Letters to the editor

Sir;- Outbreaks of ovine abortions caused by Campylobacter fetus fetus (previously C. fetus intestinalis) occur sporadically in mimy areas of New Zealand. The devastating effects on some flocks have stimulated recent studies of the disease,^(1)(3)(7) and have also led to the commercial production of a single strain vaccine (Campylovexin; Wellcome N.Z. Ltd, Auckland). However, it has been shown that single strain C. Fetus vaccines do not protect against serologically distinct strains.⁽²⁾ Since at least two different serotypes of C. fetus fetus are present in New ZealandY) it is not surprising that the commercially available vaccine is only partially effective in some outbreaks.⁽⁷⁾     

Ouabain-sensitive respiration and protein synthesis in duodenal mucosa and liver in rats fed increasing levels of pea fibre or protein and housed in 18 or 28°C environments

Seventy‐two Wistar rats were used in two studies to investigate the effect of environmental temperature (18 or 28°C), and increasing levels of dietary fibre (low, 68 g/kg dry matter (DM); medium 110 g/kg DM; high, 157 g/kg DM) and protein (low, 91 g/kg DM; medium, 171 g/kg DM; high, 262 g/kg DM) on respiration attributable to Na⁺,K⁺‐ATPase activity and protein synthesis in duodenal mucosa and liver of rats. In vitro O₂ consumption in tissues was measured polarographically using a Clark‐style YSI biological O₂ monitor. Whole‐body O₂ consumption was measured with two open‐circuit respiration chambers. Whole‐body O₂ consumption was higher (p < 0.05) at 18°C than at 28°C. Rats fed the low protein diet had significantly higher (p < 0.05) whole‐body O₂ consumption than those fed the medium or high protein diet. Compared with 28°C, the environmental temperature of 18°C caused an increase (p < 0.05) in total O₂ consumption and O₂ consumption attributable to Na⁺,K⁺‐ATPase activity in duodenal mucosa. There was no effect (p > 0.05) of environmental temperature on total O₂ consumption, Na⁺,K⁺‐ATPase activity attributable to protein synthesis dependent on O₂ consumption in the liver. Total O₂ consumption and O₂ consumption attributable to Na⁺,K⁺‐ATPase activity increased (p < 0.05) in duodenal mucosa in rats fed the low level of dietary fibre compared with rats fed the medium level of dietary fibre. In vitro O₂ consumption determined in duodenal mucosa and in liver did not always correspond to whole‐body O₂ consumption. This may indicate that respiration in the duodenum and liver adapts differently and may not reflect changes in whole‐body respiration in response to dietary modification and changes in thermal environment.     

Effects of leptin on the release of luteinizing hormone, growth hormone and prolactin from cultured bovine anterior pituitary cells

The effects of leptin on the release of luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) were studied in cultured bovine anterior pituitary (AP) cells in vitro. The AP cells were obtained from fully‐fed Japanese Black steers and were incubated for 3 h with 10^?13 to 10^?7 mol/L of leptin after incubating in Dulbecco's modified Eagle's Medium for 3 days. Leptin significantly increased the concentration of LH in the culture medium by 45 and 44% at doses of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin significantly increased the concentration of GH in the culture medium by 14 and 12% at doses of 10^?8 and 10^?7 mol/L, respectively (P < 0.05). Leptin also significantly increased the concentration of PRL in the culture medium by 26% compared with the controls at a dose of 10^?7 mol/L (P < 0.05). These results show that leptin stimulates the release of LH, GH and PRL by acting directly on bovine AP cells from fully‐fed steers.     

Prolactin assay for fescue toxicity in sheep

Fungal endophyte (Acremonium spp.) toxicity affects sheep and cattle grazing a number of forages, including ryegrasses (Lolium spp.) and tall fescue (Festuca nrundinacea). The endophyte in tall fescue is responsible for ill thrift, as indicated by low weight gains or weight loss⁽¹⁾ and decreased reproductive efficiency⁽²⁾, and is associated with increased body temperatures. Extensive research has been undertaken in the USA to determine ways of reducing the impact of fescue toxicity on the beef industry⁽³⁾, whilst New Zealand research is directed toward both ryegrass and fescue endophyte toxicity and its impact on sheep⁽⁴⁾⁽⁵⁾ and cattle⁽⁶⁾ production. Much of this work involves field evaluations of productive performance in animals fed toxic and non-toxic forage, so that it is important to be sure that treatment effects are due to the endophyte.     

