RESUMO
The objective of this study was to determine the effect of early lactation body condition (BC) loss in multiparous dairy cows on serum lipids and the effect of these changes on oocyte and cumulus cell transcriptomes. Body condition loss in dairy cattle after parturition is associated with reduced fertility and increased pregnancy loss. The complex interplay between BC, nutrition, dry matter intake, milk production, and time of calving has presented a barrier to understanding mechanisms leading to reduced fertility. We identified cows that lost BC (L group; n = 10) or maintained or gained BC (M/G group; n = 8) during the first 27 to 33 d in milk and investigated changes in serum fatty acids and oocyte and cumulus cell transcriptomes at 75 to 81 d in milk. The L group had increased serum levels of nonesterified fatty acids and mead acid, and reduced serum levels of petroselaidic acid and behenic acid. Transcriptome analyses revealed 38 differentially expressed genes (DEG) in oocytes and 71 DEG in cumulus cells of L (n = 3) compared with M/G group (n = 3). Network analysis connected serum fatty acid changes to downstream effects including reduced inflammatory response and mitochondrial membrane depolarization, increased production of reactive oxygen species, and functions related to fatty acid metabolism and cytoplasmic organization in oocytes. These effects were associated with predicted effects on signaling in oocytes through calcium, insulin, O-GlcNAcase (OGA), fibroblast growth factor receptor 4 (FGF4R), peroxisome proliferator activated receptor gamma coactivator 1 α (PPARGC1A), and phospholipase D2 (PLD2) pathways, with a connection to the cumulus cell via calcium signaling. These results connect BC loss following parturition to changes in serum lipid levels, and changes potentially affecting oocyte quality; thus, these results provide new insight into mechanism of reduced fertility.
Assuntos
Ácidos Graxos não Esterificados , Insulinas , Ácido 3-Hidroxibutírico , Animais , Cálcio/metabolismo , Bovinos , Células do Cúmulo/metabolismo , Dieta/veterinária , Ácidos Graxos/metabolismo , Feminino , Lactação/fisiologia , Leite/metabolismo , Oócitos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Período Pós-Parto/metabolismo , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , TranscriptomaRESUMO
Cloning cattle using somatic cell nuclear transfer (SCNT) is inefficient. Although the rate of development of SCNT embryos in vitro is similar to that of fertilized embryos, most fail to develop into healthy calves. In this study, we aimed to identify developmentally competent embryos according to blastocyst cell composition and perform transcriptome analysis of single embryos. Transgenic SCNT embryos expressing nuclear-localized HcRed gene at day 7 of development were imaged by confocal microscopy for cell counting and individually transferred to recipient heifers. Pregnancy rates were determined by ultrasonography. Embryos capable of establishing pregnancy by day 35 had an average of 117 ± 6 total cells, whereas embryos with an average of 128 ± 5 cells did not establish pregnancy (P < 0.05). A lesser average number of 41 ± 3 cells in the inner cell mass (ICM) also resulted in pregnancies (<0.05) than a greater number of 48 ± 2 cells in the ICM. Single embryos were then subjected to RNA sequencing for transcriptome analysis. Using weighted gene coexpression network analysis, we identified clusters of genes in which gene expression correlated with the number of total cells or ICM cells. Gene ontology analysis of these clusters revealed enriched biological processes in coenzyme metabolic process, intracellular signaling cascade, and glucose catabolic process, among others. We concluded that SCNT embryos with fewer total and ICM cell numbers resulted in greater pregnancy establishment rates and that these differences are reflected in the transcriptome of such embryos.
