Follicular waves and circulating concentrations of gonadotrophins,inhibin and oestradiol during the anovulatory season in mares

Follicular waves and associated circulating hormone concentrations were studied during the anovulatory season in pony mares (n=8). Follicles were monitored by ultrasonography and a blood sample was taken daily from 29 January until ovulation (mean, 28 April). A mid-anovulatory period (largest follicle, 16.0+/-0.5 mm in diameter) and transitional period (largest follicle, 22.4+/-0.5 mm) were distinctive in each mare. The two periods were delineated by an increase in the diameter of the largest follicle to >/=21.0 mm. Follicular waves, identified by significant increases in the mean diameter of the second to sixth largest follicles, were detected during both the mid-anovulatory and transitional periods. The mean number of follicles >/=15.0 mm in diameter and the diameter of the second to sixth largest follicles increased in association with statistically identified FSH surges. The pattern of the FSH concentration changes during surges did not change during the mid-anovulatory and transitional periods. During the declining portion of the FSH surge, follicle growth continued and circulating total inhibin increased, indicating suppression of FSH by inhibin from the growing follicles. Circulating oestradiol or LH did not change relative to wave emergence. Results indicated that follicular waves occurred during the second-half of the anovulatory season, even during the period of lowest follicular activity. On a temporal basis, follicular wave emergence was stimulated by surges in circulating FSH. However, the increase in follicle growth to >/=21.0 mm in diameter for the wave at the beginning of the transitional period and for the subsequent waves was not attributable to a change in the characteristics of the associated FSH surges.     

Characterization of morphology and angiogenesis in follicles of mares during spring transition and the breeding season

The mare is a seasonal breeder and undergoes a period of ovarian transition in spring between winter anoestrus and cyclicity. During spring transition LH concentrations are low and many mares have successive large anovulatory follicular waves which reach the size of preovulatory follicles. Follicular angiogenesis is essential for growth and health of preovulatory follicles. The aim of the present study was to investigate the morphology and vascularity of transitional anovulatory follicles. On gross inspection, the wall of transitional follicles was visibly less well vascularized than that of preovulatory follicles. Histologically, it could be seen that the theca was only poorly developed in transitional follicles. Immunostaining for factor VIII showed that there were significantly (P < 0.05) fewer blood vessels in the theca of transitional follicles. There was substantially less (P < 0.001) proliferative activity, measured by immunostaining for Ki67, in the endothelial cells and granulosa cells of transitional follicles compared with preovulatory follicles. Preovulatory follicles had a heavy band of immunostaining in the theca for vascular endothelial growth factor (VEGF), whereas staining was sparse in the transitional follicles. It was concluded that the poor vascularity and development of the theca layer in transitional follicles could be related to low circulating LH, and possibly other trophic hormones, and are likely to be the key factors in explaining the steroidogenic incompetence of transitional anovulatory follicles.     

Systemic concentrations of hormones during the development of follicular waves in mares and women: a comparative study

Changes in systemic concentrations of FSH, LH, oestradiol and progesterone during the ovulatory follicular wave were compared between 30 mares and 30 women. Based on a previous study, the emergence of the future ovulatory follicle was defined as occurring at 13.0 mm in mares and 6.0 mm in women, and deviation in diameter between the two largest follicles was expected to begin at 22.7 mm in mares and 10.3 mm in women. Mean FSH concentrations were high in mares during the luteal phase, resulting from statistically identified FSH surges occurring in individuals on different days and in different numbers (mean, 1.5 +/- 0.2 surges/mare); the internadir interval was 3.9 +/- 0.3 days. In contrast, mean FSH in women was low during the luteal phase and increased to a prolonged elevation during the follicular phase. The prolonged elevation was apparent in each individual (internadir interval, 15.2 +/- 0.4 days). Changes in LH or oestradiol concentrations encompassing deviation were not detected in mares, but both hormones increased slightly but significantly between emergence and deviation in women. The hypothesis that a greater number of growing follicles causes a greater predeviation decrease in FSH was supported for mares (r, -0.39; P< 0.04), but a similar negative correlation (r, -0.36) was not significant in women. The hypothesis that the increase in oestradiol during the luteal phase in women was at least partly attributable to luteal-phase anovulatory follicular waves was not supported. Normalization of FSH concentrations to the day of emergence showed maximum value on the day of emergence with a significant increase and decrease on each side of emergence in both species. The day of expected deviation occurred 3 days after emergence during the decline in FSH in both species. These results indicated that the previously reported striking similarities in emergence and deviation between mares and women during the ovulatory follicular wave are associated with species similarities in the temporal relationships between follicle events and FSH concentration changes. Thus, mares may be useful research models for studying the role and mechanism of the action of FSH in emergence and deviation during the ovulatory follicular wave in women.     