Sex steroid hormones do not enhance the direct stimulatory effect of kisspetin‐10 on the secretion of growth hormone from bovine anterior pituitary cells

The aims of the present study were to clarify the effect of kisspeptin10 (Kp10) on the secretion of growth hormone (GH) from bovine anterior pituitary (AP) cells, and evaluate the ability of sex steroid hormones to enhance the sensitivity of somatotrophic cells to Kp10. AP cells prepared from 8–11‐month‐old castrated calves were incubated for 12 h with estradiol (E₂, 10^?8 mol/L),progesterone (P₄, 10^?8 mol/L), testosterone (T, 10^?8 mol/L), or vehicle only (control), and then for 2 h with Kp10. The amount of GH released in the medium was measured by a time‐resolved fluoroimmunoassay. Kp10 (10^?6 or 10^?5 mol/L) significantly stimulated the secretion of GH from the AP cells regardless of steroid treatments (P < 0.05), and E₂, P₄, and T had no effect on this response. The GH‐releasing response to growth hormone‐releasing hormone (GHRH, 10^?8 mol/L) was significantly greater than that to Kp10 (P < 0.05). The present results suggest that Kp10 directly stimulates the release of GH from somatotrophic cells and sex steroid hormones do not enhance the sensitivity of these cells to Kp10. Furthermore, they suggest that the GH‐releasing effect of Kp10 is less potent than that of GHRH.     

A serological study of leptospirosis in domestic deer in New Zealand

Extract

Rape (Brassica napus) is grown in New Zealand predominantly for fattening lambs after weaning⁽¹⁾, although its use has steadily declined, especially in the North Island. One of the important precautions to be taken when using rape for supplementary feeding is that it must be mature before feeding. Rape grazed in the immature stage, before it takes on a purple tinge, can induce photosensitisation (rape scald)⁽²⁾.     

Effects of Density and Water Availability on the Behavior, Physiology, and Weight Loss of Slaughter Horses during Transport

The aim of this study was to determine the effects of density and provision of water on aggressive behavior, stress, and weight loss in slaughter horses during transport. A 16.2-m, single deck semi-trailer was divided into three compartments to create high, medium, and low density (397 ± 6.5 kg/m², 348 ± 5.2 kg/m², and 221 ± 7.6 kg/m² per compartment) groups of unrestrained horses. Six shipments containing 23 to 30 horses per shipment were transported in June and July of 2004 for 18 to 20 hours. While the truck was stopped for 1 hour after 8 hours of transport and then again just before unloading, horses in each of two compartments received water for 1 hour from six water bowls (three bowls mounted on each side of a compartment). The third, non-watered compartment served as a control. Aggressive behavior of the horses was recorded using 12 video cameras installed in the trailer. All occurrences of aggressive behavior were counted from 15-minute segments of video during 2-hour intervals for each horse that was visible in each density group. Neither density nor water significantly affected (P > 0.21) aggressive behavior, cortisol, plasma chemistry profile, dehydration, or weight loss. Aggression did not differ (P = 0.49) between the first and second halves of the shipments, indicating that fatigue had not advanced to the stage where aggression was suppressed. Individual horses, rather than density were the major cause of aggressive behavior. However, two horses went down in the high density treatment, indicating that factors in high density could lead to increased morbidity or death.     

Effect of staining and freezing media on sortability of stallion spermatozoa and their post-thaw viability after sex-sorting and cryopreservation

Sex‐sorted, frozen–thawed stallion spermatozoa remain out of reach of commercial horse breeders because of the low efficiency of the sex‐sorting process and unacceptable fertility rates after insemination. Two experiments were designed to test the effects of alternative staining and freezing media to improve the viability of sex‐sorted frozen–thawed stallion spermatozoa. Experiment 1 compared two freezing media, INRA 82^® and a modified lactose‐ethylenediaminetetraacetic acid (EDTA), for the cryopreservation of sex‐sorted stallion spermatozoa. No significant differences between the two freezing media could be identified, suggesting that both cryodiluents would be suitable for incorporation into a sex‐preselection protocol for stallion spermatozoa. Experiment 2 compared Kenney’s modified Tyrode’s (KMT) and Sperm TALP (Sp‐TALP) as the staining and incubation medium for stallion spermatozoa prior to sex‐sorting. A significant increase in the percentage of acrosome‐reacted spermatozoa occurred after staining and incubation in the clarified Sp‐TALP compared with KMT. As no improvements in sorting rates were achieved using Sp‐TALP, it was concluded that stallion sorting protocols could include KMT as the staining and incubation medium while either INRA 82^® or lactose‐EDTA could be employed as a cryodiluents.     