Assuntos
Desenvolvimento Embrionário , Transcriptoma , Gravidez , Animais , Bovinos , Feminino , Transcriptoma/genética , Desenvolvimento Embrionário/genética , Blastocisto , Técnicas de Transferência Nuclear/veterinária , Clonagem de Organismos/métodos , Contagem de CélulasRESUMO
Dairy cow infertility negatively affects profit of dairy production enterprises around the world, and enhancing conception rates of dairy cows is a critical management issue to resolve. It appears that conception rates of dairy cows are attenuated due to reduced progesterone concentrations in circulation during growth of the ovulatory follicle. It is not clear how reduced progesterone influences fertility, but data presented in this brief review suggest that it can be somewhat reversed through increasing concentrations of progesterone during the growth of the ovulatory follicle before luteolysis. Ovsynch protocols may be utilised to enhance progesterone concentrations through the induction of an accessory corpus luteum (CL) following the initial gonadotropin-releasing hormone (GnRH) treatment. Cows at Day 13 of the oestrous cycle with a 7-day-old accessory CL had ~50% more progesterone at the time of prostaglandin injection of Ovsynch compared with cows with only a Day 13 CL. Ovsynch can consistently induce an accessory CL following the initial GnRH treatment if cows are on Days 6 or 7 of the oestrous cycle at the time of treatment. Pre-synchrony strategies are critical to enhance the probability that cows will be on Days 6 or 7 at first GnRH treatement of Ovsynch.
Assuntos
Bovinos/fisiologia , Fertilidade/fisiologia , Lactação/fisiologia , Folículo Ovariano/fisiologia , Progesterona/sangue , Animais , Senescência Celular/fisiologia , Feminino , Hormônio Liberador de Gonadotropina/uso terapêutico , Infertilidade Feminina/fisiopatologia , Infertilidade Feminina/prevenção & controle , Luteólise/fisiologiaRESUMO
The objective of this study was to test the effect of low circulating concentrations of progesterone (P4) on pre-ovulatory follicle development in heifers as part of an overarching objective to develop a model to understand this phenomenon in dairy cattle without the confounding factors of lactation. Holstein heifers between 12 and 13 mo of age were pre-synchronized to ensure all heifers were on d 6 of the estrous cycle at the start of the Ovsynch program. Only heifers with CL regression and ovulation to the following pre-treatment strategy were used in the study: 0.5 mg cloprostenol (PGF2α), 2 d later, 0.1 mg GnRH, 6 d later, GnRH (G1; 1st GnRH of Ovsynch). Heifers (n = 159) responding to pre-treatment were randomly assigned to 4 groups and completed the Ovsynch program: high P4 control (HPC), low P4 control (LPC; PGF2α 24 h after G1), PG2 (PGF2α 24 and 48 h after G1) and PG3 (PGF2α 24, 48, and 96 h after G1). Only heifers that had ovulation to G1 remained in the study. Blood samples were collected in all heifers on d 7 (n = 157) and in a subset of heifers on d 1, 2, 3, 4 (n = 82) after G1 to measure serum P4. Pre-ovulatory follicle size at G1 (13.0 ± 0.1 mm; P = 0.53) and mean serum P4 24 h after G1 (d 1; 3.62 ± 0.11 ng/mL; P = 0.46) did not differ among treatments. HPC heifers had greater (P < 0.001) mean serum P4 compared to LPC, PG2 and PG3 on d 2, 3, 4, and 7. On d 2, 3 and 4, mean serum P4 of LPC, PG2 and PG3 heifers did not differ (P > 0.10). On d 7, LPC heifers had greater (P < 0.001) serum P4 compared to PG2 and PG3 heifers. Mean ± SEM serum P4 on d 7 after G1 was 8.43 ± 0.39, 2.55 ± 0.36, 1.58 ± 0.20, and 1.21 ± 0.15 ng/mL for HPC, LPC, PG2, and PG3, respectively. Percentage of heifers with P4 < 0.50 ng/mL on d 7 was greater (P < 0.05) for LPC, PG2 and PG3 (27, 32 and 26%, respectively) compared to HPC (0%). A greater (P < 0.05) proportion of heifers ovulated before G2 in the LPC, PG2 and PG3 than in the HPC. For heifers that ovulated after G2, low serum concentrations of P4 in LPC, PG2 and PG3 induced double ovulations in 6/97 heifers after the final GnRH of Ovsynch compared to 0/33 in HPC. In summary, PGF2α treatments during early CL development reduced circulating P4 concentrations 7 d after G1 compared with both HPC and LPC. However, it did not effectively control CL and follicle function to be utilized as a model to test high vs. low serum P4 on fertility parameters in Holstein heifers.