Interactions of follicular factors and season in the regulation of circulating concentrations of gonadotrophins in mares

Follicle growth and associated changes in circulating hormone concentrations were evaluated after follicle ablation in mares (n = 13) during four 13 day periods beginning at means of -98.5, -61.4, -26.0 and 10 days from the first ovulation and corresponding to mid-anovulatory, early-transitional, late-transitional and ovulatory periods, respectively. During each period, all follicles > 5 mm in diameter were ablated (day 0) followed by no subsequent ablations (all-follicle group) or the ablation of all follicles > 5 mm when a new follicle reached > 10 mm (0-follicle group). A follicular wave emerged at means of days 1.5-2.5 in all mares of the all-follicle group during each period. Follicle activity increased between the mid-anovulatory and the transitional periods, as indicated by increases in the diameter of the largest follicle and the number of follicles in the all-follicle groups; nonetheless, there were no differences in the FSH concentrations of the wave-stimulating surge throughout the study. Circulating total inhibin increased between day 3 and day 6 in the all-follicle groups but not in the 0-follicle groups, and was higher during the late-transitional and ovulatory periods than during earlier periods in the all-follicle groups. In the all-follicle groups, circulating FSH decreased between day 3 and day 6. In the 0-follicle groups, FSH did not decrease and was higher during the late-transitional and ovulatory periods than during the mid-anovulatory period. Circulating LH was higher during the late-transitional and ovulatory periods than during earlier periods and was not different between the all-follicle and 0-follicle groups. On the basis of temporal relationships, it was concluded that the follicles of a wave secreted inhibin during all periods and the follicular inhibin suppressed circulating FSH. An increase in circulating inhibin induced by the growth of follicles > 21 mm in diameter during the late-transitional and ovulatory periods counteracted a stimulatory effect on FSH mediated by season.     

In vivo effects of pregnancy-associated plasma protein-A, activin-A and vascular endothelial growth factor on other follicular-fluid factors during follicle deviation in mares

During a follicular wave in mares, the two largest follicles (F1 and F2) begin to deviate in diameter when F1 is a mean of 22.5 mm. The intrafollicular effects of pregnancy-associated plasma protein-A (PAPP-A), IGF-I, activin-A and vascular endothelial growth factor (VEGF) on other follicular-fluid factors during deviation were studied. In four treated groups (n = 7/group), a single dose of one of the four factors was injected into F2 when F1 was > or = 20.0 mm (expected beginning of deviation). In a control group (n = 7), F2 was injected with vehicle. One day after treatment, a sample of follicular fluid was taken from F1 and F2 of the control group and from F2 of the treated groups and was assayed for free IGF-I, oestradiol, androstenedione, activin-A, inhibin-A, follistatin and VEGF. In the control group, the means for all end points were significantly greater in F1 than in F2, except that concentrations of androstenedione were lower in F1 than in F2. The treatment effects for F2 were significant as follows: PAPP-A increased the concentrations of free IGF-I, inhibin-A, follistatin and VEGF and decreased the concentrations of androstenedione; IGF-I increased the concentration of inhibin-A and decreased the concentration of androstenedione; activin-A decreased the concentrations of follistatin and androstenedione and increased the diameter of F2; and VEGF increased the concentration of IGF-I and decreased the concentration of androstenedione. These results support the hypotheses that during deviation in mares PAPP-A increases the follicular-fluid concentrations of free IGF-I, follistatin responds to changes in follicular-fluid concentrations of activin-A, and VEGF affects the concentrations of other follicular-fluid factors.     

Plasma FSH, inhibin A and inhibin isoforms containing pro- and -alphaC during winter anoestrus, spring transition and the breeding season in mares

