Contribution of key elements of nutritional metabolism to the development of cystic ovarian disease in dairy cattle.

The alteration of signaling molecules involved in the general metabolism of animals can negatively influence reproduction. In dairy cattle, the development of follicular cysts and the subsequent appearance of ovarian cystic disease (COD) often lead to decreased reproductive efficiency in the herd. The objective of this review is to summarize the contribution of relevant metabolic and nutritional sensors to the development of COD in dairy cows. In particular, we focus on the study of alterations of the insulin signaling pathway, adiponectin, and other sensors and metabolites relevant to ovarian functionality, which may be related to the development of follicular persistence and follicular formation of cysts in dairy cattle. The results of these studies support the hypothesis that systemic factors could alter the local scenario in the follicle, generating an adverse microenvironment for the resumption of ovarian activity and possibly leading to the persistence of follicles and to the development and recurrence of COD.

A review on inflammation and angiogenesis as key mechanisms involved in the pathogenesis of bovine cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of reproductive failure in dairy cattle. The main aim of this review is to discuss some aspects related to inflammation and angiogenesis that seem to be involved in the development of follicular cysts in domestic animals, with special emphasis on the bovine species, in an attempt to elucidate the relationship between these two processes in the early stages of persistence and in the development of bovine COD. We describe the changes in the expression of cytokines and angiogenic factors that seem to generate disturbances in the intraovarian component underlying the aberrant persistence of follicular cysts. Results show that pro-inflammatory and anti-inflammatory cytokines behave as regulators of angiogenesis through direct and indirect effects, like overexpression of pro-angiogenic factors, particularly in bovine ovarian cells from follicular cysts and persistent follicles. We conclude that, in dairy cattle, an imbalance in the expression of cytokines and pro-angiogenic growth factors related to ovulation and the processes associated with it would contribute to follicular persistence and to the recurrent appearance of COD.

Association between phagocytic activity of monocytes and days to conception after parturition in dairy cows when considering the hormonal and metabolic milieu.

The nutritional conditions and immune status of dairy cows affect reproductive performance. This study was conducted with the aim to analyze the phagocytic activity (PA) and phagocytic capacity (PC) of circulating monocytes after the period of transition from pregnancy to lactation, to evaluate possible associations with duration of time period to conception following parturition. Results indicated PA was not associated with duration of time period to conception following parturition. In contrast, cows with a lesser PC conceived earlier (98 ± 9 days in milk, DIM) than those with a greater PC (168 ± 15 DIM). Based on these results, to analyze the association of the hormonal and metabolic milieu with the PA and PC, the animals were grouped considering the days to conception following parturition. In the group with the greater number of days to conception (>168 DIM), the PA was associated with concentrations of progesterone and beta-hydroxybutyrate (BHB) at 90 DIM and glucose at 120 DIM, whereas PC was associated with the concentrations of progesterone, cortisol and glucose at 90 DIM, non-esterified fatty acids (NEFA) at 120 DIM, 17ß-estradiol at 150 DIM, and 17ß-estradiol and BHB at 180 DIM. Overall, these results represent a new perspective related to the reproductive performance of dairy cows. The modifications of cellular functions may be useful for predicting the onset of health complications in dairy cows and to manage cows in ways that result in an enhanced fertility during the subsequent lactational period.

Contribution of the VEGF system to the follicular persistence associated with bovine cystic ovaries.