Assuntos
Sincronização do Estro , Progesterona , Animais , Bovinos , Corpo Lúteo , Dinoprosta/farmacologia , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Inseminação Artificial/veterinária , Lactação , OvulaçãoRESUMO
The overarching objective of this study was to develop an alternative strategy for first and greater services that will improve fertility in lactating dairy cows for dairy operations limited by labor or other logistical constraints that make it difficult to use Presynch-11, G6G, or Double-Ovsynch. Our overall hypothesis was that simplification of a Presynch program through the combination of PGF2α and GnRH on the same day (PG + G), 7 days before the first GnRH of Ovsynch, would allow for similar ovulation and luteolysis rate and pregnancies per AI (P/AI) compared with G6G. Lactating dairy cows 58 to 64 days in milk (first service; n = 114), and cows diagnosed not pregnant 39 days after previous AI (second + service; n = 122) were blocked by parity and service and randomly assigned to control or PG + G. Control cows received G6G (n = 116) that consisted of PGF2α, 2-day GnRH, 6-day GnRH, 7-day PGF2α, 56-hour GnRH, and 16-hour AI. Treated cows (PG + G; n = 121) received PGF2α and GnRH, 7-day GnRH, 7-day PGF2α, 56-hour GnRH, and 16-hour AI. All cows received a second PGF2α 24 hours after the PGF2α of Ovsynch. First service cows received AI between 76 and 82 days in milk and second + service received AI 57 days after previous AI. Pregnancies/AI (n = 230) were similar in controls compared with treated cows on Day 35 (57 vs. 50%; P = 0.27) and Day 49 (54 vs. 47%; P = 0.33), respectively. Percent of cows ovulating after GnRH of the presynchronization was greater (P = 0.002) for controls vs. treated (80 vs. 58%); however, ovulation after first GnRH of Ovsynch was similar (67 vs. 68%; P = 0.86). Serum concentrations of progesterone were similar (P = 0.78) at the time of first GnRH of Ovsynch for control and treated cows (2.22 vs. 2.14 ng/mL). However, serum progesterone at the time of PGF2α of Ovsynch was greater (P = 0.002) for control cows compared with treated cows (5.75 vs. 4.64 ng/mL). In summary, administering both PGF2α and GnRH on the same day, 7 days before the start of Ovsynch, appears to be a simple alternative that results in acceptable P/AI but potentially less progesterone during the growth of the ovulatory follicle.
Assuntos
Bovinos , Dinoprosta/administração & dosagem , Sincronização do Estro/métodos , Hormônio Liberador de Gonadotropina/administração & dosagem , Animais , Feminino , Fertilidade , Inseminação Artificial/veterinária , Lactação , Luteólise/efeitos dos fármacos , Ovulação , Gravidez , Progesterona/sangueRESUMO
Timed-AI after synchronization of ovulation has become one of the most used reproductive technologies developed during the past 40 years. Various adaptations of this technology are now extensively used worldwide, in the beef and dairy cattle industry. Our well-cited report, published in Theriogenology in 1995, presented a method termed Ovsynch, that used GnRH and PGF2α to perform synchronization of ovulation and timed AI in lactating dairy cows. This report introduced Ovsynch, more as a concept of induced ovulation, and demonstrated the ovarian dynamics during the protocol. Validation and improvements on this method were subsequently performed in numerous university studies and on commercial dairies, worldwide. This review will provide a brief historical background, some personal recollections, and certain modifications that have been made in synchronization of ovulation protocols. Each section emphasizes the physiology that underlies the most widely-used synchronization of ovulation protocols and key modifications and some practical application of these protocols on commercial operations. Finally, the effect of timed AI in the US dairy industry and in the Brazilian beef cattle industry are compared. Although numerous studies have been done using these protocols, there is still substantial need for research to improve the synchronization, efficacy, simplicity, and practical application of these protocols.