Ten mares were studied from February (winter anoestrus) to their second ovulation in the breeding season to investigate the relationship between resumption of ovarian cyclicity in the spring and circulating concentrations of FSH, inhibin A and inhibin isoforms containing pro- and -alphaC immunoreactivity. An additional four mares were studied during one oestrous cycle. Growth and regression of ovarian follicles were monitored by transrectal ultrasonography. The frequency of blood sampling varied from three times a week to once a day, depending on the follicular activity present. Concentrations of FSH, oestradiol, inhibin A and pro- and -alphaC isoforms were low during deep winter anoestrus when minimal follicular activity was present in the ovaries. During spring transition, an increase in FSH concentration preceded the emergence of each follicular wave. Concentrations of inhibins were significantly higher (P < 0.05) during growth of anovulatory follicles in spring transition than during winter anoestrus. Plasma concentrations of oestradiol and inhibin A were significantly higher (P < 0.001, P < 0.05, respectively) during the growth of preovulatory follicles than during the growth of transitional anovulatory follicles, but concentrations of inhibin pro-alphaC isoforms did not differ between the two types of follicle. During the oestrous cycle, there was a significant inverse relationship (P < 0.001) between concentrations of FSH and the inhibins. Plasma inhibin pro-alphaC isoforms, but not inhibin A, reached a peak on the day of ovulation. The results strongly indicate that FSH regulates growth of spring anovulatory and preovulatory follicles. Inhibins are likely to contribute to negative feedback on the release of FSH from the pituitary gland both during the transitional period and the breeding season in mares.     

Secretion of matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinases into follicular fluid during follicle development in equine ovaries

Extensive tissue remodelling is required in equine ovaries for follicle growth and development and also migration of the follicle to the ovulatory fossa, where ovulation occurs. The mechanisms for these processes are largely unexplored. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) are important for control of breakdown of extracellular matrix during tissue remodelling. The aims of this study were to determine the pattern and sites of secretion of the gelatinases MMP-2 and -9 and TIMPs into follicular fluid during follicle development in mare ovaries. The predominant gelatinase detected in follicular fluid was MMP-2, which was present in similar amounts throughout follicular development, as demonstrated by zymography. MMP-9 was also present in follicular fluid and secretion increased significantly (P < 0.05) with development of follicles from < 10 mm to 11-20 mm in diameter. Follicular fluid also contained TIMP-1, TIMP-2, unglycosylated and glycosylated TIMP-3, and TIMP-4, as shown by reverse zymography. The abundance of TIMPs remained largely unchanged during follicle development. MMP-2 and -9 were localized by immunohistochemistry to stromal cells and granulosa and theca cells. TIMP-1, -2, -3 and -4 were present in granulosa and theca cells of the follicle and in stromal cells and also associated with extracellular matrix of the ovarian stromal tissue. The MMPs and TIMPs are likely to be involved in the regulation of the breakdown of extracellular matrix during tissue remodelling for follicle development and migration to the ovulation fossa in mares.     

Relationship between metabolic hormones and ovulation of dominant follicle during the first follicular wave post-partum in high-producing dairy cows

Recent studies suggest that IGF-I is a crucial regulatory factor in follicular growth during early post-partum period. The aim of the present study was to determine in detail the changing profiles of metabolic and reproductive hormones in relation to ovulation of the dominant follicle (DF) of the first follicular wave post-partum in high-producing dairy cows. Plasma concentrations of related hormones in 22 multiparous Holstein cows were measured from 4 weeks pre-partum to 3 weeks post-partum, and the development of DF was observed with colour Doppler ultrasound. Thirteen cows showed ovulation by 15.2 days post-partum. Anovulatory cows showed higher GH and lower IGF-I levels than those in ovulatory cows during the peri-partum period. Each DF developed similarly, and a clear blood flow in the follicle wall was observed despite ovulation or anovulation. In addition, detailed endocrine profiles were analyzed in 9 out of the 22 cows. Five cows showed an increase in plasma oestradiol-17beta (E2) with follicular growth followed by E2 peak, LH surge and ovulation. In these cows, plasma IGF-I concentrations remained high until 10 days post-partum followed by a gradual decrease. Subsequently, the insulin level increased together with the E2 peak towards ovulation. These profiles were not observed in anovulatory cows. In conclusion, our data strongly support the concept that IGF-I and insulin represent 'metabolic signals' of the resumption of ovarian function post-partum in high-producing dairy cows. Moreover, we provide the first visual evidence that both ovulatory and anovulatory DFs of the first follicular wave post-partum are similarly supplied with active blood flow.     

The effect of marine algae in the ration of high-yielding dairy cows during transition on metabolic parameters in serum and follicular fluid around parturition

