Profile of Steroid Receptors and Increased Aromatase Immunoexpression in Canine Inflammatory Mammary Cancer as a Potential Therapeutic Target.

Canine inflammatory mammary cancer (IMC) has been proposed as a model for the study of human inflammatory breast cancer (IBC). The aims of this study were to compare the immunohistochemical expression of aromatase (Arom) and several hormone receptors [estrogen receptor α (ERα), estrogen receptor ß (ERß), progesterone receptor (PR) and androgen receptor (AR)], in 21 IMC cases vs 19 non-IMC; and to study the possible effect of letrozole on canine IMC and human inflammatory breast cancer (IBC) in vitro using IPC-366 and SUM-149 cell lines. Significant elevations of the means of Arom Total Score (TS), ERß TS and PR TS were found in the IMC group (p = 0.025, p = 0.038 and p = 0.037, respectively). Secondary IMC tumours expressed higher levels of Arom than primary IMC (p = 0.029). Non-IMC PR- tumours contained higher levels of Arom than non-IMC PR+ tumours (p = 0.007). After the addition of letrozole, the number of IMC and IBC cells dropped drastically. The overexpression of Arom found and the results obtained in vitro further support canine IMC as a model for the study of IBC and future approaches to the treatment of dogs with mammary cancer, and especially IMC, using Arom inhibitors.

Isoflavones and their effects on the onset of puberty in male Wistar rats.

This study was performed to determine how two of the most important isoflavones, genistein and daidzein, affect the gonadal axis in male prepuberal rats. One hundred and seventy-five prepuberal male Wistar rats were allocated into seven groups: one control group and six experimental groups that were orally administered a high or low dose of genistein, daidzein or a mixture of both. Testosterone determination was assayed by EIA. The testes and body weights were measured, and the histology of the epididymis with the sperm content and epididymal sperm count were evaluated. In the control group, we observed an increase in the serum testosterone levels (>2.5 ng ml(-1) ) at the third week (52 days), which corresponded to the onset of puberty in these rats. The same increase in serum testosterone levels was observed at the fourth week in rats that received low doses of isoflavones; therefore, we concluded that the onset of puberty was delayed. At high doses, there was no significant increase in testosterone levels, which could be related to the fact that these male rats did not reach puberty. These findings were supported by the results obtained from the analysis of the epididymal content as well as the testes/body weight ratio.

Serum and Tissue Steroid Hormone Levels in Canine Mammary Tumours: Clinical and Prognostic Implications.

Hormonal dependency of canine mammary tumours (CMT) has been studied over the last few decades. However, studies assessing the prognostic and predictive potential of serum and/or tissue steroid hormone levels are still scarce in CMT. To the best of our knowledge, this is the first report relating serum and tissue levels of steroid hormones and prognosis in dogs. Serum and tumour tissue from 45 female dogs with spontaneous CMT were included in the study. Moreover, serum and normal mammary tissue from 13 healthy female dogs were also included as controls. Steroid hormones were determined by competitive enzyme immunoassay. Overall, levels of steroid hormones in serum and tissue homogenates were significantly different between malignant and benign mammary tumours (p < 0.01), except for progesterone (P4) serum levels that revealed no statistical differences between groups. In malignant tumours, oestrone sulphate (SO4E1), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T) and P4 elevated tissue concentrations were significantly associated with tumour relapse and/or distant metastasis during follow-up. A significant association was found between elevated tissue SO4E1 (p = 0.003), 17ß-oestradiol (E2) (p = 0.036), DHEA (p = 0.022), A4 (p = 0.001) and P4 (p = 0.013) concentrations and shorter disease-free survival and overall survival in female dogs with malignant mammary tumours. The high levels of tissue steroids found in cases of poor prognosis open the possibility of additional new therapeutic approaches. Future clinical trials will be needed to clarify the usefulness of targeting steroid hormones in the treatment of this neoplastic disease.

Immunohistochemical vascular factor expression in canine inflammatory mammary carcinoma.