Assuntos
Bovinos/fisiologia , Sincronização do Estro/métodos , Inseminação Artificial/veterinária , Animais , Indústria de Laticínios , História do Século XX , História do Século XXI , Inseminação Artificial/história , Inseminação Artificial/métodos , Ovulação , Progesterona/uso terapêuticoRESUMO
The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are potential regulators of the focalized extracellular matrix degradation required for ovulation. The objectives of the present study were to determine localization and temporal regulation of TIMP-3 and TIMP-4 mRNA and protein in bovine preovulatory follicles. Ovaries containing preovulatory follicles were collected at 0, 12 and 20 h after GnRH injection for real-time PCR quantification of TIMP-3 and TIMP-4 mRNAs and immunohistochemical localization studies. Additional samples collected at 0, 6, 12, 18 and 24 h post GnRH injection were subjected to Western analysis to determine temporal changes in TIMP-3 and TIMP-4 proteins in the apex and base of preovulatory follicles. Results indicate the gonadotropin surge regulates TIMP-3 and TIMP-4 expression. TIMP-3 and TIMP-4 mRNAs increased within 12 h after GnRH injection. TIMP-3 protein was localized to granulosal and thecal layers of preovulatory follicles and adjacent ovarian stroma, whereas TIMP-4 immunoreactivity was localized to granulosal and thecal cells and ovarian blood vessels. Amounts of TIMP-3 and TIMP-4 proteins in the follicular apex peaked within 12 h post GnRH injection and subsequently declined by 24 h. However, amounts of TIMP-3 and TIMP-4 proteins in the base were not elevated after GnRH administration. Results demonstrate that mRNA and protein for both TIMP-3 and TIMP-4 are increased in bovine preovulatory follicles following the gonadotropin surge. Coordinate expression of TIMPs and MMPs may help regulate the extracellular matrix remodeling characteristic of the ovulatory process.
Assuntos
Fase Folicular/metabolismo , Folículo Ovariano/química , Inibidor Tecidual de Metaloproteinase-3/análise , Inibidores Teciduais de Metaloproteinases/análise , Animais , Western Blotting/métodos , Bovinos , Feminino , Hormônio Liberador de Gonadotropina/farmacologia , Imuno-Histoquímica/métodos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Inibidor Tecidual de Metaloproteinase-3/genética , Inibidor Tecidual de Metaloproteinase-3/metabolismo , Inibidores Teciduais de Metaloproteinases/genética , Inibidores Teciduais de Metaloproteinases/metabolismo , Inibidor Tecidual 4 de MetaloproteinaseRESUMO
The ovulatory process is characterized by focalized extracellular matrix degradation at the apex of preovulatory follicles. Many studies have implicated the matrix metalloproteinases (MMPs) as potential mediators of follicle rupture. Objectives of this study were to determine localization and effect of the gonadotropin surge on temporal expression of MMP-1 and MMP-13 in bovine preovulatory follicles. Samples were collected at 0, 6, 12, 18, 24, and 48 h (corpora lutea) after GnRH injection (n = 5-6 per time point) and amounts of MMP-1 and MMP-13 mRNA and protein determined using dot blot or semiquantitative RT-PCR and Western blot analyses. Samples were also collected at 0 and 20 h after GnRH injection for immunohistochemical localization of MMP-1 and MMP-13. Results indicate that follicular expression of MMP-1 and MMP-13 increased following the gonadotropin surge. Abundance of MMP-1 mRNA increased at 6, 12, and 48 h post-GnRH injection. Immunoreactive MMP-1 was localized to granulosal and thecal layers of preovulatory follicles. Amounts of MMP-1 protein increased in both the apex and the base of preovulatory follicles. Abundance of MMP-13 mRNA increased at 6, 24, and 48 h post GnRH injection. Amounts of MMP-13 protein also increased in the follicular apex and base. Immunoreactive MMP-13 was localized to granulosal and thecal layers of preovulatory follicles. Results indicate MMP-1 and MMP-13 are increased in bovine preovulatory follicles following the gonadotropin surge but do not support a requirement for differential up-regulation of MMP-1 and MMP-13 (follicular apex vs. base) for the preovulatory collagenolysis required for follicle rupture.