Cystic ovaries (CO) characterize a disorder frequently found in dairy cattle. However, despite the contributions by several researchers, the mechanism that leads to ovulatory failure has not yet been completely elucidated. Thus, the aim of this study was to examine the mRNA expression of bovine vascular endothelial growth factor (VEGFA)-164, VEGFA-164b and VEGF receptors (VEGFR1 and VEGFR2) by real-time PCR and protein expression by immunohistochemistry, immunofluorescence and Western blot in follicular fluid from dairy cows with spontaneous CO and in an experimental model of follicular persistence induced by prolonged treatment with progesterone. Results showed that both VEGFA isoforms and receptors were coexpressed in granulosa and theca interna cells and in follicular fluid of ovaries from all the groups evaluated. VEGFA-164, VEGFA-164b and VEGFR2 protein expression was higher in theca cells of persistent follicles from group P0 (expected time of ovulation) than in those from dominant follicles (as reference structure) from the control group (p < 0.05). Also, VEGFA-164 expression was higher in theca cells of cysts than in those of dominant follicles of the control group (p < 0.05). In follicular fluid, VEGFA-164 expression was higher in persistent follicles from group P5 (5 days of follicular persistence) than in the control, P0 and P15 groups, and higher in cysts than in dominant follicles from the control group (p < 0.05). This study provides evidence of an altered expression of VEGFA-164, VEGFA-164b and VEGFR2 during the formation of persistent follicles and cysts in cows. Together, these results evidence that early development of CO in cows is concurrent with an altered expression of these growth factors and that these alterations may contribute to the follicular persistence, angiogenic dysregulation and ovulatory failure found in cows with follicular cysts.

Immune status during postpartum, peri-implantation and early pregnancy in cattle: An updated view.

Throughout the estrous cycle the mammalian endometrium undergoes morphological and functional changes that are essential for the establishment of pregnancy and proper ovarian and uterine functions. Among these changes, the most important are alterations in both inter- and intracellular signalling molecules, many of which modulate immune processes. In the endometrial tissue there are local innate (nonspecific) and adaptive (specific/acquired) response mechanisms which vary because of the endocrine status during the estrous cycle, pregnancy and postpartum period. Endometrial cells have responses that support the immune system by producing pro-inflammatory factors such as cytokines, sensors, effector molecules and chemokines. This response is important during gestation, pregnancy, and fetal growth, as well as in preventing infection, and immuno-rejection of the semi-allogeneic embryo. In dairy cows, both before and immediately after calving, there are marked changes in the values for hormonal and metabolic variables and the immune status is impaired. Thus, in several studies there has been assessment of the physiological and/or abnormal maternal immune changes and possible effects on dairy cow reproductive performance. The objective with this review is to summarize the novel information about the immune mechanisms involved during the postpartum period, subsequent peri-implantation period and pregnancy in dairy cows, and the possible effects on reproductive performance. This information provides for an enhanced understanding of the local and systemic immune responses associated with the metabolic and hormonal status of dairy cows, and alterations in the immune system of high producing cows and the possible effects on subsequent fertility.

Protein and gene expression of relevant enzymes and nuclear receptor of hepatic lipid metabolism in grazing dairy cattle during the transition period.

We aimed to study the protein and gene expression of some hepatic enzymes of lipid metabolism along with plasma biomarkers in grazing dairy cattle during the transition period. Blood and liver biopsies from a group of eight multiparous cows were sampled at -28, -14, +4, +14, +28 and +56 days relative to parturition. Peak concentrations of NEFA and beta-hydroxybutyric acid with high triacylglycerol content in the liver were recorded on day 4 postpartum. Consistent with blood biomarkers, the gene expression of carnitine palmitoyltransferase 1A (CPT1A) and acyl-CoA oxidase 1 (ACOX1) increased, whereas that of diacylglycerol O-acyltransferase 1 (DGAT1) decreased. Nevertheless, CPT1A protein expression did not change during all the period evaluated and ACOX1 protein expression increased on day 56 postpartum. In addition, the protein expression of peroxisome proliferator-activated receptor alpha (PPAR-alpha) increased on day 28 postpartum. On the other hand, DGAT1 protein expression decreased on day 14 postpartum. As expected, the expression of genes associated with fatty acid oxidation increased on the first days postpartum but, notably, protein expression was highest after transition. Since most infectious diseases and metabolic disorders in dairy cattle occur particularly on the first days postpartum, it is not so clear whether an increase in the oxidation capacity of the liver at that time could help to prevent disease and improve dairy production. The valuable results about protein expression of enzymes involved in liver lipid metabolism could help to better characterize the metabolism of dairy cattle during the transition period.

