Effect of Parenteral Supplementation of Minerals and Vitamins on Oxidative Stress Biomarkers and Hepatic Fatty Acid Metabolism in Dairy Cows During the Transition Period.

In the present work we aimed to study the effects of parenteral vitamin and mineral supplementation on hepatic fatty acid metabolism as well as on the oxidative stress biomarkers in biological samples of transition cows. The supplemented group (SG, n = 11) received a subcutaneous injection of 5 mL of vitamin A palmitate 35 mg/mL, vitamin E acetate 50 mg/mL plus other injection of 5 mL of copper edetate 10 mg/mL, zinc edetate 40 mg/mL, manganese edetate 10 mg/mL, and sodium selenite 5 mg/mL on days - 60, - 30, and 7 (± 3) relative to calving. The control group (CG, n = 11) received two subcutaneous injections of 5 mL of 9 mg/mL sodium chloride at the same times of the SG. Blood, urine, and liver biopsies were sampled 21 (± 3) days before the expected calving date and 7 and 21 (± 3) days after calving. Results revealed that supplemented animals had higher glutation peroxidase (GSH-Px) activity, lower and higher concentration of 3-nitrotyrosine (3-NT) in the liver and plasma, respectively, higher expression of the mitochondrial beta-oxidation enzyme carnitine palmitoyltransferase 1 in the liver, and lower content of hepatic triacylglycerol, mirroring plasma liver function parameters. No differences between groups were found in the superoxide dismutase activity, MDA concentrations, the protein abundance of peroxisomal acyl-CoA oxidase 1, diacylglycerol O-acyltransferase 1, and peroxisome proliferator-activated receptor alpha. These results suggest that the vitamin and mineral supplementation provided to dairy cows had a beneficial effect on GSH-Px activity, hepatic 3-NT concentration, and on the metabolic adaptation during the peripartum period.

Antioxidant Interactions between Citrus Fruit Carotenoids and Ascorbic Acid in New Models of Animal Cell Membranes.

The regular consumption of citrus fruits by humans has been associated with lower incidence of chronic-degenerative diseases, especially those mediated by free radicals. Most of the health-promoting properties of citrus fruits derive from their antioxidant content of carotenoids and ascorbic acid (ASC). In the current work we have investigated the scavenging (against hydroxyl radical) and quenching capacities (against singlet oxygen) of four different carotenoid extracts of citrus fruits in the presence or absence of ASC (µM range) in organic solvent, aqueous solution, micelles and in an innovative biomimicking liposomal system of animal cell membrane (AML). The fruits of four varieties of citrus were selected for their distinctive carotenoid composition (liquid chromatography characterization): 'Nadorcott' mandarin and the sweet oranges 'Valencia late', 'Ruby Valencia' and 'Pinalate' mutant. The quenching activity of citrus carotenoids strongly depended on the biological assemblage: freely diffusible in organic solvent, 'Ruby Valencia' carotenoids (containing lycopene) showed the highest quenching activity, whereas 'Nadorcott' mandarin extracts, rich in ß-cryptoxanthin, prevailed in micellar systems. Interestingly, the addition of 10 µM ASC significantly increased the quenching activity of all citrus extracts in micelles: 'Valencia' orange (+53%), 'Pinalate' (+87%), 'Ruby' (4-fold higher) and 'Nadorcott' mandarins (+20%). Accurate C11-BODIPY581/591 fluorescence assays showed solid scavenging activities of all citrus extracts against AML oxidation: 'Valencia' (-61%), 'Pinalate' (-58%) and 'Ruby' oranges (-29%), and 'Nadorcott' mandarins (-70%). Indeed, all four citrus extracts tested here have balanced antioxidant properties; extracts from the 'Nadorcott' mandarin slightly prevailed overall, due, at least in part, to its high content of ß-cryptoxanthin. This study depicts some of the antioxidant interactions between citrus fruit carotenoids and ascorbic acid in models of animal cell membranes and reinforces the contribution of them in promoting health benefits for humans.