The In Vitro Secretion of Acetylcholinesterase by Adult Stages of Heligmosomoides polygyrus: the Effects of Broad‐Spectrum Anthelmintics

The secretion of acetylcholinesterase (AChE) by female and male Heligmosomoides polygyrus was studied in different in vitro culture media. AChE secretion was increased in the presence of fetal calf serum or bovine serum albumin (BSA). In the absence of crowding effects, specific AChE activity in excretion/secretion products was higher for male (2.41 ± 0.07 µmol min^?1 l^?1 mg^?1) than for female (0.56 ± 0.04 µmol min^?1 mg^?1) worms but on a per nematode basis both sexes showed comparable rates of secretion. Acetylthiocholine iodide was the favoured substrate of the enzyme. When the nematodes were incubated in vitro with albendazole (ABZ), ricobendazole (RBZ), mebendazole (MBZ), levamisole (LVM), morantel (MRT) or ivermectin (IVM), at concentrations from 1 mM to 10 nM, in RPMI medium for 2 or 6 h and then transferred to a drug‐free medium (RPMI medium supplemented with 0.5 % BSA) for 24 h or continuously exposed to the drugs in supplement‐free medium (24 h), the concentration‐ and time‐dependent inhibitory effects on AChE secretion were observed. The continued exposure to the drugs for all incubation periods (with a single exception for LVM 1 mM) produced the highest levels of inhibition. Under these conditions, the concentrations inhibiting the secretion of AChE by 50 % (IC₅₀) relative to drug‐free controls were estimated. The IC₅₀ values ranged from 0.012 µM (IVM) to 2.96 µM (MRT). The potential of this bioassay for the selective primary evaluation of new compounds with broad‐spectrum anti‐nematodal activity is discussed.     

Effects of leptin and leptin peptide amide on the release of luteinizing hormone, growth hormone and prolactin from cultured porcine anterior pituitary cells

The present study was carried out to determine whether leptin or leptin (116–130) peptide amide (lep (116–130)), an active fragment of the native protein in rats, is able to stimulate the release of luteinizing hormone (LH), growth hormone (GH) or prolactin (PRL) from cultured porcine anterior pituitary (AP) cells in vitro. The AP cells were obtained from 6 month‐old pigs and were incubated for 3 h with 10^?11?10^?7 mol/L leptin or lep (116–130) after being cultured in Dulbecco's modified Eagle's medium for 3–4 days. Leptin significantly increased the concentration of LH and GH in the culture medium at concentrations of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin did not increase the concentration of PRL in the culture medium. In contrast to these results, no effects of lep (116–130) on the release of LH, GH or PRL were seen in the cultured cells. These results suggest that leptin stimulates the release of LH and GH by acting directly on porcine AP cells, and that a fragment of leptin protein comprising amino acids 116–130 is not associated with the secretion of hormones in pigs.     

Feline Leukaemia Virus Testing

Sir, — Cat owners, cat breeders and veterinarians are now well aware that feline leukaemia virus (Felv) is a cause of serious disease in cats and also that Felv is involved in the pathogenesis of many diseases other than lymphosarcoma⁽⁸⁾. Epidemiological studies using a variety of techniques to detect virus and antibodies have defined populations of cats that are at risk from the effects of the virus and methods of control have been suggested^(2)(3)(4). All methods of control are based on separation cf Felv-infected cats from susceptible animals. To do this, viraemic cats need to be identified either by the demonstration of Felv-specific antigen in the cytoplasm of circulating leucocytes or platelets (by immunofluorescence on blood smears), demonstration of viral antigen in serum or plasma, or by isolation of virus from the plasma in feline cell cultures.     