Assuntos
Colagenases/genética , Fase Folicular/fisiologia , Gonadotropinas/metabolismo , Folículo Ovariano/enzimologia , Animais , Bovinos , Colagenases/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica/fisiologia , Hormônio Liberador de Gonadotropina/farmacologia , Metaloproteinase 13 da Matriz , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
The matrix metalloproteinases (MMPs) have been implicated in the ovulatory process, but the specific roles of individual MMPs are unclear. This study examined the effect of the preovulatory gonadotropin surge on localization and regulation of MMP-2, MMP-14, and tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA and MMP-2 and TIMP-2 activity in bovine preovulatory follicles and new corpora lutea (CL). Ovaries containing ovulatory follicles or new CL were collected at approximately 0, 6, 12, 18, 24, and 48 h (CL) after a GnRH-induced gonadotropin surge. Messenger RNA for TIMP-2 and MMP-14 increased within 6 and 24 h of the gonadotropin surge, respectively, whereas MMP-2 mRNA was constitutively expressed. Activity for MMP-2 in follicular fluid and follicle homogenates was not changed, but follicular fluid TIMP-2 activity increased in response to the gonadotropin surge. Messenger RNA for MMP-2 was localized to the thecal layer of bovine preovulatory follicles, whereas MMP-14 mRNA was localized primarily to the thecal layer and adjacent ovarian stroma. Expression of MMP-14 was also observed in the granulosal layer after the gonadotropin surge. In contrast, TIMP-2 mRNA was localized predominantly to the granulosal layer with intense expression in the antral portion of the granulosal layer in response to the gonadotropin surge. These data support the hypothesis that increased expression of MMP-14 and TIMP-2 may help regulate follicle rupture and/or the ovulatory follicle-CL transition in cattle.
Assuntos
Bovinos/metabolismo , Hormônio Luteinizante/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloendopeptidases/genética , Ovulação/fisiologia , Inibidor Tecidual de Metaloproteinase-2/genética , Animais , Corpo Lúteo/metabolismo , Feminino , Expressão Gênica , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinases da Matriz Associadas à Membrana , Metaloendopeptidases/metabolismo , Folículo Ovariano/metabolismo , RNA Mensageiro/análise , Inibidor Tecidual de Metaloproteinase-2/metabolismoRESUMO
This study examined the effect of the preovulatory gonadotropin surge on the temporal and spatial regulation of tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and uPA receptor (uPAR) mRNA expression and tPA, uPA, and plasmin activity in bovine preovulatory follicles and new corpora lutea collected at approximately 0, 6, 12, 18, 24, and 48 h after a GnRH-induced gonadotropin surge. Messenger RNAs for tPA, uPA, and uPAR were increased in a temporally specific fashion within 24 h of the gonadotropin surge. Localization of tPA mRNA was primarily to the granulosal layer, whereas both uPA and uPAR mRNAs were detected in both the granulosal and thecal layers and adjacent ovarian stroma. Activity for tPA was increased in follicular fluid and the preovulatory follicle apex and base within 12 h after the gonadotropin surge. The increase in tPA activity in the follicle base was transient, whereas the increased activity in the apex was maintained through the 24 h time point. Activity for uPA increased in the follicle apex and base within 12 h of the gonadotropin surge and remained elevated. Plasmin activity in follicular fluid also increased within 12 h after the preovulatory gonadotropin surge and was greatest at 24 h. Our results indicate that mRNA expression and enzyme activity for both tPA and uPA are increased in a temporally and spatially specific manner in bovine preovulatory follicles after exposure to a gonadotropin surge. Increased plasminogen activator and plasmin activity may be a contributing factor in the mechanisms of follicular rupture in cattle.