Human inflammatory breast carcinoma (IBC) and canine inflammatory mammary carcinoma (IMC) are considered the most malignant types of breast cancer. IMC has similar characteristics to IBC; hence, IMC has been suggested as a model to study the human disease. To compare the angiogenic and angioinvasive features of IMC with non-IMC, 3 canine mammary tumor xenograft models in female SCID mice were developed: IMC, comedocarcinoma, and osteosarcoma. Histopathological and immunohistochemical characterization of both primary canine tumors and xenografts using cellular markers pancytokeratin, cytokeratin 14, vimentin, and α-smooth muscle actin and vascular factors (VEGF-A, VEGF-D, VEGFR-3, and COX-2) was performed. Tumor cell proliferation index was measured by the Ki-67 marker. The xenograft models reproduced histological features found in the primary canine tumor and preserved the original immunophenotype. IMC xenografts showed a high invasive character with tumor emboli in the dermis, edema, and occasional observations of ulceration. In addition, compared with osteosarcoma and comedocarcinoma, the IMC model showed the highest vascular factor expression associated with a high proliferation index. Likewise, IMC xenografts showed higher COX-2 expression associated with VEGF-D and VEGFR-3, as well as a higher presence of dermal lymphatic tumor emboli, suggesting COX-2 participation in IMC lymphangiogenesis. These results provide additional evidence to consider vascular factors, their receptors, and COX-2 as therapeutic targets for IBC.

The effects of isoflavones on androgens and glucocorticoids during puberty on male Wistar rats.

Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy-five 30-day-old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low-dose groups, the same peak was found at the fourth week (p < 0.05), indicating a delay in the onset of puberty in these groups. Moreover, high doses groups serum androgen levels were significantly lower (p < 0.05) than the control group from the first week until fifth week. This fact was supported by a epididymal histological analysis that indicate in low doses there were several content of spermatozoa at fourth week and in high doses there were few content of spermatozoa. Besides, corticosterone levels followed the same pattern of androgens in all groups. We can conclude that oral administration of isoflavones in male rats decreased the secretion of androgens and glucocorticoids causing a delay in the onset of puberty and may cause physiological and developmental problems.

Local climate determines intra- and interspecific variation in sexual size dimorphism in mountain grasshopper communities.

The climate is often evoked to explain broad-scale clines of body size, yet its involvement in the processes that generate size inequality in the two sexes (sexual size dimorphism) remains elusive. Here, we analyse climatic clines of sexual size dimorphism along a wide elevation gradient (i) among grasshopper species in a phylogenetically controlled scenario and (ii) within species differing in distribution and cold tolerance, to highlight patterns generated at different time scales, mainly evolutionary (among species or higher taxa) and ontogenetic or microevolutionary (within species). At the interspecific level, grasshoppers were slightly smaller and less dimorphic at high elevations. These clines were associated with gradients of precipitation and sun exposure, which are likely indicators of other factors that directly exert selective pressures, such as resource availability and conditions for effective thermoregulation. Within species, we found a positive effect of temperature and a negative effect of elevation on body size, especially on condition-dependent measures of body size (total body length rather than hind femur length) and in species inhabiting the highest elevations. In spite of a certain degree of species-specific variation, females tended to adjust their body size more often than males, suggesting that body size in females can evolve faster among species and can be more plastic or dependent on nutritional conditions within species living in adverse climates. Natural selection on female body size may therefore prevail over sexual selection on male body size in alpine environments, and abiotic factors may trigger consistent phenotypic patterns across taxonomic scales.

The inhibition of steroid hormones determines the fate of IPC-366 tumor cells, highlighting the crucial role of androgen production in tumor processes.

Inflammatory mammary cancer (IMC) is a disease that affects female dogs. It is characterized by poor treatment options and no efficient targets. However, anti-androgenic and anti-estrogenic therapies could be effective because IMC has a great endocrine influence, affecting tumor progression. IPC-366 is a triple negative IMC cell line that has been postulated as a useful model to study this disease. Therefore, the aim of this study was to inhibit steroid hormones production at different points of the steroid pathway in order to determine its effect in cell viability and migration in vitro and tumor growth in vivo. For this purpose, Dutasteride (anti-5αReductase), Anastrozole (anti-aromatase) and ASP9521 (anti-17ßHSD) and their combinations have been used. Results revealed that this cell line is positive to estrogen receptor ß (ERß) and androgen receptor (AR) and endocrine therapies reduce cell viability. Our results enforced the hypothesis that estrogens promote cell viability and migration in vitro due to the function of E1SO4 as an estrogen reservoir for E2 production that promotes the IMC cells proliferation. Also, an increase in androgen secretion was associated with a reduction in cell viability. Finally, in vivo assays showed large tumor reduction. Hormone assays determined that high estrogen levels and the reduction of androgen levels promote tumor growth in Balb/SCID IMC mice. In conclusion, estrogen levels reduction may be associated with a good prognosis. Also, activation of AR by increasing androgen production could result in effective therapy for IMC because their anti-proliferative effect.