Effect of pH on rheotaxis of bull sperm using microfluidics

The aim of the present research is to study the effect of pH values on the sperm rheotaxis properties. Semen collected from bulls was diluted with SOF medium (1:10). pH of the medium was adjusted using a digital pH meter to the following pH values: 6.0, 6.2, 6.4, 6.4, 6.8, 7.0. All kinetic parameters of sperm (n = 3,385) were determined through a computer‐assisted sperm analysis (CASA) system using microfluidic devices with controlled flow velocity. The following parameters were determined: total motility (TM%), positive rheotaxis (PR%), straightline velocity (VSL, μm/s), average path velocity (VAP, μm/s), linearity (LIN, as VSL/VCL, %), beat cross‐frequency (BCF, Hz) and curvilinear velocity (VCL, μm/s). Nitric oxide, calcium and potassium were estimated in semen at different pH values. To confirm the effect of nitric oxide and K⁺, we used sodium nitroprusside (an NO donor) and KCL as (a K⁺ donor) to see their effect on sperm PR%. The results showed no difference in TM% at pH (6–7). The PR% was the lowest at pH 6 and 7. The best parameters for the PR% were at pH 6.4–6.6. The concentration of Ca⁺² did not change at different pH values. The mean NO values decreased with the increase of pH; however, the mean values of K⁺ increased with the increase of pH. Addition of high concentration of NO and K⁺ to the semen media at fixed pH level had a negative effect on TM% and PR%. In conclusion, the bull sperm had the best rheotaxis properties at pH 6.4–6.6 and sensitive to the change of seminal NO and K⁺.     

Effect of transport enrichment medium, transport time, and growth medium on the detection of Campylobacter fetus subsp. venerealis.

The combination of medium and growth conditions, including transport enrichment medium (TEM), transport time, TEM incubation time, and growth medium, that best support Campylobacter fetus subsp. venerealis while inhibiting contaminants was studied. The 3 TEMs evaluated, Weybridge, Cary-Blair, and 0.85% saline solution, were inoculated with preputial smegma spiked with C. fetus subsp. venerealis and held in the laboratory for 4 or 24 hours before inoculation onto growth medium. The effect of overnight incubation at 37 C of the TEM was also evaluated. Median scores of C. fetus subsp. venerealis growth and microbial contaminant inhibition were compared within TEM, transport time, overnight incubation, and growth medium groups using the Mann-Whitney U-test and the Kruskal-Wallis test. The proportion of samples with any growth or contamination within each group was also compared using the chi-square test. Results suggest that the growth of C. fetus subsp. venerealis was influenced by 3 of the 4 criteria evaluated. Weybridge TEM more effectively maintained the organism than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time of 4 hours rather than 24 hours (P < 0.001) and avoiding overnight incubation of TEM at 37 C (P < 0.001) were associated with improved growth. Significant differences were not identified among growth media; however, Skirrow Campylobacter agar appeared to yield slightly better growth than did either blood agar or Greenbriar Plus agar. Contaminant growth was also influenced by 3 of the 4 variables. Weybridge TEM inhibited contaminant growth more effectively than did either Cary-Blair or 0.85% saline solution (P < 0.001). Transport time was not associated with contaminant growth. Eliminating overnight incubation of TEM reduced contamination (P < 0.01). Skirrow agar was preferred to both blood agar and Greenbriar Plus agar for suppression of contaminants on solid medium (P < 0.001). These results suggest that the detection of C. fetus subsp. venerealis is enhanced when preputial smegma samples arrive at the diagnostic laboratory within 4 hours after inoculation into Weybridge TEM and are transferred to Skirrow agar the same day they arrive in the laboratory.     

Detection of Coxiella burnetii, the agent of Q fever, in oviducts and uterine flushing media and in genital tract tissues of the non pregnant goat

The aim of the present study was the detection and quantification of Coxiella burnetii DNA in the flushing media (oviducts and uterine horns) and genital tract tissues of non pregnant goats from 20 goats chosen at random from 86 goats originating from 56 different breeding herds in south-west France. The serological prevalence rate of C. burnetii in the study population was 70.3%.The DNA of C. burnetii was identified using conventional PCR in the flushing media from the oviducts and uterus in 8/20 goats (40%) and in genital tract tissues (oviduct, uterus and ovary) in 5/20 goats (25%). This study clearly shows for the first time that the media used to flush the oviducts or uterine horns, collected using the standard embryo harvesting technique in goats, are susceptible to infection with C. burnetii. The 16 conventional PCR-positive samples were also analyzed using real-time PCR. The bacterial load of the oviduct and uterine flushing media varied from 2.9 × 10⁴ to 7.5 × 10⁶ bacteria per flushing medium, while the bacterial load of the tissue samples varied from 1.0 × 10² to 1.5 × 10⁵ bacteria per mg of tissue. The infection of genital tract flushing media and tissues is a risk factor for the transmission of C. burnetii from donor to recipient during embryo transfer or to the embryo and fetus when gestation is pursued to term.     