Estrogens receptors, nuclear coactivator 1 and ligand-dependent corepressor expression are altered early during induced ovarian follicular persistence in dairy cattle.

Failure of ovulation can lead to follicular persistence, one of the main components of the pathogenesis of cystic ovarian disease (COD) in dairy cattle. Follicular persistence causes the permanence of a functional follicular structure in the ovary, which alters the cyclicity of the female and causes infertility. The aim of the present study was to evaluate the expression of estrogen receptors (ESR) 1 and 2, and the coregulatory proteins NCOA1, NRIP1 and LCOR by immunohistochemistry, in antral and preovulatory/persistent follicles in a model of follicular persistence induced by low levels of progesterone, to detect incipient changes during COD development, on the expected day of ovulation (P0) and after 5 (P5), 10 (P10) and 15 (P15) days of follicular persistence. Twenty-five Holstein cows were used, which were distributed in 5 groups: control group (n = 5), group P0 (n = 5), group P5 (n = 5), group P10 (n = 5), group P15 (n = 5). ESR1 expression was lower in antral follicles of the P5 (theca), P10 and P15 (theca and granulosa) groups relative to the control group (p < 0.05), and also lower in granulosa cells of persistent follicles of the P5, P10 and P15 groups than in dominant follicles of the control group (p < 0.05), without differences in theca cells. ESR2 expression showed no differences between groups. The ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as from 5 days of persistence (p < 0.05). NCOA1 expression was higher in granulosa cells of both antral and persistent follicles from the P0 group relative to the P5 and P10 groups, but showed no differences with the control and P15 groups (p < 0.05). Theca cells of antral and persistent follicles showed higher expression in the P0 and P15 groups in relation to the control, P5 and P10 groups (p < 0.05). No differences were detected for NRIP1 in antral, dominant and persistent follicles between groups. LCOR expression showed a decrease in granulosa cells of antral follicles from all persistence groups relative to the control group (p < 0.05). In theca cells, antral follicles of the P10 group showed lower LCOR expression than the control group (p < 0.05). LCOR expression was similar for dominant and persistent follicles. Considering that the ESR1:ESR2 balance favored ESR2 expression along the development of persistent follicles, as well as the decreased LCOR and NCOA1 expression, we may assume that, at the early stages of persistence, there is a negative regulation of ESR transcription. This coincides with the effects of estrogens through ESR on proliferation and apoptosis among other processes that favor follicular persistence. The results obtained provide relevant information in the knowledge of local events during the development of follicular persistence that could explain the failures in the reversion of the disease through hormonal treatments and the high recurrence rates reported for COD. In addition, it contributes to the study and identification of possible therapeutic targets, for the design of new treatments.

Biomarkers of oxidative stress and liver function in early lactation and their relationship with the reproductive efficiency of multiparous grazing dairy cows in Argentina. A retrospective study.

This study aimed to analyze the possible relationship between days to conception and different oxidative stress (OS) biomarkers and liver functional parameters in multiparous dairy cows. Besides, a fast reliable method for the accurate measurement of malondialdehyde (MDA) by liquid chromatography-tandem mass spectrometry was developed in several matrices. During lactation, the days to conception of 28 cows were determined for a retrospective study. According to this parameter, cows were divided into two groups: high and low days to conception (HDC and LDC, respectively). Blood, urine and liver biopsies were sampled 21 days before the expected calving date, and 7 and 21 days after calving. The method developed for MDA was validated according to international requirements. The lower limit of quantification was 0.25 µmol/L for plasma and urine and 10.00 µmol/L for liver tissue. No differences between groups were observed in the systemic concentration of non-esterified fatty acids, ß-hydroxybutyric acid and liver triacylglycerol content (P > 0.05). Cholesterol concentration was higher in the LDC than in the HDC group (P < 0.05). Plasma 3-nitrotyrosine (3-NT) concentration was lower in the LDC than in the HDC group on day 21 post-calving (P < 0.05). Superoxide dismutase activity was higher in the LDC than in the HDC group (P < 0.05). Particularly, in the liver, 3-NT and MDA concentrations were lower in the LDC than in the HDC group (P < 0.05). These results allow inferring that the amelioration of OS biomarkers in plasma and liver could be related to a better reproductive performance of dairy cows.

