between the two seasons in the fresh and frozen-thawed semen. Our data suggest that semen can be effectively collected and cryopreserved throughout the year within central regions of Brazil.
Assuntos
Análise do Sêmen , Preservação do Sêmen , Masculino , Animais , Cavalos , Análise do Sêmen/veterinária , Sêmen/fisiologia , Hidrocortisona , Testosterona , Motilidade dos Espermatozoides , Espermatozoides/fisiologia , Criopreservação/veterinária , Preservação do Sêmen/veterináriaRESUMO
Introduction: The aim of this study was to evaluate potential effects of diflubenzuron on the production and quality of gametes, and on in vitro embryo production (IVEP) outcomes, in cattle. Methods: Two experiments were performed, the first to evaluate effects on semen, and the second on cumulus-oocyte complexes (COC) and on IVEP. Nelore (Bos taurus indicus) bulls (n = 14) or heifers (n = 16) were allocated into control (CG) or treatment (DIF) groups. All groups received a mineral mix supplement added (DIF) or not (CG) with diflubenzuron (30 mg/head/day), during 8 weeks. Animals were weighed and blood samples were collected throughout the experimental period. Every other week, bulls were subjected to semen collection and heifers to transvaginal ultrasound-guided follicle aspiration sessions. Semen underwent physical and morphological evaluation, and samples were stored for further computer-assisted sperm analysis. The COC recovered were evaluated according to morphology and those classified as viable were sent to an IVEP laboratory. Results: Diflubenzuron had no effect (P > 0.05) on average body weight or in any blood hematological or biochemical endpoints, regardless of gender. In experiment 1, there was no difference (P > 0.05) between DIF and CG groups for sperm concentration, morphology, or kinetics. In experiment 2, there was also no effect of diflubenzuron on the number of total, viable, or grade I oocytes, as well as on cleavage or blastocyst rates (P > 0.05). Discussion: In summary, the oral administration of diflubenzuron, within the recommended dose, has no short-term negative effects on sperm production and quality or on oocyte yield and developmental potential in vitro, in cattle.
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The purpose of this study was to characterize the reproductive physiology, oocyte competence, and chromatin compaction in Nelore calves in the early-prepubertal period (EPP) and the intermediate-prepubertal period (IPP). Calves aged 2-5 (EPP) and 8-11 months old (IPP) were assigned to Trial 1 (morpho-physiological-endocrine evaluations, n = 8) or Trial 2 (oocyte donors, n = 8) vs. the respective control groups of cows (n = 8, each). All morphological endpoints, except the antral follicle count, increased from the EPP to the IPP. The EPP LH-FSH plasma concentrations were similar to cows, whereas LH was lower and FSH was higher in the IPP than in cows. . Cows produced more Grade I (12.9% vs. 4.1% and 1.7%) and fewer Grade III COC (30.1% vs. 44.5% and 49.0%) than the EPP and IPP calves, respectively. The IPP calves' oocyte diameter was similar to those from cows but greater than those from EPP females (124.8 ± 8.5 and 126.0 ± 7.5 µm vs. 121.3 ± 7.5 µm, respectively). The expression of the chromatin compaction-related gene HDAC3 was downregulated in calves. The proportion of the blastocyst rate to the controls was lower in EPP than in IPP calves (43.7% vs. 78.7%, respectively). Progressive oocyte competence was found during the prepubertal period, which can help to decide whether to recover oocytes from calves.
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The aim of this study was to evaluate the effect of culture media and stage of development in the osmotic ability of in vitro-fertilized bovine embryos and the expression of aquaporin 3 (Aqp3) and Na/K ATPase isoform 1 (ATPAse1) genes in embryos (i) with different ability to undergo rehydration and (ii) following vitrification. In experiment 1, in vitro fertilized presumptive zygotes were co-cultured in SOFaac or modified CR2aa medium and embryos at blastocyst and expanded blastocyst stages at day 7 post-insemination were exposed to NaCl hypertonic medium (900 mOsm) for 5 min following 120 min of culture in isotonic medium in order to evaluate dehydration and rehydration, respectively. No difference (P>0.05) on blastocyst rate was found between CR2aa and SOFaac medium but embryos co-cultured in SOFaac medium underwent greater (P<0.05) dehydration. Embryos at expanded blastocyst stage underwent greater dehydration but slower rehydration than embryos at blastocysts stage (P<0.05). In the experiment 2, the amount of Aqp3 and ATPase1 transcripts were quantified in blastocysts with high or low rehydration after exposure to hypertonic medium. No difference (P>0.05) on relative amount of transcripts was found in either genes. In the experiment 3, expanded blastocysts produced in a co-culture system were vitrified, warmed and then cultured for 72 h for analysis of embryo survival and amount of Aqp3 and ATPase1 transcripts. Lower (P<0.05) embryo survival rate was found for vitrified-warmed embryos (57.9%) than for their fresh counterparts (84.6%). There was no difference on expression of ATPase1 gene but lower (P<0.01) amount of Aqp3 transcripts was found in the vitrified-warmed embryos. In conclusion, embryo ability to undergo shrinkage and swelling is influenced by medium used in a co-culture system and by embryo stage. Rehydrating ability of embryos after exposure to NaCl hypertonic medium is not associated with variations on expression of Aqp3 and ATPase1 genes, but the vitrification can alter gene expression of in vitro-fertilized bovine embryos produced in a co-culture system.