Comparative in vitro efficacy of antimicrobial shampoos: a pilot study

This study compared the antimicrobial efficacy of shampoos against meticillin‐sensitive Staphylococcus pseudintermedius (MSSP), meticillin‐resistant S. pseudintermedius (MRSP), antibiotic‐sensitive Pseudomonas aeruginosa (PA), multidrug‐resistant P. aeruginosa (MDR‐PA) and Malassezia pachydermatis. Three isolates were incubated for 10, 30 and 60 min with each shampoo diluted in phosphate‐buffered saline. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud’s dextrose agar (Malassezia). The minimal bactericidal concentrations (MBCs) for chlorhexidine products (Malaseb^®, Pyoderm^®/Microbex^® and Hibiscrub^®) were 1:1,024–1:2,048 for MSSP and MRSP, 1:512–1:1,024 for PA and MDR‐PA, and 1:2,048–1:5,096 for Malassezia at all time points. The MBCs for benzoyl peroxide (Paxcutol^®) for MSSP and MRSP were 1:2–1:8 at 10 min, and 1:256 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and 1:512–1:1,024 dilutions were effective against Malassezia at all time points. The MBCs for ethyl lactate (Etiderm^®) for MSSP and MRSP were 1:2 at 10 min, and 1:2–1:16 after 30 and 60 min. A 1:2 dilution was effective against Pseudomonas, and a 1:512 dilution was effective against Malassezia at all time points. Chloroxylenol (Coatex^®) and acetic acid–boric acid (Malacetic^®) were not effective against MSSP, MRSP or Pseudomonas. Both were effective against Malassezia at 1:8–1:16 dilution at 10 min, and at 1:8–1:32 dilution after 30 and 60 min. In conclusion, chlorhexidine appeared to be the most effective topical biocide, and MRSP and MDR‐PA were no less susceptible than antibiotic‐sensitive organisms. These results should, however, be confirmed with larger numbers of isolates.     

Growth of Yersinia pseudotuberculosis on selective media

As a prelude to experiments designed to evaluate the growth of Yersinia pseudotuberculosis on frozen vacuum-packed venison, we wished to assess the sensitivity of the commonly used selective medium, Yersinia selective agar (YSA) with the added antibiotics cefsu- lodin, irgasan and novobiocin (CIN agar), for recovery and enumeration of this organism⁽¹⁾.     

Microflora in traditional starter cultures for fermented milk, hurunge, from Inner Mongolia, China

Microflora were investigated in traditional starter cultures for fermented milk, hurunge, which are used for fermented dairy products by nomadic families in the Inner Mongolia Autonomic Region, China. The acid‐forming bacteria and yeast counts ranged from 1.8 × 10⁵ to 5.3 × 10⁸ c.f.u./g and from 6.1 × 10⁵ to 3.2 × 10⁶ c.f.u./g, respectively. Sixty‐six strains of lactic acid bacteria and 30 strains of yeasts were isolated and identified from three hurunge samples collected from the nomadic families. Lactococcus raffinolactis was the most predominant lactococci isolated from these samples. The other lactococci were Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Leuconostoc mesenteroides ssp. cremoris. Two major lactobacilli strains, Lactobacillus plantarum and Lactobacillus casei, were identified. In addition, Lactobacillus kefiranofaciens, Lactobacillus acetotolerans, which grew in 11% acetic acid culture medium, and Lactobacillus homohiochii, which grew in the culture medium containing 16% ethanol, were also identified. The isolated yeast strains were identified as Candida kefyr, Saccharomyces cerevisiae, Kluyveromyces marxianus var. lactis, Candida krusei and Candida valida.     

Intra-uterine transmission of Mycobacterium avium subsp paratuberculosis in subclinically affected red deer (Cervus elaphus)

AIM: To determine the rate of transmission of Mycobacterium avium subsp paratuberculosis (M. ptb) from hind to fetus in utero, and the risk of transmission from dam to fawn via infected colostrum and milk in subclinically affected red deer hinds.