Cell adhesion molecules E-cadherin and CADM1 are differently expressed in canine inflammatory mammary cancer.

Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most aggressive and lethal types of mammary tumors with specific characteristics such as exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism. E-cadherin expression is another specific feature of IBC not previously studied in canine IMC. In this study, the expression of E-cadherin and CADM1 (Cell Adhesion molecule 1) and their possible role as key molecules involved in the pathogenesis of IMC were immunohistochemically analyzed in 19 canine IMC and 15 grade III non-IMC cases. E-cadherin and CADM1 expression was higher in IMC cases (p = 0.002, p = 0.008, respectively). In the IMC group, E-cadherin cytoplasmic immunolabeling was more frequent (p = 0.035) and it was associated to the expression of the angiogenic and lymphangiogenic factors COX-2 (p = 0.009), VEGF-A (p = 0.031) and VEGF-D (p = 0.008). The differential mRNA expression between IMC and non-IMC was studied by microarray analysis in 6 cases. E-cadherin gene (CDH1) was not up-regulated in IMC cases at a transcriptional level; interestingly CADM1 was 7-fold upregulated. The differential expression of E-cadherin protein in IMC suggests a possible role of E-cadherin in the characteristic exacerbated angiogenesis and lymphangiogenesis and further support IMC as a natural model for the study of human IBC. Future studies in IBC and IMC including a broad panel of adhesion molecules are necessary to elucidate their role in the metastatic process and angiogenesis.

Histological, immunohistological, and ultrastructural description of vasculogenic mimicry in canine mammary cancer.

Canine inflammatory mammary cancer (IMC) and human inflammatory breast cancer (IBC) are the most aggressive and lethal type of mammary cancer in female dogs and in women. The generation of microvascular channels by malignant tumor cells (endothelial-like cells [ELCs]) without endothelial cell participation (vasculogenic mimicry) has been reported in human breast cancer, including IBC, and is considered a new type of tumor angiogenesis. The aim of this study was to investigate the presence of ELCs in highly malignant canine mammary tumors (IMC and non-IMC) by histology, inmunohistochemistry (pancytokeratin, cytokeratin 14, vimentin, actin, desmin, vWF, CD31, and CD34), and electron microscopy. This retrospective study included 21 female dogs with diagnoses of IMC and 20 animals with metastatic grade III noninflammatory malignant mammary tumors (MMT). IMC tumors (33.33%) and MMT (5%) showed ELCs forming structures similar to small capillaries. The histological, immunohistochemical (positive to AE1/AE3 and cytokeratin 14, mostly negative to endothelial markers), and ultrastructural characteristics of these cells indicated vasculogenic mimicry. The higher frequency of this phenomenon in inflammatory versus noninflammatory canine mammary cancer is in agreement with previous studies in experimental and spontaneous human IBC, and it could be in relation with the extremely high lymphangiogenic capacity and metastatic lymphangiotropism characteristics of inflammatory breast cancer.

Changes in steroid hormone profile and tumour progression after genistein treatment of canine inflammatory mammary cancer xenotransplanted mice.

Isoflavones, such as genistein, have been proposed to have beneficial effects on health, including preventive or therapeutic actions in carcinogenesis. Their structural similarity to oestrogens allows them to bind at the cellular level with oestrogen receptors. Therefore, this study attempted to determine the antitumoural effects of genistein administered in a canine inflammatory mammary cancer xenograft model, in terms of tumour proliferation, appearance of metastases and steroid hormone regulation. Using histology and immunohistochemical analyses as well as the EIA technique for hormonal determinations, the antitumoural effects of genistein on an inflammatory mammary cancer xenograft model were assessed for 3 weeks. Mice treated with genistein showed higher Ki-67 levels than the control group. There were significantly more distant metastases in the genistein-treated xenografts versus the control group. Intratumoural and serum progesterone, androstenedione and oestrogen levels in treated mice were elevated, whereas intratumoural testosterone levels were decreased compared to the control group. These results revealed that genistein ingestion promotes tumour proliferation and elevates metastatic rates by increasing intratumoural and circulating oestrogen levels in a mammary cancer xenograft model.