Alterations in key metabolic sensors involved in bovine cystic ovarian disease.

High-producing dairy cows frequently suffer metabolic alterations that cause different diseases, which could decrease the reproductive efficiency of the herd. Among these reproductive disorders, cystic ovarian disease (COD) has been related to alterations in metabolites and hormonal factors such as insulin, adiponectin and leptin. The aim of this study was to determine the protein expression of adiponectin and some of its downstream targets in ovarian follicles of control cows and cows with clinical diagnosis of COD. We also analyzed some key metabolic sensors in plasma and follicular fluid from both groups. In follicular cysts, we detected higher protein expression of adiponectin receptor 2 (AdipoR2), 5' adenosine monophosphate-activated protein kinase (AMPK), carnitine palmitoyl transferase 1 (CPT1) and acyl-coenzyme A oxidase 1 (ACOX1) relative to control antral follicles (p < 0.05). This was related to higher plasma adiponectin concentration in cows with COD than in control cows (p < 0.05). On the other hand, insulin concentrations showed an opposite pattern (p < 0.05). Furthermore, we found alterations in local and systemic concentrations of several metabolites. In this regard, in follicular fluid of cystic cows, the concentrations of non-esterified fatty acids and beta-hydroxybutyrate were higher (p < 0.05), whereas the concentrations of glucose and triacylglycerol were lower than in follicular fluid from control cows (p < 0.05). Besides, in both follicular fluid and plasma of cows with COD, the concentration of cholesterol was higher than in control animals (p < 0.05). These results evidence a local altered scenario of some metabolic sensors in cystic follicles, which could generate an adverse microenvironment for the resumption of ovarian activity, possibly causing the persistence of follicles and the recurrence of COD.

Impaired insulin signaling pathways affect ovarian steroidogenesis in cows with COD.

Cystic ovarian disease (COD) represents an important cause of infertility in dairy cattle and is associated with multiple physiological disorders. Steroidogenesis, which is necessary to ensure normal ovarian functions, involves multiple enzymatic pathways coordinated by insulin and other proteins. We have previously shown that cows with COD have an altered insulin response. Therefore, in the present study, we evaluated further alterations in intermediates downstream of the PI3K pathway and pathways mediated by ERK as critical signals for the expression of steroidogenic enzymes in the ovaries of control cows and cows with spontaneous COD. To this end, we evaluated the gene and protein expression of pan-AKT, mTOR, ERK1/2, and steroidogenic enzymes by real-time PCR and immunohistochemistry. Steroid hormone concentrations were assessed at systemic and intrafollicular level. Results showed altered expression of intermediate molecules of the insulin signaling pathway, whose action might modify the synthetic pathway of steroidogenic hormones. Similarly, the expression of steroidogenic enzymes and the concentration of progesterone in serum and follicular fluid were altered. These alterations support the hypothesis that systemic factors contribute to the development and/or maintenance of COD, and that metabolic hormones within follicles such as insulin exert determinant effects on ovarian functionality in cows with COD.

Altered Expression of Anti-Müllerian Hormone during the Early Stage of Bovine Persistent Ovarian Follicles.

Anti-Müllerian hormone (AMH) is a homodimeric glycoprotein expressed exclusively in the gonads. This hormone is an important regulator of the early growth of follicles through inhibitory effects on the recruitment of primordial follicles into the pool of growing follicles and on granulosa cell proliferation. Cystic ovarian disease (COD) is an important disorder affecting the fertility of dairy cattle. In the present study, we evaluated the expression of AMH in granulosa cells and AMH secretion into follicular fluid in pre-ovulatory follicles from control cows, animals with spontaneously arising COD and during the development of the disease, at 5, 10 and 15 days of follicular persistence. To this end, after an oestrous synchronization protocol, low doses of progesterone was administered for 5, 10 and 15 days after the expected day of ovulation (day 0 of follicular persistence) in treated cows (groups P5, P10 and P15, respectively), using an intravaginal progesterone-releasing device. Results showed a decrease in the expression of AMH in granulosa cells throughout folliculogenesis (P <0.05) and in the spontaneously arising follicular cysts and persistent follicles related to the control group (P <0.05). There was also a higher concentration of AMH in the follicular fluid of persistent follicles at 10 and 15 days of follicular persistence (P <0.05). Together, these results may indicate an alteration in AMH expression and secretion, which occurs early in folliculogenesis and incipiently during the development of COD, and which could contribute to the recurrence of this disease in cattle.