Assuntos
Blastocisto/fisiologia , Criopreservação , Meios de Cultura/farmacologia , Técnicas de Cultura Embrionária/veterinária , Fertilização in vitro , Técnicas de Maturação in Vitro de Oócitos , Vitrificação , Animais , Aquaporina 3/genética , Aquaporina 3/metabolismo , Biomarcadores/metabolismo , Bovinos , Criopreservação/métodos , Criopreservação/veterinária , Células do Cúmulo/fisiologia , Dessecação , Desenvolvimento Embrionário , Feminino , Fertilização in vitro/métodos , Fertilização in vitro/veterinária , Expressão Gênica , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Oócitos/fisiologia , Osmose , Salinidade , Sêmen/fisiologia , ATPase Trocadora de Sódio-Potássio/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Zigoto/fisiologiaRESUMO
The aim of this study was to evaluate the outcomes of an early resynchronization protocol (Resynch) initiated at different timepoints after timed artificial insemination (TAI) and with unknown pregnancy status. Holstein cows (n = 164) were submitted to the following TAI protocol: D0, insertion of an intravaginal progesterone (P4) device and 2 mg im estradiol benzoate (EB); D8, removal of P4 device and treatment with 0.5 mg im sodium cloprostenol (PGF); D9, 0.1 mg im Lecirelin (LEC); and D10, TAI1. Cows were then randomly assigned to Resynch protocols starting either on day 20 (Resynch20D, n = 82) or 25 after TAI1 (Resynch25D, n = 82) with the insertion of a new P4 device and EB treatment. In both groups, P4 device was removed on day 8 after the beginning of Resynch, the same day of pregnancy diagnosis by ultrasonography. In pregnant cows there was no further action. Non-pregnant cows were treated with 0.5 mg im PGF, had a blood sample collected for serum P4 analysis and we measured and recorded the size of the largest follicle and the presence or absence of a corpus luteum (CL). One day later, cows were treated with 0.1 mg im LEC and TAI2 occurred 12-14 h later. The diameter of the largest follicle and serum P4 were compared between groups by ANOVA for the main effects of treatment, presence of a CL, and their interaction, whereas pregnancy per artificial insemination (P/AI) and the percentage of cows with a CL on the day of ultrasonography were analyzed using the Chi-square test. Follicle diameter on day 8 of Resynch was greater for cows in the Resynch20D group compared with Resynch25D (15.9 ± 3.9 vs 12.2 ± 2.5 mm, respectively; P = 0.046). The Resynch25D group had a greater percentage of cows with a CL (51.9 vs 18.9%, respectively; P = 0.0008) and higher serum P4 (2.8 ± 1.1 vs 1.7 ± 0.8 ng/mL; P = 0.041) at the end of the protocol compared with Resynch20D. P/AI at TAI1 was 35.4 and 36.6% (P > 0.10) for cows enrolled in Resynch20D and Resynch25D groups, respectively. P/AI to TAI2, after Resynch protocols, was greater in Resynch25D than Resynch20D (44.2 vs 22.6%, respectively; P < 0.05). In conclusion, starting an early resynchronization protocol 25 days after TAI increases P/AI compared with starting 20 days after TAI, and this was associated with a presumed greater proportion of cows with a functional CL at the moment of P4 device removal.