Quantification of sexual steroid hormones in faeces of Iberian wolf (Canis lupus signatus): a non-invasive sex typing method.

The determination of gender in wild animals is essential for behavioural and ecological studies, and also for conservation. The objectives of this study were (i) the determination of gender in faecal samples of Iberian wolf based on the differential concentrations of sexual steroid hormones (SSH) and (ii) to analyse the profiles of SSH in males and females (considering the gender determination carried out previously) during the non-reproductive and reproductive periods. The quantification of androgens (testosterone, T), progestin (progesterone, P) and oestrogen (oestradiol, E) was conducted by means of enzyme immunoassay. The k-means conglomerate analysis showed that the 59 faecal samples grouped into three different conglomerates, considering SSH levels. Groups 1 and 2 showed higher levels of T than group 3. Therefore, the faecal samples included in groups 1 and 2 (17 samples) corresponded to males and those of group 3 (42 samples) to females. The levels of T + P + E and T/P were higher in the group of males than in the group of females. The results of this study also showed that levels of T in males were higher during the reproductive period than in the non-reproductive period. However, the concentrations of P and E turned out to be higher during the non-reproductive season. In females, the levels of the three hormones (T, P and E) were higher during the reproductive period.

In vitro and in vivo effect of flutamide on steroid hormone secretion in canine and human inflammatory breast cancer cell lines.

The aim was to study the effects of flutamide on cell proliferation, in vivo tumour growth and steroid production in canine and human IBC cell lines. IPC-366 and SUM149 cell cultures were exposed to flutamide concentrations for 72 hours. Additionally, IPC-366 and SUM149 xenotransplanted mice were treated subcutaneously with flutamide 3 times a week for 2 weeks. Steroid hormones determination in culture media, serum and tumour homogenates (pregnenolone, progesterone, androstenedione, testosterone, dihydrotestosterone, 17ß-oestradiol and oestrone sulphate) were assayed by EIA. in vitro cell proliferation percentages showed a decrease in all flutamide dosages in IPC-366 and SUM149. in vivo flutamide reduced tumour size by 55% to 65%, and metastasis rates decreased. In treated groups, androgen levels in culture media, serum and tumour homogenates were increased as oestrogen levels decreased. These results suggest that flutamide treatment inhibits cell proliferation and promotes tumour reduction by increasing androgen levels and also support future therapy approaches.

Quantification of epidermal growth factor receptor (EGFR) in canine mammary tumours by ELISA assay: clinical and prognostic implications.

The involvement of epidermal growth factor receptor (EGFR) is well established in human breast cancer, however, in canine mammary tumours (CMT), including inflammatory mammary carcinomas (IMC), still needs to be clarified. Enzyme immune assay techniques were used for EGFR determinations in tumour tissue from 45 bitches with CMT and in normal mammary glands from eight control dogs. Higher tissue EGFR levels were found in CMT compared with controls (P < 0.05). In malignant CMT, tissue EGFR elevated concentrations were statistically significantly associated with tumour relapse and/or distant metastasis during follow-up and with reduced disease-free and overall survival times. The IMC cases had the highest tissue EGFR levels compared with other malignant non-IMC tumours (P < 0.001). The results support the hypothesis that EGFR levels influence prognosis in malignant CMT, suggesting that EGFR may represent a therapeutic target in cases of high histological aggressiveness and especially in cases of metastatic phenotype and poor prognosis.

Canine cell line, IPC-366, as a good model for the study of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is an aggressive type of cancer with poor survival in women. Inflammatory mammary cancer (IMC) in dogs is very similar to human IBC and it has been proposed as a good surrogate model for study the human disease. The aim was to determine if IPC-366 shared characteristics with the IBC cell line SUM149. The comparison was conducted in terms of ability to grow (adherent and nonadherent conditions), stem cell markers expression using flow cytometry, protein production using western blot and tumorigenic capacity. Our results revealed that both are capable of forming long-term mammospheres with a grape-like morphology. Adherent and nonadherent cultures exhibited fast growth in vivo. Stem cell markers expressions showed that IPC-366 and SUM149 in adherent and nonadherent conditions has mesenchymal-like characteristics, E-cadherin and N-cadherin, was higher in adherent than in nonadherent cultures. Therefore, this study determines that both cell lines are similar and IPC-366 is a good model for the human and canine disease.