Association between heat stress during intrauterine development and the expression and regulation of ovarian steroid hormone receptors in adult Holstein cows.

CONTEXT: Dairy cattle experience stressful environmental situations that affect production. Heat stress during gestation can influence the intrauterine development of offspring, resulting in long-term damage that can affect the reproductive life of the adult offspring. AIM: The aim of the present study was to evaluate changes in the expression and regulation of steroid hormone receptors in the ovary of Holstein cows gestated under different temperature-humidity index (THI) during their in utero development. METHODS: Animals were classified by their exposure to temperature-humidity index (THI) ≥72 during their development in utero according to date of birth or date of effective service of their mother. This study was not carried out under controlled conditions, but the conditions to which the cows were naturally exposed during their development were considered retrospectively, controlling the variables in the statistical analyses (age as a covariate, dairy farm as a random factor). Gestation was divided into two periods (P1=days 0-150; and P2=day 151 to calving) and three trimesters (T1=days 0-90; T2=days 91-180; and T3=day 181 to calving), and the exposure to THI ≥72 was calculated in each one. The following characteristics were evaluated: gene expression of estrogen receptor (ESR) 1, ESR2 and progesterone receptor (PGR), CpG methylation in the 5'UTR of ESR1 and ESR2, and protein expression of ESR1, ESR2, PGR and coregulatory proteins in the dominant follicles of daughter cows in adulthood. KEY RESULTS: We found associations between heat stress variables during gestation and the methylation status of CpG sites in the 5'UTR of ESR1 and ESR2 in dominant follicles. Results also showed association between exposure to high THI values during intrauterine development and expression of ESR1, ESR2 and PGR and coregulatory proteins in dominant follicles of adult cows. CONCLUSIONS: These results provide novel information about the impact of prenatal heat stress on molecular aspects at the ovary level in the offspring, during their adult life, which probably impacts the reproductive aspects of the herd.

An altered expression of components of the IGF system could contribute to follicular persistence in Holstein cows.

In dairy cows, reproductive diseases such as cystic ovarian disease (COD) represent a major problem that impacts on dairy production. It has been postulated that the insulin-like growth factor (IGF) system may contribute to follicular persistence and development of COD. Thus, the aim of the present study was to analyze relevant members of the IGF system in a critical period immediately after the expected time of ovulation, to obtain information about their role in follicular persistence in dairy cows. 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 previously detected in cows with COD occur early in COD pathogenesis. The serum concentration of IGF1 was higher in cows with 10 and 15 days of follicular persistence than in control cows. IGF1 expression in granulosa cells was similar in the follicles analyzed. In contrast, in theca cells, persistent follicles of days 5 and 10 showed the lowest IGF1 expression. IGF binding protein (IGFBP) 2 and 3 expression was lower in persistent follicles than in dominant follicles of the control group. Although IGF receptor (IGFR) 1 expression was similar in the groups analyzed, p-IGFR1 expression was significantly higher in dominant follicles of the control group than in persistent follicles. These data suggest alterations in the IGF system at the early stages of follicular persistence. The evidences obtained allow supporting that the IGF system could plays a key role in dairy cattle reproduction.

Ascorbic Acid Content and Transcriptional Profiling of Genes Involved in Its Metabolism during Development of Petals, Leaves, and Fruits of Orange (Citrus sinensis cv. Valencia Late).

