The cytokine context present in the reproductive tract of cows is closely involved in normal uterine functions, including the estrous cycle and the establishment and maintenance of pregnancy. However, the roles of some cytokines in the uterus, and their relation with reproductive performance remain to be elucidated. Thus, this study aimed to examine the protein expression of several cytokines such as TNFα, IL-6, IL-8, IFNγ, IL-4, and TGF-β3 in endometrial biopsies previous to conception, to evaluate the possible association with delayed conception in dairy cows. Protein expression levels were evaluated by immunohistochemistry. Results showed that the protein expression levels of TNFα, IL-6, IL-4 and TGF-β3 were not associated with the parturition-conception interval, whereas the high protein expression levels of IFNγ were associated with the parturition-conception interval. Finally, the low protein expression of IL-8 showed a statistical tendency to be associated with delayed conception. This is the first report about the protein expression of IFN-γ in the endometrium of dairy cows and also, this cytokine could enhance the favorable conditions to achieve an early pregnancy.

In dairy cows, the processes involved in the resolution of uterine inflammation during the postpartum are closely related to improved fertility during the subsequent lactation period. Little is known, however, about the role and distribution of endometrial immune cell populations during the pre-implantation period. This study was aimed to analyze the endometrial distribution of several mononuclear immune cells (T cells, γδ T cells, B cells and macrophages) in healthy dairy cows during the postpartum, beyond the transition period, looking for its possible association with the parturition-conception interval (PCI) and delayed conception. The quantification of immune cells was evaluated by immunohistochemistry (IHC), and the expression of hormone receptors in immune cells was evaluated by double IHC. Dairy cows were grouped according to their PCI: PCI shorter than or equal to 90 DIM (PCI≤90), PCI between 90 and 120 DIM (PCI90-120), and PCI greater than 150 DIM (PCI≥150). The distribution of endometrial mononuclear immune cells was analyzed by a Generalized Linear Model, and the association of the distribution of mononuclear immune cells with delayed conception was evaluated with a Kaplan-Meier test. The cows from the PCI90-120 group showed the highest number of endometrial macrophages, and a lower number of B cells than the PCI≤90 group. Results also showed an association between the lower number of B cells in the endometrium during the pre-implantation period and earlier conception. Also, the present findings indicates that ESR and PR are expressed in the endometrial MØ, T cells, γδ T cells and B cells.

Before ovulation, the ovary exhibits signs of local inflammation. However, the effects of adrenocorticotropin (ACTH) on the complexity of this inflammatory response are not yet well described. Thus, the aim of this study was to evaluate the effects of ACTH administered to dairy cows during the preovulatory period on the local distribution of different subsets of leukocytes infiltrated in the ovary, along with the gene expression of relevant chemokines (C-C motif chemokine ligand-2 (CCL2), C-X-C motif chemokine ligand-8 (CXCL8), CCL25 and CXCL1) involved in leukocyte chemotaxis and blood perfusion on the follicular wall of dominant follicles. Also, the direct effect of ACTH on chemokine gene expression was addressed in cultured antral follicular walls. For this purpose, both an in vivo and an in vitro experiment were performed. For the in vivo experiment, exogenous ACTH (100 IU) was administered intramuscularly to Holstein cows (n = 12) during proestrus every 12 h for four days before ovulation, when ovariectomy was performed (day 18). Daily ovarian Doppler ultrasonography was used to evaluate the percentage of irrigated area, the pulsatility index and the resistance index in the dominant follicles. The distribution of monocytes-macrophages (CD14), T- (CD2) and B-lymphocytes (CD79a) and granulocytes (CH138A) in the ovary was analyzed by immunohistochemistry. In follicular wall samples, gene expression of CCL2, CXCL8, CXCL1 and CCL25 was evaluated, whereas IL-17A expression was analyzed by Western blot. The total number of CD14, CD79a and CD2 infiltrated cells was lower in the ACTH-treated group than in the control group (p < 0.05). Chemokine gene expression showed lower mRNA of CCL2, CCL25 and CXCL1 (p < 0.05) in the ACTH-treated group. Meanwhile, IL-17A protein expression and hemodynamic parameters were similar between groups (p > 0.05). In the in vitro assay, antral follicular walls were stimulated with ACTH to corroborate the gene expression profile of chemokines. mRNA expression of CCL2 tended to be lower in the stimulated follicular walls (p = 0.092). Our results suggest that exogenous ACTH stimulus during the preovulatory period reduces the number of infiltrated leukocytes in the bovine ovary and this could be due to a lower chemotaxis capacity of the ovary.

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.