Nutritional evaluation of raw and extruded kidney bean (Phaseolus vulgaris L. var. pinto) in chicken diets.

An experiment was conducted to study the effect of inclusion of different concentrations (0, 100, 200, and 300 g/kg) of raw kidney bean and extruded kidney bean in broiler chick (0 to 21 d of age) diets on performance, digestive organ sizes, protein and amino acid digestibilities, intestinal viscosity, cecal pH, and blood parameters. Data were analyzed as a 3 x 2 factorial arrangement with 3 levels of kidney bean with and without extrusion. Positive control without kidney bean was used. Increasing the kidney bean content in the diet reduced weight gain and consumption, and increased the feed-to-gain ratio. Relative pancreas, liver, and jejunum weights, and intestinal viscosity were increased in response to increasing kidney bean concentration in the diet. The inclusion of different concentrations of kidney bean did not affect the apparent ileal digestibility of essential and nonessential amino acids, except for Met, Phe, and Cys, which were increased. Increasing kidney bean in the diet did not affect blood parameters, except for total protein, which was increased, and for androstenedione and testosterone, which were reduced. Extrusion significantly improved weight gain, feed consumption, and feed conversion. Relative pancreas, liver, and jejunum weights were reduced and spleen weight, cecal and intestinal viscosity were increased by extrusion. Apparent ileal digestibility of crude protein and all essential and nonessential amino acids were improved by extrusion. Like-wise, extrusion increased significantly the concentrations of cholesterol, triglycerides, glucose, and testosterone. We concluded that the inclusion of kidney bean in chicken diets cause a negative effect on performance and CP and amino acid digestibilities, and modified digestive organ sizes, intestinal viscosity, cecal pH, and some blood parameters. These effects were counteracted by the extrusion of kidney bean. However, the inclusion of extruded kidney bean in a chick diet resulted in poorer performance compared with that obtained with a corn-soybean diet.

Effect of Ovariohysterectomy at the Time of Tumor Removal in Dogs with Mammary Carcinomas: A Randomized Controlled Trial.

BACKGROUND: Ovarian hormones play crucial roles in mammary carcinogenesis. However, whether ovarian ablation by ovariohysterectomy (OHE) improves the prognosis in dogs with mammary carcinomas is unclear. OBJECTIVES: Determine if OHE at the time of mastectomy improves the prognosis in dogs with mammary carcinomas and evaluate if hormonal factors influence the effect of OHE. ANIMALS: Sixty intact dogs with mammary carcinomas. METHODS: Dogs were randomly assigned in a 1:1 ratio to undergo OHE (n = 31) or not (n = 29) at the time of tumor removal. Peri-surgical serum estradiol (E2) and progesterone concentrations were measured, tumor diagnosis was confirmed histologically, and tumor estrogen and progesterone receptor status was immunohistochemically determined. The dogs were monitored for recurrence and metastases every 3-4 months for at least 2 years. Uni- and multivariable survival analyses were performed with relapse and all-cause death as endpoints in addition to univariable subgroup analyses. RESULTS: Overall, OHE did not significantly decrease hazard of relapse (hazard ratio [HR], 0.64; P = .18) or all-cause death (HR, 0.87; P = .64) in univariable analyses. In multivariable analysis OHE did not significantly influence the hazard of relapse (HR, 0.54; P = .12), but an interaction effect was identified between ER status and E2 (P = .037). Subgroup analysis identified decreased hazard of relapse in the OHE group compared to the non-OHE group in the subsets of dogs with increased E2 (HR, 0.22; P = .012) or grade 2 tumors (HR, 0.26; P = .02). CONCLUSION: Dogs with grade 2, ER-positive tumors, or with increased peri-surgical serum E2 concentration represent a subset of dogs with mammary carcinomas likely to benefit from OHE.

Role of steroid hormones and prolactin in canine mammary cancer.