Citrus fruit is one of the most important contributors to the ascorbic acid (AsA) intake in humans. Here, we report a comparative analysis of AsA content and transcriptional changes of genes related to its metabolism during development of petals, leaves and fruits of Valencia Late oranges (Citrus sinensis). Petals of close flowers and at anthesis contained the highest concentration of AsA. In fruits, AsA content in the flavedo reached a maximum at color break, whereas the pulp accumulated lower levels and experienced minor fluctuations during development. AsA levels in leaves were similar to those in the flavedo at breaker stage. The transcriptional profiling of AsA biosynthetic, degradation, and recycling genes revealed a complex and specific interplay of the different pathways for each tissue. The D-galacturonic acid pathway appeared to be relevant in petals, whereas in leaves the L-galactose pathway (GGP and GME) also contributed to AsA accumulation. In the flavedo, AsA content was positively correlated with the expression of GGP of the L-galactose pathway and negatively with DHAR1 gene of the recycling pathway. In the pulp, AsA appeared to be mainly controlled by the coordination among the D-galacturonic acid pathway and the MIOX and GalDH genes. Analysis of the promoters of AsA metabolism genes revealed a number of cis-acting elements related to developmental signals, but their functionalities remain to be investigated.

Regulation of Tocopherol Biosynthesis During Fruit Maturation of Different Citrus Species.

Tocopherols are plant-derived isoprenoids with vitamin E activity, which are involved in diverse physiological processes in plants. Although their biosynthesis has been extensively investigated in model plants, their synthesis in important fruit crops as Citrus has scarcely been studied. Therefore, the aim of this work was to initiate a physiological and molecular characterization of tocopherol synthesis and accumulation in Citrus fruits during maturation. For that purpose, we selected fruit of the four main commercial species: grapefruit (Citrus paradisi), lemon (Citrus limon), sweet orange (Citrus sinensis), and mandarin (Citrus clementina), and analyzed tocopherol content and the expression profile of 14 genes involved in tocopherol synthesis during fruit maturation in both the flavedo and pulp. The selected genes covered the pathways supplying the tocopherol precursors homogentisate (HGA) (TAT1 and HPPD) and phytyl pyrophosphate (PPP) (VTE5, VTE6, DXS1 and 2, GGPPS1 and 6, and GGDR) and the tocopherol-core pathway (VTE2, VTE3a, VTE3b, VTE1, and VTE4). Tocopherols accumulated mainly as α- and γ-tocopherol, and α-tocopherol was the predominant form in both tissues. Moreover, differences were detected between tissues, among maturation stages and genotypes. Contents were higher in the flavedo than in the pulp during maturation, and while they increased in the flavedo they decreased or were maintained in the pulp. Among genotypes, mature fruit of lemon accumulated the highest tocopherol content in both the flavedo and the pulp, whereas mandarin fruit accumulated the lowest concentrations, and grapefruit and orange had intermediate levels. Higher concentrations in the flavedo were associated with a higher expression of all the genes evaluated, and different genes are suitable candidates to explain the temporal changes in each tissue: (1) in the flavedo, the increase in tocopherols was concomitant with the up-regulation of TAT1 and VTE4, involved in the supply of HGA and the shift of γ- into α-tocopherol, respectively; and (2) in the pulp, changes paralleled the expression of VTE6, DXS2, and GGDR, which regulate PPP availability. Also, certain genes (i.e., VTE6, DXS2, and GGDR) were co-regulated and shared a similar pattern during maturation in both tissues, suggesting they are developmentally modulated.

Association between heat stress during intrauterine development and the calving-to-conception and calving-to-first-service intervals in Holstein cows.

Heat stress has been widely studied in relation to its effects on the production and reproduction of dairy cattle. However, the long-term effects of heat stress during intrauterine development on adult cows have been scarcely considered. Thus, the aim of this study was to evaluate possible changes in the reproductive performance of Holstein cows gestated under different values of the Temperature-Humidity Index (THI) during their intrauterine development. Data collected from a database of reproductive and productive records of 10,790 Holstein cows from the central region of Argentina and the THI data from the agrometeorological station of the Instituto Nacional de Tecnología Agropecuaria (INTA) EEA Rafaela, Santa Fe, Argentina, were used. The gestation of the cows evaluated was divided into trimesters, in which the highest THI cycles (sum of three or more consecutive days of exposure to a THI ≥72 during each trimester) and number of days with a THI ≥ 72 were calculated. The calving-to-conception and calving-to-first-service intervals of the cows evaluated were considered as reproductive variables associated with their first lactation. Generalized Linear Mixed Models were used, considering the cow's dairy farm as a random factor within the model. The exposure of the animals to environments with a THI ≥72 during the first trimester of gestation had a negative impact on the reproductive efficiency parameters analyzed. The results obtained indicate that the exposure of pregnant females to high THI values has a long-term impact on their daughters, which may contribute to a decrease in their reproductive performance, possibly through inherited epigenetic characteristics that remain in later generations through fetal programming.