METHODS: Hinds were sourced from farms in Otago or Southland and selected for the study if they were positive to the immunoglobulin G1 (IgG1) modified enzyme-linked immunosorbent assay (ELISA) (Paralisa) and exhibited no clinical signs of Johne's disease. The hinds (n=35) were sent to a deer slaughter premises (DSP; n=31) or were killed on-farm (n=4). All post-mortem samples were collected from the fetus first and then from the dam, taking care to avoid cross contamination between samples. Fresh samples (n=185) were collected for culture, and tissue samples (n=72) were collected from 24 hinds and their fetuses for histopathological examination.

RESULTS: A total of 24/35 hinds selected were suitable for inclusion in the study. Eighteen of these pregnant hinds were culture-positive for M. ptb, and 14 of these had culture-positive fetuses, representing a transmission rate of 78% (95% confidence interval (CI) =0.58–0.98) from dam to fetus. Of the 16 mammary glands sampled, 11 (69%) were culture-positive for M. ptb while 12/15 (80%) mammary lymph nodes sampled were also culture-positive.

CONCLUSIONS: This study demonstrated a high rate of transmission of M. ptb from dam to fetus in red deer, and a potential risk of transmission to fawns suckling from mothers that are subclinically affected with Johne's disease.     

Addition of erythrocytes to in vitro culture medium attenuates the detrimental effects of reactive oxygen species on bovine preimplantation embryo development

Erythrocytes were recently found to improve the early development of mice embryos by their antioxidant effect. The purpose of the present study was to examine the effect of erythrocytes on the in vitro development of bovine in vitro fertilized (IVF) embryos in medium supplemented with reactive oxygen species (ROS). IVF embryos were cultured in CR1aa medium supplemented with oxidizing agents, 0.5 mmol/L hypoxanthine and 0.01 U/mL xanthine oxidase (HX/XOD), in the presence and absence of erythrocytes (5 × 10⁴, 5 × 10⁵, 5 × 10⁶ and 5 × 10⁷ erythrocytes/mL). After 8 days, blastocysts were examined with a stereomicroscope. HX/XOD blocked development to the blastocyst stage (HX/XOD: 0%, control: 33%), but in the presence of both erythrocytes and HX/XOD, blastocyst development was restored to about one‐third to two‐thirds the normal rate (5 × 10⁵ to 5 × 10⁷ erythrocytes/mL: 12 to 23%). Furthermore, adding erythrocytes or erythrocyte hemolysate to medium without HX/XOD increased the blastocyst rate. These results suggest that the addition of erythrocytes can attenuate the detrimental effects of ROS on embryo development in bovine species as well as in mice.     

Effects of VEGF and bFGF on Proliferation and Production of Steroids and Nitric Oxide in Porcine Granulosa Cells

Ovarian angiogenesis, which is currently considered to be of crucial importance in controlling the growth of developing follicles, is a physiological process driven by a variety of angiogenic factors. Among these, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been recognized as key players in promoting cell growth and differentiation. Porcine granulosa cells from small (<3 mm), medium (3–5 mm) and large (>5 mm) follicles were seeded at different densities in DMEM:Ham's F12 (1:1) with or without different concentrations of VEGF or bFGF. After 48 h of culture, media were assayed for oestradiol (E2) 17β, progesterone (P4), nitric oxide (NO) and VEGF levels; in addition, cell proliferation was evaluated by ³H‐thymidine incorporation assay. Both bFGF and VEGF effects on E2 and P4 production by cultured granulosa cells resulted to be dependent on follicle size. The bFGF was always ineffective in modulating cell proliferation, while VEGF exerted an inhibitory effect on the proliferation in the small follicle group and a stimulatory one in the medium and large follicle groups. The bFGF consistently reduced NO levels in culture media. The VEGF appeared to be ineffective in modifying NO production in the small follicle group, while it was stimulatory in the medium follicle group and inhibitory in the large follicle group. Basal VEGF production was higher in cells from the large follicle as compared with the small and medium follicle groups, and it was unaffected by bFGF. These results suggest that VEGF plays a modulatory role in granulosa cell functional activity and it is possibly involved in the regulation of follicle growth; on the contrary, bFGF does not appear to represent a significant regulatory factor in our cellular model, except for an inhibitory action on the production of NO, whose anti‐angiogenic properties need to be further substantiated.