Role of Components of the Insulin-like Growth Factor System in the Early Stages of Ovarian Follicular Persistence in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been postulated that the insulin-like growth factor (IGF) system may contribute to follicular persistence and development of COD. The initiation of the IGF response is a result of interactions between IGF-binding proteins (IGFBPs) and IGFBP proteases, mainly pregnancy-associated plasma protein A (PAPP-A). IGFBPs bind IGFs with high affinity and consequently regulate their access to IGF receptors (IGFRs). The aim of this research was to determine variations in components of the IGF system in the ovaries of cows with persistent follicles induced by long-term administration of progesterone. Proteins of the IGF system were evaluated at 0 (expected day of ovulation), 5, 10 and 15 days of follicular persistence to determine whether the changes occur early in the development of COD. The concentrations of IGF1 and IGFBP4 in follicular fluid were similar in all groups with follicular persistence and in control antral follicles. IGFR1 and IGFBP4 expression in situ were higher in granulose cells in persistent follicles than in control follicles. No differences were found in PAPP-A concentration within follicular fluid in persistent follicles relative to control antral follicles. These data support the hypothesis that the IGF system is altered in the initial stages of development of follicular persistence and has a determinant role in ovarian function in cattle.

Altered expression of cytokines IL-1α, IL-6, IL-8 and TNF-α in bovine follicular persistence.

In dairy cattle, cystic ovarian disease (COD) is an important cause of subfertility, and two of the main signs are ovulation failure and follicular persistence. The aim of this study was to examine the expression of the cytokines IL-1α, IL-6, IL-8 and TNF-α in ovarian follicular structures at different times of persistence in a model of follicular persistence induced by prolonged treatment with progesterone in dairy cows. Protein expression of IL-1α, IL-6, IL-8 and TNF-α was evaluated by immunohistochemistry. Additionally, IL-6 concentration in follicular fluid and serum was determined by ELISA. IL-1α, IL-6, IL-8 and TNF-α expression was increased in follicles with different persistence times in relation to the control dominant follicles, in granulosa cells. For IL-6, IL-8 and TNF-α, this increase was detected early (P0: expected time of ovulation and/or P5: 5 days of follicular persistence). Additionally, theca cells showed an increase in IL-6 in antral (groups P10 and P15) and persistent follicles (group P10) related to dominant follicles from the control group (p < 0.05). Serum concentration of IL-6 was higher in groups P5, P10 and P15 than in control cows (p < 0.05). The results show evidence that early development of COD in cows is concurrent with altered expression of these cytokines in different ovarian follicular structures and may contribute to the follicular persistence and endocrine changes found in cattle with follicular cysts.

Detection and activity of 11 beta hydroxylase (CYP11B1) in the bovine ovary.

Glucocorticoids (GCs) such as cortisol and corticosterone are important steroid hormones with different functions in intermediate metabolism, development, cell differentiation, immune response and reproduction. In response to physiological and immunological stress, adrenocorticotropic hormone (ACTH) acts on the adrenal gland by stimulating the synthesis and secretion of GCs. However, there is increasing evidence that GCs may also be synthesized by extra-adrenal tissues. Here, we examined the gene and protein expression of the enzyme 11ß-hydroxylase P450c11 (CYP11B1), involved in the conversion of 11-deoxycortisol to cortisol, in the different components of the bovine ovary and determined the functionality of CYP11B1 in vitro CYP11B1 mRNA was expressed in granulosa and theca cells in small, medium and large antral ovarian follicles, and CYP11B1 protein was expressed in medium and large antral follicles. After stimulation by ACTH, we observed an increased secretion of cortisol by the wall of large antral follicles. We also observed a concentration-dependent decrease in the concentration of cortisol in response to metyrapone, an inhibitor of CYP11B1. This decrease was significant at 10-5 µM metyrapone. In conclusion, this study demonstrated for the first time the presence of CYP11B1 in the bovine ovary. This confirms that there could be a local synthesis of GCs in the bovine ovary and therefore a potential endocrine responder to stress through these hormones.