Transcriptional Analysis of C-Repeat Binding Factors in Fruit of Citrus Species with Differential Sensitivity to Chilling Injury during Postharvest Storage.

Citrus fruit are sensitive to chilling injury (CI) during cold storage, a peel disorder that causes economic losses. C-repeat binding factors (CBFs) are related to cold acclimation and tolerance in different plants. To explore the role of Citrus CBFs in fruit response to cold, an in silico study was performed, revealing three genes (CBF1, CBF2, and CBF3) whose expression in CI sensitive and tolerant cultivars was followed. Major changes occurred at the early stages of cold exposure (1-5 d). Interestingly, CBF1 was the most stimulated gene in the peel of CI-tolerant cultivars (Lisbon lemon, Star Ruby grapefruit, and Navelina orange), remaining unaltered in sensitive cultivars (Meyer lemon, Marsh grapefruit, and Salustiana orange). Results suggest a positive association of CBF1 expression with cold tolerance in Citrus cultivars (except for mandarins), whereas the expression of CBF2 or CBF3 genes did not reveal a clear relationship with the susceptibility to CI. Light avoidance during fruit growth reduced postharvest CI in most sensitive cultivars, associated with a rapid and transient enhance in the expression of the three CBFs. Results suggest that CBFs-dependent pathways mediate at least part of the cold tolerance responses in sensitive Citrus, indicating that CBF1 participates in the natural tolerance to CI.

Antioxidant capacity in fruit of Citrus cultivars with marked differences in pulp coloration: Contribution of carotenoids and vitamin C.

The purpose of this study was to evaluate the specific contribution of carotenoids and vitamin C to the lipophilic and hydrophilic antioxidant capacity, respectively, of the pulp of citrus fruits using the genetic diversity in pigmentation and in the carotenoid complement. To this end, six citrus varieties were selected: two mandarins, Clemenules (Citrus clementina) and Nadorcott (C. reticulata); two grapefruits (C. paradisi), Marsh and Star Ruby; and two sweet oranges (C. sinensis), Valencia late and Valencia Ruby. Total carotenoid content and composition in the pulp of fruits were very different, in relation to their color singularities. Valencia Ruby and Nadorcott had the highest carotenoid content, accumulating the former large amounts of linear carotenes (phytoene, phytofluene, and lycopene) and Nadorcott of ß-cryptoxanthin. Orange fruits contained the highest amount of vitamin C while in Nadorcott mandarin it was substantially lower. Analysis of antioxidant capacity, evaluated by 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, in the pulp of the different fruit varieties indicated a high and positive correlation between vitamin C content and hydrophilic antioxidant capacity. Nevertheless, a weak correlation was observed between carotenoids content and lipophilic antioxidant capacity in the pulp extracts assayed by ABTS. Overall, vitamin C in the pulp of citrus fruit had an important contribution to the hydrophilic antioxidant capacity, whereas that of carotenoids to lipophilic antioxidant capacity was very variable, being the highest that of Valencia Ruby orange, with large concentrations of lycopene and phytoene, followed by Nadorcott mandarin, with high ß-cryptoxanthin content.

Effect of fruit shading and cold storage on tocopherol biosynthesis and its involvement in the susceptibility of Star Ruby grapefruit to chilling injury.