Assuntos
Sincronização do Estro , Resultado da Gravidez , Animais , Bovinos , Corpo Lúteo , Estradiol , Feminino , Hormônio Liberador de Gonadotropina , Inseminação Artificial/veterinária , Lactação , Gravidez , ProgesteronaRESUMO
The aim of this study was to evaluate the effect of active immunization against GnRH in Nelore (Bos indicus) cows repeatedly used as oocyte donors that developed chronic cystic ovarian disease (COD). In the first experiment, ovarian and uterine features were first compared between COD cows (n = 15) and healthy cows (n = 22, cycling control group) from the same breed and herd. Cows with COD had a greater number of large (P < 0.0001) and medium follicles (P < 0.01) but lesser small follicles (P < 0.05) than cycling controls. Mucometra was diagnosed in 73.3% of COD cows, but in none of the controls. No difference in average thickness of the endometrium was detected between groups; however, endometrial thickness and mucometra score were negatively correlated (R = -0.73, P = 0.0029) in COD cows. In the second experiment, COD cows were randomly allocated into two experimental groups, which received two 1.0 mL SC injections of either an anti-GnRH vaccine (COD immunized group, n = 8) or saline (COD control group, n = 7), given 28 days apart. Cows were examined weekly by transrectal ultrasonography during nine consecutive weeks after the first injection to evaluate the number and distribution of follicles among size classes, endometrial thickness, and presence of clinical mucometra. Vaccination against GnRH resulted in a progressive suppression of follicle growth and a reduction in the average size of the largest follicle, as well as in the number of large follicles (P < 0.01) in COD immunized cows compared with COD controls from week 7 onwards. Conversely, the number of small follicles in the COD immunized group increased after week 5 and was greater (P = 0.0023) than controls on week 9. Endometrial thickness and mucometra score were not affected (P > 0.05) by immunization against GnRH. In the third experiment, the COD immunized cows with effective suppression of follicle growth four weeks after the 2nd injection (n = 6) were submitted to three consecutive ovum pick-up (OPU) sessions (weeks 10, 11, and 12) for in vitro embryo production (IVEP). Cumulus-oocyte complexes (COC) collected from slaughterhouse ovaries were used as controls for IVEP. COD cows with produced 25.0 ± 3.8 COC per OPU session with no apparent detrimental effect of anti-GnRH vaccine on oocyte developmental potential in vitro, i.e., we observed similar cleavage rate (P = 0.5914) and greater blastocyst rate (P = 0.0177) in immunized cows compared with COC from slaughterhouse controls. Finally, in the fourth experiment wave emergence and follicular dynamics after follicle ablation were compared between COD immunized cows with effective suppression of follicle growth and a subset (n = 6) of the cycling, control group. No follicles grew beyond 4 mm diameter after follicle ablation in the COD immunized group, whereas a normal follicular wave emergence occurred in cycling controls. Antral follicle count was similar between cycling controls and COD immunized groups at 24 h and 96 h post-follicle ablation (P > 0.05), but greater in cycling controls at 48 h and 72 h post-follicle ablation (P < 0.05). In summary, our results suggest that active immunization against GnRH is effective to induce the regression of follicular cysts as well as increase the number of small follicles growing on the ovaries, in oocyte donors diagnosed with chronic COD, with no apparent negative effect on oocyte developmental potential in vitro.
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Doenças dos Bovinos , Doenças Ovarianas , Animais , Bovinos , Doenças dos Bovinos/prevenção & controle , Feminino , Fertilização in vitro/veterinária , Hormônio Liberador de Gonadotropina , Oócitos , Doenças Ovarianas/veterinária , Vacinação/veterináriaRESUMO
The objective of this work was to evaluate the selection of immature bovine oocytes by brilliant cresyl blue dye (BCB) and expression of transcripts MATER and ZAR1. Cumulus-oocyte complexes (COCs) from slaughterhouse ovaries were exposed to BCB diluted in mDPBS and incubated for 60 min at 38.5 degrees C in humidified air. After exposure those COCs were distributed in two groups, according to their cytoplasm colour: BCB+ (coloured cytoplasm) or BCB- (colourless cytoplasm). The control group was submitted to in vitro maturation (IVM) immediately after morphological selection and holding control group COCs were exposed to mDPBS without BCB but in the same incubation conditions of BCB+ and BCB- group. The COCs of all groups were submitted to IVM, in vitro fertilization (IVF) and in vitro culture (IVC). Cleavage rate (72 h post-insemination) was similar between control (65.3%) and BCB+ (64.4%) groups, but greater than (p < 0.05) holding control (49.8%) and BCB- (51.3%) groups. Blastocyst rate (192 h post-insemination) was not different between BCB+ (18.5%) and control (16.3%) groups, but greater (p < 0.05) than BCB- (8.4%) group. No difference was found for blastocyst rate between holding control group (14.2%), control and BCB+ groups. The relative expression of MATER and ZAR1 genes was evaluated by real-time PCR in immature oocytes collected from the control, holding control, BCB+ and BCB- groups. Despite the relative expression of MATER in holding control, BCB+ and BCB- were down regulated in comparison to control group there was no statistical difference (p > 0.05) in the relative expression of MATER and ZAR1 transcripts among groups. The results indicate that the BCB dye detects immature oocyte populations with different developmental competence, although no improvement in in vitro embryo production using oocytes exposed or not to BCB was observed. Development competence of immature oocytes exposed to BCB does not seem to be associated with variations in the expression of MATER and ZAR1 transcripts.