Involvement of PAPP-A and IGFR1 in Cystic Ovarian Disease in Cattle.

Cystic ovarian disease (COD) is one of the main causes of infertility in dairy cattle. It has been shown that intra-ovarian factors, such as members of the insulin-like growth factor (IGF) system, may contribute to follicular persistence. The bioavailability of IGF to initiate its response by binding to specific receptors (IGFRs) depends on interactions with related compounds, such as pregnancy-associated plasma protein A (PAPP-A). The aim of this study was to determine IGFR1 and PAPP-A expression both in follicles at different stages of development and in cysts, to evaluate the roles in the etiopathogenesis of COD in cattle. The mRNA expression of PAPP-A was higher in granulosa cells of large tertiary follicles than in cysts, whereas the protein PAPP-A present in the follicular fluid from these follicles showed no differences. Although no PAPP-A mRNA expression was detected in smaller tertiary follicles, in their follicular fluid, this protease was detected in lesser concentration than in cysts. The mRNA expression of IGFR1 was lower in granulosa cells from cystic follicles than in those from tertiary ones. However, the protein expression of this receptor presented the highest levels in cystic structures, probably to increase the possibility of IGF response. The data obtained would indicate that animals with COD have an altered regulation of the IGF system in the ovary, which could be involved in the pathogenesis of this disease in cattle.

Impaired insulin signaling pathway in ovarian follicles of cows with cystic ovarian disease.

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. Follicular cell steroidogenesis and proliferation in ovulatory follicles is stimulated by hormones such as insulin and its necessary post-receptor response. The aim of this study was to determine the expression of insulin receptor (IR), IR substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K), key intermediates in the insulin pathway, in control cows and cows with spontaneous COD and ACTH-induced COD. IR and IRS1 mRNA levels were greater in granulosa cells and lower in follicular cysts than in control tertiary follicles. PI3K mRNA levels were similar in all follicles evaluated, whereas the expression of IR, IRS1 and PI3K was similar in theca cells. Protein expression of IR was higher in control tertiary follicles than in the same structures in animals with COD and with cysts. IRS1 and PI3K protein expression showed the same pattern in tertiary and cystic follicles. However, the protein expression of subunit alpha p85 of PI3K was greater in theca cells from tertiary follicles than in cystic follicles. These results provide new insights into the insulin response in cows with COD. The lower gene and protein expressions of some insulin downstream effectors at an early stage of the signaling pathway could negatively influence the functionality of ovaries and contribute to follicle persistence.

Epidemiological description of cystic ovarian disease in argentine dairy herds: risk factors and effects on the reproductive performance of lactating cows.

To describe the epidemiology of cystic ovarian disease (COD), to find possible risk factors associated with the incidence of cysts and to analyse the impact of COD on the reproductive performance of dairy cows, databases from 22 dairy herds from the main dairy region in Argentina were retrospectively evaluated throughout a 3-year period (2009-2011). A total of 248 COD cases over 9156 parturitions were recorded, resulting in a cumulative incidence rate of 2.7%. Cystic ovarian disease incidence density was lower during the first 100 days post-partum (DPP) than during later stages of lactation. Seasonality had a significant influence on the disease presentation with higher incidence rates during winter and spring. Cows with a previous diagnosis of clinical mastitis showed 2.72 times more chances of developing ovarian cysts. Cystic cows had longer calving to first service and calving to conception intervals and lower conception rate than controls.