The aim of this study was to investigate the role of tocopherols in the susceptibility of Star Ruby grapefruit to postharvest chilling injury (CI). Fruit exposed to normal sunlight (NC, non-covered) and deprived of light (C, covered) in the last stages of development were used. Tocopherol contents increased in the flavedo of both NC and C fruit during development, concomitantly with the up-regulation of TAT1 and most genes of the tocopherol-core pathway. Fruit shading reduced total contents by repressing γ-tocopherol accumulation, associated to a down-regulation of GGDR and VTE1 and, to a lesser extent, of VTE2, VTE3a and VTE4. During cold storage, total and α-tocopherol contents increased in NC and C fruit, and no direct relationship between tocopherol accumulation and CI tolerance was found. Cold stress up-regulated most genes involved in the synthesis of tocopherol precursors and down-regulated those of the tocopherol-core pathway, but changes seemed to be cold-mediated and not related to CI development.

Carotenoids, Vitamin C, and Antioxidant Capacity in the Peel of Mandarin Fruit in Relation to the Susceptibility to Chilling Injury during Postharvest Cold Storage.

Chilling injury (CI) is a postharvest disorder occurring in the fruit of cold-sensitive Citrus species during storage at low temperatures. This study investigated the involvement of carotenoids and vitamin C, two major antioxidants of citrus peel, and the antioxidant capacity in the CI susceptibility of mandarin fruit. To that end, the fruit of three commercial varieties, Fortune, Nova, and Nadorcott, with significant differences in CI susceptibility, were selected. By on-tree fruit bagging, carotenoids and vitamin C contents were modified, and a differential effect of each cultivar on CI was observed. Carotenoid analysis in the peel revealed a strong negative correlation between total carotenoid concentration (TCC) at harvest, and specifically of ß-cryptoxanthin and violaxanthin, and CI index at the end of storage. In contrast, vitamin C content was significantly and positively correlated with CI susceptibility. The antioxidant activity assessed by the DPPH• and FRAP reflected the contribution of vitamin C to the antioxidant system, while the SOAC assay correlated positively with TTC, ß-cryptoxanthin, and violaxanthin. Collectively, the antioxidant capacity of carotenoids at harvest, as efficient singlet oxygen quenchers, suggests a protective role against the development of CI in mandarin fruit, while vitamin C is not likely playing a critical role.

Alterations in the insulin signaling pathway in bovine ovaries with experimentally induced follicular persistence.

Reproductive diseases in dairy cows as cystic ovarian disease (COD) represent a major problem that impacts on dairy production. COD is characterized by anovulation, persistence of the dominant follicle, and interruption of normal estrous cycles. Anovulation is attributable to a failure in the LH surge, due to endocrine imbalances and alterations in local factors, such as the insulin signaling pathway. Thus, the aim of this study was to examine the expression of critical nodes of the insulin pathway, including insulin receptor (IR), IR substrate (IRS), phosphatidylinositol-3-kinase (PI3K), and protein kinase B (also known as Akt or pan-Akt), in ovarian follicular structures of cows during the development of follicular persistence induced by long-term progesterone administration. Immunoexpression of IR, p-IR, IRS1, p-IRS1, PI3K, pan-Akt and p-pan-Akt was evaluated in situ by immunohistochemistry and the concentration of insulin in serum and follicular fluid was determined by radioimmunoassay. p-IR, p-IRS1, PI3K and p-pan-Akt expression was decreased in follicles at different times of persistence in relation to the control dominant follicles, in both granulosa and theca cells, whereas IR and IRS1 immunoexpression was decreased in persistent follicles at 5 and 15 days of persistence in granulosa cells. Serum and follicular fluid insulin concentration was higher in cows with persistent follicles than in control cows. These results show that decreased expression and/or activation of the receptors and other intermediates of the insulin signaling pathway in persistent follicles indicates that reduced response/resistance to insulin rather than the concentration of insulin per se may be one of the important molecular mechanisms in the development of persistent follicles in dairy cows.

Abundance of insulin-like growth factors 1 and 2, and type 1 insulin-like growth factor receptor in placentas of dogs.