In several animal studies, prolactin has been found to be essential for mammary epithelial development, and its administration has been consistently shown to increase the rate of mammary tumours. High levels of steroid hormones have also been suggested to enhance mammary cancer development. The present study investigates the levels of the following hormones in serum and in tissue homogenates in dogs bearing canine mammary tumours: prolactin (PRL), progesterone (P4), dehydroepiandrosterone (DHEA), androstenedione (A4), testosterone (T), 17beta-estradiol (17beta-E2) and estrone sulfate (S04E1). Eighty mammary tumours (40 dysplasias and benign and 40 malignant tumours) from 32 female dogs, and 10 normal mammary glands from eight female dogs without history of mammary tumours, were analysed. Prolactin and steroid hormones in serum and tissue homogenates, were analysed by enzyme immunoassays (EIA) techniques, previously validated for this animal species. Levels of prolactin in tissue homogenates were significantly different between malignant and benign mammary tumours (p<0.01). Serum prolactin concentrations were lower in the control group as compared with the group of dogs with benign tumours and in dogs with malignant tumours (p=0.01). Serum prolactin levels in dogs with benign lesions were not significantly different than those obtained from dogs with malignant tumours. Levels of steroid hormones were significantly higher in malignant tumours compared with the benign tumours and normal mammary glands (p<0.01) both in serum and homogenate determinations. Our results suggest that the canine neoplastic mammary gland could be a source of prolactin. Our hypothesis is that both prolactin and steroid hormones are involved in the growth of canine mammary cancer, and that they might have an autocrine/paracrine role in the maintenance of this disease.

The effect of dexamethasone on disruption of ovarian steroid levels and receptors in female rats.

This study was conducted to investigate if the injection of a single dose of dexamethasone may cause disruption of adult female rat gonadal function in terms of plasma and ovarian level of both androgen and estrogen, ovarian morphology, and changes in localization of androgen, estrogen and glucocorticoid receptors. Adult female Long Evans rats (n=50, 250-300 g) were used. At day 0 rats received subcutaneously 1 ml of saline (n=25; control group) or dexamethasone at 0.1 mg/kg (n=25, treated group). Rats were sacrificed in groups of five on days 10, 15, 20, 25 and 30 after injection. Blood samples and one ovary were collected to analyze dexamethasone, 17beta-estradiol (E2), testosterone (T) and androstenedione (A4) concentrations by amplified EIA. The remaining ovary was removed and processed for histopathology and immunocytochemistry. Differences between individual means were analyzed by Pairwise t-test and Bonferroni post test to asses whether values presented statistical significance. Increased E2, T and A4 levels were observed both in plasma and ovary samples in treated group when comparing with control (p< 0.01) at all days post-injection even when dexamethasone was undetectable. Ovarian morphology of treated group showed features compatible with female infertility. Inmmunolocalization of androgen and estrogen receptors showed that both were negative in treated group while controls showed highest positivity (AR +++, ER ++). Glucocorticoid receptor showed higher positivity in dexamethasone treated rats (GR ++) than in controls (GR +). Obtained results showed clear evidence that a single dose of dexamethasone may disrupt gonadal function in rats, and that possibly leads to infertility.

Developmental competence of immature pig oocytes under the influence of EGF, IGF-I, follicular fluid and gonadotropins during IVM-IVF processes.

Epidermal growth factor (EGF) and insulin like growth factor-I (IGF-I) were evaluated for their effects on in vitro maturation and fertilization in presence or absence of gonadotropin and porcine follicular fluid. Four groups were made with the addition of growth factors: none (control), EGF, IGF-I or EGF+IGF-I. Each group underwent four predefined treatments with gonadotropin (FSH and LH), follicular fluid, a combination of both, or none (as control). Porcine cumulus-oocyte complexes (COCs) were matured in media containing the above-mentioned treatments for 42-44 h prior to fertilization with fresh sperm capacitated for 2.5 h. At the end of the fertilization period, the presumable embryos were fixed, stained and examined as whole-mounts to ascertain their nuclear status. The addition of EGF alone or in combination with IGF-I, significantly increased the proportion of monospermic oocytes forming 2 normal pronuclei. Also, supplementation with both growth factors together enhanced the percentages of pronucleus formation and total penetration. In addition, treatments with EGF+IGF-I significantly decreased (P<0.01) the incidence of degeneration in fertilized oocytes. However, no significant differences in the proportions of COCs undergoing polyspermy were observed among all treatments. These results suggest a stimulatory effect of tested growth factors in maturation and fertilization of pig oocytes. Furthermore, gonadotropins and follicular fluid can be replaced by the addition of EGF and IGF-I to the maturation media with positive effects on fertilization rate.