Sixteen Holstein cows were assigned to 2 groups to evaluate the caloric and metabolic effect of feeding marine algae (ALG) from 3 wk prepartum until 12 wk postpartum. Milk production characteristics and the profiles of hormones and metabolites in the serum were monitored from −7 to 46 d in milk (DIM) and in follicular fluid (FF) from 14 to 46 DIM. All cows received a corn- and grass silage-based partially mixed ration supplemented with concentrate and protein supplement. In the diet of the ALG group, 2 kg of the concentrate was replaced by a concentrate containing ALG (44 g/d of docosahexaenoic acid). Diets were isocaloric (net energy basis) and equal in intestinal digestible protein. The ALG diet increased milk yield (41.2 vs. 38.2 kg/d) and decreased milk fat yield (1.181 vs. 1.493 kg/d) and milk fat content (31.6 vs. 40.7 g/kg). Protein yield (1.336 vs. 1.301 kg/d) was not affected but a tendency toward decreased milk protein content (32.8 vs. 34.7 g/kg) was observed. Marine algae supplementation increased the β-hydroxybutyric acid (BHBA) concentration in FF of the ALG cows compared with that in the controls (0.992 vs. 0.718 mmol/L). The total protein concentration in FF was decreased in ALG (62.9 vs. 67.6 g/L). Plasma and serum metabolites did not significantly differ between treatments except for a tendency toward a lower concentration of urea in the serum of the control compared with ALG (4.69 vs. 5.13 mmol/L). Based on metabolizable energy calculations, a daily energy-sparing effect of 3.48 Mcal was obtained due to milk fat depression (MFD). The concomitant increase in milk yield suggests that at least part of this spared energy is used to stimulate milk production. Theoretically, 3.48 Mcal of ME could lead to an increase in milk yield of 7.43 kg/d, which is higher than the observed 3 kg/d. However, when evaluating nutrient requirements during MFD in early lactation, we calculated that increased milk production is caused by a propionate-saving effect of 2.71 mol in the udder when milk fat is depressed. Concurrent increased BHBA concentrations in FF in the ALG group cannot be attributed to a worsened energy status of the animals because all other indicators contradict any change in energy balance, indicating that BHBA might not be an appropriate metabolic parameter to estimate the energy balance in early lactating dairy cows during MFD.     

Use of a direct-fed microbial product as a supplement during the transition period in dairy cattle

Two studies were conducted. The objective of the first study was to assess the effects of a direct-fed microbial (DFM) product on dry matter intake, milk yield, milk components, disease incidence, and blood metabolites in dairy cattle. The objective of the second study was to assess the effects of DFM on apparent total-tract nutrient digestibility (ATTD). One hundred twenty primiparous and multiparous Holstein cows housed in a tiestall facility at the University of Guelph were used in study 1, and a subset (21) of the same cows participated in study 2. Cows were blocked by anticipated calving date (6 blocks) and then randomly assigned within parity to receive either a DFM supplement (Chr. Hansen Ltd., Milwaukee, WI) or placebo (control). The DFM supplement provided cows with 5.0 × 10⁹ cfu/d of 3 strains of Enterococcus faecium and 2.0 × 10⁹ cfu/d of Saccharomyces cerevisiae. The DFM supplement was mixed with 0.5 kg of ground dry corn and top-dressed during the morning feeding. The placebo supplement contained the corn only. Individual feed intakes and milk yields were recorded daily. The experiment commenced 3 wk before calving and ended 10 wk postcalving. Milk samples for component analysis were collected on 3 d per week and pooled by week. Body weights and body condition scores were assessed 1 d before enrollment in the study (wk –3), postcalving (wk 1), and at the end of wk 3, 6, and 9. Blood samples were collected before calving (wk –3) and the end of wk 1 and 3. Study 1 showed that treatment had no effect on average dry matter intake or milk yield (kg/d) over the duration of the experiment. The changes in body weights and body condition scores and net energy balance over the duration of the experiment did not differ due to treatment. Treatment had no effect on plasma concentrations of β-hydroxybutyrate, nonesterified fatty acids, glucose, or haptoglobin. Study 2 investigated the effects of DFM on ATTD of starch and neutral detergent fiber (NDF) using insoluble NDF and lignin as internal markers. Study 2 used 21 cows (block 6) from the cows that participated in study 1 while the cows were between 60 and 70 d in milk. Cows receiving DFM had lower fecal starch content (0.88 ± 0.10 vs. 1.39 ± 0.25) and greater ATTD for starch (98.76% ± 0.28 vs. 97.87% ± 0.24) compared with those receiving placebo, and the AATD of NDF did not differ. Additionally, we detected no difference between internal markers for the measurement of ATTD. In conclusion, we were unable to detect a change in overall dry matter intake, milk yield, or milk and blood parameters with DFM supplementation. However, our results demonstrated that DFM can have a positive effect on total-tract starch digestibility. More studies are needed to investigate the effects of DFM and their modes of action under multiple management conditions.     