Insulin-like growth factors (IGFs) are among the primary compounds regulating placental development. In bitches, relative abundance of IGF1, IGF2 and IGFR1 mRNA transcripts have been studied in the pre-implantation uterus and early endotheliochorial placentas. The IGF2 and IGFR1 distribution has also been previously described in the uterus before embryo implantation. The aim of this study was to detect, characterize, and localize the presence of IGF1, IGF2, and IGFR1 in early-developing and mature placentas of dogs. Placentas of 15 bitches were analyzed using immunohistochemistry. The IGFs were located in endometrial epithelium and glands, with the staining pattern and intensity being less in mature placentas. Cytotrophoblast cells (CTB) and syncytiotrophoblast (STB) cells contained both IGFs; the labeling was greater in CTB of the early-developing than mature placentas. The maternal endothelium was positively stained for both IGFs, while the vascular endothelium of the chorioallantoic membrane were only stained for IGF2. The IGFR1 was detected in all cell populations evaluated. Results regarding trophoblastic IGF are quite consistent with those reported in human placentas. Spatiotemporal IGFs/IGFR1 pattern might reflect the occurrence of autocrine and paracrine signaling during placentation in bitches, and the involvement in early placental developmental processes. Furthermore, it is hypothesized that, besides hemotrophic actions of plasma IGFs, endometrial secreted IGFs may promote early placental development through histotrophic signaling.

Liver fatty acid metabolism associations with reproductive performance of dairy cattle.

The peri-calving period is characterized by a negative energy balance, which leads to lipid mobilization. Thus, during this period, the liver has important functions related to optimizing milk yield, preventing metabolic and infectious diseases, and improving fertility. To clarify the relationship between liver fatty acid metabolism and reproductive performance, the present study was conducted to assess the abundance of specific hepatic proteins related to lipid metabolism in both plasma and follicular fluid in dairy cattle with different days to conception (DC). Sixteen animals were grouped according to DC, as more and fewer DC (MDC and FDC, respectively). Blood and liver biopsies were sampled 14 days before the expected calving date and 4, 14 and 28 days after calving. The plasma beta-hydroxybutyric acid (BHBA) concentrations and the liver triacylglycerol (TAG) content were greater in the MDC group (P <  0.05), whereas the protein abundance of carnitine palmitoyl transferase 1 was greater in the FDC group (P < 0.05). Additionally, total bilirubin (TBil) concentration was less in the FDC than MDC group on day 28 (P < 0.05). These results indicate lipid mobilization and liver fatty acid oxidation capacity in dairy cows could contribute to the adaptations and reproductive performance.

Follicular structures of cows with cystic ovarian disease present altered expression of cytokines.

Ovulation is considered an inflammatory, cytokine-mediated event. Cytokines, which are recognized as growth factors with immunoregulatory properties, are involved in many cellular processes at the ovarian level. In this sense, cytokines affect fertility and are involved in the development of different ovarian disorders such as bovine cystic ovarian disease (COD). Because it has been previously demonstrated that ovarian cells represent both sources and targets of cytokines, the aim of this study was to examine the expression of several cytokines, including IL-1ß, IL-1RA, IL-1RI, IL-1RII, IL-4 and IL-8, in ovarian follicular structures from cows with spontaneous COD. The protein expression of these cytokines was evaluated by immunohistochemistry. Additionally, IL-1ß, IL-4 and IL-8 concentrations in follicular fluid (FF) and serum were determined by enzyme-linked immunosorbent assay (ELISA). In granulosa and theca cells, IL-1RI, IL-1RII, IL-1RA and IL-4 expression levels were higher in cystic follicles than in the control dominant follicles. The serum and FF concentrations of IL-1ß and IL-4 showed no differences between groups, whereas IL-8 concentration was detected only in FF of cysts from cows with COD. The FF and serum concentrations of IL-1ß and IL-8 showed no significant differences, whereas IL-4 concentration was higher in FF than in serum in both the control and COD groups. These results evidenced an altered expression of cytokines in ovaries of cows with COD that could contribute to the pathogenesis of this disease.

Expression of TGFBR1, TGFBR2, TGFBR3, ACVR1B and ACVR2B is altered in ovaries of cows with cystic ovarian disease.