Assuntos
Bovinos/crescimento & desenvolvimento , Corantes/química , Proteínas do Ovo/metabolismo , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Oxazinas/química , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Bovinos/embriologia , Bovinos/metabolismo , Corantes/metabolismo , Proteínas do Ovo/genética , Feminino , Oócitos/citologia , Oxazinas/metabolismoRESUMO
The chemokine receptor 2 (CCR2) was first described as a chemotactic factor involved in immune responses, but it also plays an essential function in several biological processes. The chemokine (C-C motif) ligand 2 (CCL2) binds to CCR2 triggering G protein-coupled receptor (GPCR) signaling in leukocytes, including activation of PI3K/Akt/mTOR, a key pathway that is also related to follicular activation and survival. However, the potential role of CCR2 in ovarian follicular physiology remain unexplored. Thus, we investigated the role of CCR2 on follicular growth during adult life and aging. Ovaries and oocytes were collected from wild type (WT) mice at 1.5 months old (mo), and CCR2 expression was observed predominantly in oocytes included in growing follicles, as well as after ovulation. Follicle populations were assessed in WT and CCR2-/- mice at 1.5 mo, and CCR2-/- mice had more primordial and less primary and secondary follicles, while there were no differences in antral follicle numbers. Pro-apoptotic genes Bax and Casp3 were downregulated, while anti-apoptotic Bcl2 was upregulated in CCR2-/- mice. To further characterize the role of CCR2 in ovarian aging, follicle populations were assessed in WT and CCR2-/- mice at 1.5, 2.5, 6, 10, and 12 mo. A larger ovarian follicular reserve at 1.5-6 mo was observed in CCR2-/- mice. Finally, CCR2-/- aged mice (6-12 mo) ovulated more oocytes than WT mice. Altogether, these data suggest that CCR2 plays an important role in the regulation of murine folliculogenesis, potentially affecting the reproductive lifespan.
Assuntos
Fertilidade/fisiologia , Atresia Folicular/fisiologia , Oogênese/fisiologia , Folículo Ovariano/crescimento & desenvolvimento , Receptores CCR2/deficiência , Animais , Apoptose/fisiologia , Caspase 3/metabolismo , Feminino , Camundongos , Camundongos Knockout , Modelos Animais , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores CCR2/genética , Fatores de TempoRESUMO
The potential of dams as oocyte donors can be a selection criterion for animal breeding programs, but also an involuntary driver of the process. In both cases, it is important to determine genetic components influencing the outcome of in vitro embryo production (IVEP). The objective of the present study was to perform a detailed genetic analysis for in vitro embryo production traits in Dairy Gir cows. A dataset containing 11,450 records of ovum pick-up (OPU) and in vitro fertilization (IVF) procedures from 2684 Dairy Gir donors was evaluated. Analyzed traits were number (NOV) and percentage (POV) of viable oocytes; number (NGI) and percentage (PGI) of grade I oocytes; number (NEMB) and percentage (PEMB) of viable embryos. All analyzes were performed using animal models by a Bayesian framework. Heritability estimates varied from 0.16 to 0.32 for count traits and from 0.01 to 0.06 for percentage traits. The proportion of the total variation represented by the additive genetic effect of sire (semen used in IVF) for NEMB and PEMB was 7% and 5% respectively. Associations between estimated breeding values from progeny tested bulls for IVEP traits, milk production, age at first calving and conformation traits were mainly low or close to zero. Results indicate that selection for IVEP traits is possible in Dairy Gir cattle and would not impair genetic progress for traits already considered as selection criteria. The NOV seems to be a promising target trait. However, a selection index could help to avoid the use of sires with negative genetic merit for percentage traits, minimizing possible deterioration in the long term.