Chromosome translocation induced by the insertion of the URA blaster into the major repeat sequence (MRS) in Candida albicans

Electrophoretic karyotype studies have shown that clinical isolates of Candida albicans have extensive chromosome length polymorphisms. Chromosome translocation is one of the causes of karyotypic variation. Chromosome translocation events have been shown to occur very frequently at or near the major repeat sequence (MRS) on chromosomes. The MRS consists of the repeated sequences RB2, RPS and HOK, and the repeated sequences are considered to be the template for recombination. To investigate which element of the MRS is important for chromosome translocation, we constructed three cassettes, each containing a URA blaster and sequences homologous to one of the repeats, for insertion into the MRS region on the chromosomes. The ura3 strain STN22u2, which shows a stable, standard karyotype, was transformed with each construct. Insertion events with each cassette occurred at almost all chromosomes. Insertion into the RB2 repeat, but not into the RPS repeat, was accompanied by chromosome translocation in some transformants: chromosome translocations between chromosomes R and 7 and chromosomes 1 and 7 were found, as well as deletions of 7A and 7C from chromosome 7. We conclude that the insertion at the RB2 region may initiate chromosome translocation in C. albicans.     

Some insight into the mechanism of inhibition of psychrotrophic bacterial growth in raw milk by lactic acid bacteria

Addition of lactic acid bacteria to refrigerated raw milk inhibited growth of Gram-negative psychrotrophic bacteria. In order to obtain complete inhibition, population levels of lactic acid bacteria approaching 1× 10₈ cfu/ml were required. At this inoculum level a substantial pH change was observed in the milk during storage. The degree of inhibition was related to the final pH of the milk. Inhibition of the psychrotrophs was brought about by adjustment of pH with L(+) lactic acid but not by HCI. Inhibition by the lactic acid bacteria was observed in heat-treated milks seeded with a small volume of raw milk, but no inhibition of Gram-negative psychrotrophic growth was observed when cell-free preparations of lactic acid bacteria were used. A cell-free ultrafiltrate of one lactic acid culture did inhibit a strain of Bacillus circulans.     

Identification by epr spectroscopy of carvacrol and thymol as the major sources of free radicals in the oxidation of plant essential oils

The formation of stable free radicals upon reaction of the essential oils of 10 plant species with ultraviolet radiation and the superoxide radical anion, O₂^?, has been investigated by electron paramagnetic resonance (EPR) spectroscopy. Only the oils from oregano, summer savory and thyme produced EPR spectra and the results can be accounted for in terms of free radicals produced by the closely related phenolics, carvacrol and thymol. These observations are interpreted in terms of the known antioxidant properties of these molecules.     

虾类保藏过程中酚氧化酶酶促黑变作用机理及其抑制方法的研究进展

在虾类贮藏过程中,虾体的黑变严重影响了虾的感官品质,酚氧化酶是催化酪氨酸等色原物质转化为黑色素造成虾体黑变的主要因素。文章综述了国内外酚氧化酶的形成机制研究现状,重点叙述了虾类中酚氧化酶的特性及抑制虾类黑变方法的研究进展,为虾类贮藏保鲜技术的研发与改进提供参考。      

Quantification of milk yield and composition changes as affected by subclinical mastitis during the current lactation in sheep

The aim of this work was to quantify, on a half-udder basis, the changes in ewe milk yield and composition caused by unilateral subclinical mastitis within the current lactation. Fluctuations due to production level, infection severity, time from the onset of infection, and lactation curves were also studied. Yield and composition of milk from half-udders of unilateral infected ewes were compared between them and with a set of healthy halves using a mixed model. The experiment was completed with a whole-udder approach on the same animals. To test the effect of intramammary infection (IMI) in the 7 wk following the onset of infection, 20 ewes that acquired unilateral subclinical mastitis during lactation and 40 healthy ewes were used. Another group of 20 unilaterally infected ewes from wk 1 of lactation and other 40 healthy ewes were studied to test the effect of IMI on lactational milk yield and composition. The individual milk loss in ewes infected during lactation was 15% for the 7 wk following the onset of infection, and 6.6% more milk was produced by the uninfected half to compensate milk lost by the infected half. Lactational milk yield loss in ewes infected from wk 1 postpartum was 17%. The changes in milk yield were noticed from the week of infection diagnosis. The production level of animals influenced the milk yield changes caused by IMI in such a way that the more productive ewes lost more milk, although these losses were proportional to their production level. On the other hand, infection severity affected milk loss between glands, being more pronounced as somatic cell count increased. A clear decrease of lactose content and casein:protein ratio due to subclinical IMI was observed and it remained throughout the postinfection period. Improving udder health status is necessary to maintain milk production and quality in dairy ewes during lactation.