The objective of this study was to examine the expression of transforming growth factor beta receptor (TGFBR)1, TGFBR2, TGFBR3, activin receptor (ACVR)1B and ACVR2B in ovaries of cows with cystic ovarian disease (COD). The expression of the selected receptors was determined by immunohistochemistry in sections of ovaries from cows with ACTH-induced and spontaneous COD. Expression of TGFBR1 and TGFBR3 was higher in granulosa cells of cysts from cows with spontaneous COD than in tertiary follicles from the control group. Additionally, TGFBR3 expression was higher in granulosa cells of cysts from cows with ACTH-induced COD than in those from the control group and lower in theca cells of spontaneous and ACTH-induced cysts than in tertiary control follicles. There were no changes in the expression of TGFBR2. ACVR1B expression was higher in granulosa cells of tertiary follicles of cows with spontaneous COD than in the control group, whereas ACVR2B expression was higher in cysts of the spontaneous COD group than in tertiary follicles from the control group. The alterations here detected, together with the altered expression of the ligands previously reported, indicate alterations in the response of the ligands in the target cells, modifying their actions at cellular level.

Production and validation of a polyclonal serum against bovine FSH receptor.

In ovarian granulosa cells, follicle-stimulating hormone (FSH) regulates the proliferation and differentiation events required for follicular growth and oocyte maturation. FSH actions are mediated exclusively through the FSH receptor (FSHR). In cattle, the FSHR gene expression pattern during folliculogenesis and the implications of this receptor in reproductive disorders have been extensively studied. However, the limited availability of specific antibodies against bovine FSHR has restricted FSHR protein analysis. In the present study, we developed an anti-FSHR polyclonal serum by using a 14-kDa peptide conjugated to maltose binding protein. The antiserum obtained was characterized by western blot of protein extracts from bovine follicles, BGC-1 cells and primary cultures of granulosa cells stimulated with testosterone. Also, the blocking effect of serum on estradiol secretion and cell viability after gonadotropin stimulus was characterized in a functional in vitro assay. A 76-kDa protein, consistent with the predicted molecular size of full-length FSHR, was detected in ovarian tissue. Besides, two immunoreactive bands of 60-kDa and 30-kDa (only in cultured cells) were detected. These bands would be related to some of the isoforms of the receptor. Therefore, immunohistochemical assays allowed detecting FSHR in the cytoplasm of granulosa cells and an increase in its expression as follicles progressed from primordial to large preantral follicles. These results suggest that the anti-FSHR serum here developed has good reactivity and specificity against the native FSHR. Therefore, this antiserum may serve as a valuable tool for future studies of the biological function of FSHR in physiological conditions as well as of the molecular mechanism and functional involvement of FSHR in reproductive disorders.

Changes in the Proliferation/Apoptosis Balance in the Bovine Ovary: A Key Early Event in Follicular Persistence.

The objective of this work was to evaluate proliferation and apoptosis in the bovine ovary in a model of follicular persistence induced by low levels of progesterone to detect incipient changes during cystic ovarian disease development on the expected day of ovulation (day 0) and after 5, 10, and 15 days of follicular persistence. We analyzed cell proliferation by evaluating the expression of Ki-67 and apoptosis by evaluating caspase-3, BAX, and BCL2 expression. Proliferation was similar in the granulosa and theca cells of antral follicles in the P0 group (treated with progesterone up to the expected day of ovulation) and in the control group. A decrease in cell proliferation was detected after 5 days of persistence (P5) in relation to P0 (p < 0.05). Similar changes were found in the granulosa cells of the persistent follicles in relation to the control group (p < 0.05). Caspase-3 expression was similar in granulosa cells of antral follicles at early stages of persistence, with an increase after 15 days of persistence (p < 0.05). In the granulosa cells of group P10 (10 days of persistence), caspase-3 expression was reduced relative to that of antral follicles from the control group (p < 0.05). BCL2 expression was higher in granulosa cells of the persistent follicles of group P0 relative to the control follicles, with no changes in BAX expression, which was increased in persistent follicles of group P15 (p < 0.05). Similar results were observed in theca cells at initial stages of persistence. The results show that, initially, proliferation is maintained with low apoptosis and an increase in cell survival.