Assuntos
Bovinos/genética , Técnicas de Cultura Embrionária/veterinária , Embrião de Mamíferos/fisiologia , Fertilidade/genética , Animais , Bovinos/fisiologia , Feminino , Variação Genética , Masculino , Oócitos/fisiologiaRESUMO
Ovarian tissue cryopreservation is emerging as a promising alternative for fertility preservation of cancer survivors. To date, more than a hundred couples have successfully had babies using this procedure, although it is still considered experimental and demands further investigation. In this work, we evaluated the effects of vitrification, warming and autotransplantation procedures on the morphology and gene expression of murine ovaries. Ovaries were removed from adult female C57BL6 mice (n = 15), vitrified, warmed and autotransplanted (vitrified group), additionally, ovaries were autotransplanted without vitrification (control group, n = 15). After twenty days, grafted ovaries were harvested and used for histological and ultrastructural analysis, germinal vesicle (GV) oocyte collection, RNA sequencing, and Transmission Electron Microscopy (TEM). All classes of follicles and GV were observed in both control and vitrified/warmed transplanted ovaries, and the numbers of primordial, antral and atretic follicles were not different (p > 0.05). Using RNA-seq, we detected 16,602 vs 13,527 expressed genes in vitrified and control ovaries, respectively; and 623 significantly dysregulated genes (fold change >1.5; 332 up-regulated and 291 down-regulated). Cellular membranes, cytoskeletons, and extracellular matrices were found as the main functions of the differentially expressed genes. Moreover, vitrified samples also presented ultrastructural alterations in the cytoskeleton, cell junctions, and endoplasmic reticulum. Taken together, this work showed for the first time that ovarian cells might trigger a compensatory gene regulation mechanism to maintain cellular structure and folliculogenesis progression after vitrification and autotransplantation.
Assuntos
Criopreservação/veterinária , Folículo Ovariano/fisiologia , Ovário/fisiologia , Preservação de Tecido/veterinária , Transcriptoma , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Preservação de Tecido/métodos , VitrificaçãoRESUMO
OBJECTIVE: In vitro maturation has been shown to influence gene expression in oocytes, but a common shortcoming in reports on the matter has been the use of different donors in each experimental group thus disregarding donor effects. This study aimed to investigate the abundance of mRNA in oocytes matured in vivo and in vitro obtained from the same group of donors. METHODS: A bovine model was used to assess the relative abundance of specific transcripts in in vitro-matured (IN VITRO-OPU) and in vivo-matured (IN VIVO-OPU) oocytes collected from the same donors by transvaginal ovum pick-up (OPU). Transcript abundance in oocytes from the IN VIVO-OPU group and oocytes matured in vitro but retrieved from different cows slaughtered at a commercial abattoir (IN VITRO-Abattoir group) was also compared. Total RNA was extracted from denuded oocytes and cDNA was produced via reverse transcription using an oligo(dT) primer for relative quantification of eight target transcripts by real-time PCR. RESULTS: Oocytes in the IN VITRO-OPU group had lower (p<0.05) abundance of peroxiredoxin 1 (Prdx1), heat shock protein 70.1 (Hsp70.1), growth and differentiation factor 9 (Gdf9), and maternal antigen that embryo requires (Mater) transcripts than the oocytes in the IN VIVO-OPU group, all obtained from the same pool of donor cows. Similar results were seen in the comparisons involving the IN VIVO-OPU and IN VITRO-Abattoir groups (p<0.05). CONCLUSION: In vitro maturation affected the abundance of polyadenylated transcripts in the oocyte cytoplasm when compared to in vivo maturation induced by exogenous hormones in oocytes collected from the same donor pool.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos , Transcriptoma/genética , Animais , Bovinos , Feminino , Oócitos/química , Oócitos/fisiologia , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Livestock production is of great importance for the economy of most South American countries, a region that accounts for 23.0% of the world cattle population (Food Agriculture Organization - FAO, 2017). Not surprisingly, the embryo industry is historically very active in this region, particularly in Argentina and Brazil. The field of bovine embryo transfer underwent a remarkable change in the past two decades in Brazil, mainly due to in vitro embryo production (IVEP). Total embryo production increased dramatically, along with constant changes in the main features of the embryo industry - from market niches to mass production, from beef to the dairy sector, from zebu to European breeds. Recently, IVEP has also emerged in other South American countries. This review summarizes and describes factors driving the changes in the Brazilian embryo industry and discusses some of the impacts upon other embryo-related technologies.