The cytokines of the interleukin-1 (IL-1) family are closely involved in the resolution of inflammation in cows with metritis and endometritis. However, little is known about the role of these cytokines beyond uterine regression in the absence of disease, especially around conception. Thus, the aim of this study was to examine the gene and protein expression of IL-1α, IL-1β, IL-1RI, IL-1RII and IL-1Ra in endometrial biopsies previous to conception, to evaluate the possible association of these cytokines with delayed conception in dairy cows. Gene and protein expression levels were evaluated by real-time PCR and immunohistochemistry, respectively. The gene expression levels of cytokines were not associated with the duration of the period to conception following parturition. However, high protein expression of IL-1β and low protein expression of IL-1Ra were significantly associated with early conception. These results suggest that an imbalanced protein expression of IL-1β and IL-1Ra in the endometrium of dairy cows could be part of the maternal immune response mechanism necessary to propitiate early conception and probably to maintain pregnancy.

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.

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, 17β-estradiol at 150 DIM, and 17β-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.

Cattle undergo numerous environmental and management stressors that reduce fertility and affect ovulation. The extracellular matrix of the follicle wall can be altered by matrix metalloproteinases (MMPs), the activities of which are regulated by interleukins and tissue-specific inhibitors of metalloproteinases (TIMPs), especially during ovulation. The aims of the present study were to: (1) evaluate changes in the hormone milieu, the localisation and activity of MMP2 and MMP9 and the localisation of MMP14, TIMP1 and TIMP2 in response to adrenocorticotrophic hormone (ACTH) during the preovulatory period in cows; and (2) determine the direct effects of ACTH on the mRNA expression of MMP2 and MMP9 in the cultured follicle wall of bovine ovaries obtained from an abattoir. 100IU ACTH was administered during pro-oestrus every 12h until ovariectomy, which was performed before ovulation. Cortisol concentrations in the plasma and follicular fluid (FF) of preovulatory follicles were higher in ACTH-treated than control cows. Progesterone presented subluteal concentrations in plasma of ACTH-treated cows (P<0.05). MMP2 immunostaining and activity in ovaries were higher in ACTH-treated than control cows (P<0.05), whereas MMP9 immunostaining was similar between the two groups. However, unlike in control cows, MMP9 activity was absent in the FF of ACTH-treated cows. These results suggest that the administration of ACTH during the preovulatory period in cows could cause changes that culminate in modifications in the content and activation of MMPs and TIMPs in the ovary, which could interfere with the ovulation process.

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.

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.

Cystic ovarian disease (COD) is an important cause of infertility in dairy cattle. The main signs of this infertility are ovulation failure and follicular persistence. The aim of this study was to examine the expression of the cytokines IL-1β, IL-1RI, IL-1RII, IL-1RA and IL-4 in ovarian follicular structures at different times of persistence in a model of follicular persistence induced by prolonged administration of progesterone in dairy cows. Protein expression of IL-1β, IL-1RI, IL-1RII, IL-1RA and IL-4 was evaluated by immunohistochemistry. Additionally, IL-1β and IL-4 concentrations in follicular fluid and serum were determined by ELISA. In granulosa cells, IL1-RII and IL-4 expression was higher in follicles with different persistence times than in the control dominant follicles. IL-1RA expression was higher in persistent follicles of the P15 group (15 days of follicular persistence) than in those of the control group. In theca cells, IL-1RII expression was higher in persistent follicles of the P0 group (expected time of ovulation) than in dominant follicles from the control group (p < .05) and the other persistence groups, whereas IL-4 expression was higher in persistent follicles of groups P0 and P15 than in the dominant follicles of the control group (p < .05). Differences between serum and follicular fluid within each group were detected only in P0 for IL-1β, and in the control, P10 and P15 groups for IL-4 (p < .05). These results complement previous results, evidencing that early development of COD in cows is concurrent with an altered expression of cytokines in different ovarian follicular structures and may contribute to the follicular persistence and ovulation failure found in cattle with follicular cysts.

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.

The most important regulators of tissue remodelling during ovarian follicular growth, development, ovulation and atresia are gonadotropins, steroid hormones, growth factors and different proteolytic enzymes. Matrix metalloproteinases (MMPs) such as collagenase or gelatinase (i.e. MMP-1, -8, -2 and -9) and associated tissue inhibitors of metalloproteinases (TIMP-1, -2, -3 and -4) control connective tissue remodelling during follicular rupture. In this study, we hypothesized that an imbalance in the MMP-TIMP system may be an intra-ovarian component that contributes to the pathogenesis of cystic ovarian disease (COD) in cows. Taking into account that the control of MMP activity by TIMPs could determine their effects in both physiological and pathological conditions, MMP and TIMP mRNA and protein expression was examined by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC), respectively, in ovaries from control cows and cows with COD. Expression of mRNA encoding MMP-2, TIMP-1 and TIMP-2 was lower in follicular cysts than in control pre-ovulatory follicles, while the results by IHC showed this imbalance only for TIMP-2 protein expression. Additional analysis by zymography to evaluate the gelatinase activity of MMP-2 and MMP-9 demonstrated higher MMP-2 activity in follicular fluid (FF) of cysts than in FF of pre-ovulatory follicles. On the other hand, MMP-9 activity was increased in follicular cysts and absent in the FF of pre-ovulatory follicles. These findings suggest that the altered mRNA expression and activities of the MMP-TIMP system may be related to the failure in ovulation and follicular development observed in COD.

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.

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.