Effects of dietary supplementation of resveratrol on performance, egg quality, yolk cholesterol and antioxidant enzyme activity of laying hens.

1. This experiment was conducted to evaluate the effects of dietary supplementation of resveratrol on laying performance, egg quality, egg yolk cholesterol and antioxidant enzyme activities of laying hens. 2. A total of 360 Beijing PINK-1 laying hens (60 weeks old) were randomly distributed among five dietary treatments, each of which included 6 replicates of 12 hens. Dietary treatments were basal diet supplemented with 0 (control), 0.5, 1.0, 2.0 and 4.0 g/kg diet resveratrol. The study lasted for 9 weeks including 1 week of adaptation and 8 weeks of the main experimental period. 3. The results indicated that dietary resveratrol significantly improved feed conversion ratios during 5-8 weeks and 1-8 weeks of the trial. Increasing dietary concentrations of the resveratrol linearly improved Haugh unit and albumen height of eggs. 4. The content of total cholesterol (TC), total triglyceride (TG), low density lipoprotein cholesterol (LDL-C), very low density lipoprotein cholesterol (VLDL-C) in serum and cholesterol in yolk was significantly decreased by dietary resveratrol, and there were significant linear correlations between these indexes and resveratrol supplemental levels. 5. Dietary resveratrol supplementation significantly improved serum Glutathione peroxidase (GSH-Px) enzyme activity and decreased serum malondialdehyde (MDA) content in groups with 2.0 and 4.0 g/kg resveratrol as compared to the control, respectively. However, supplementation of resveratrol did not affect the activity of serum superoxide dismutase (SOD). 6. It is concluded that resveratrol supplementation has a positive effect on performance, lipid-related traits and antioxidant activity of laying hens.

Nutrition, metabolism, and fertility in dairy cows: 3. Amino acids and ovarian function.

Plasma insulin concentrations influence resumption of ovarian activity in postpartum dairy cows, and plasma insulin can be manipulated by changing dietary starch and fat supply. The objective of this experiment was to investigate the role of dietary amino acids in altering peripheral metabolic hormones and ovarian function. Thirty-two cows were fed a standard diet from calving until 40 d in milk (DIM), and then 8 cows were transferred to each of 4 dietary treatments until 70 DIM. The 4 diets were designed to supply either low (diets 1 and 2) or high (diets 3 and 4) levels of metabolizable protein (MP), containing either low (diet 1 and 3) or high (diets 2 and 4) proportions of Leu. Leucine was manipulated with heat-treated lupins and corn gluten meal. Estrus was synchronized at 60 DIM. Between 60 and 70 DIM, energy intake and energy balance were similar among diet groups, although cows receiving high MP containing high Leu had a greater milk yield than other groups (means: 37.8, 37.1, 37.4, 39.4 +/- standard error 0.85 kg/d for diets 1 to 4, respectively). Interactions between MP and Leu were found for insulin, glucagon, and the ratio between them. Insulin was not affected by Leu in diets with low MP but was decreased by greater Leu in diets with high MP (means: 0.37, 0.32, 0.46, 0.39 +/- SE 0.031 ng/mL for diets 1 to 4, respectively). Glucagon was not affected by MP in diets with low Leu but was increased by greater MP in diets with high Leu (means: 92, 81, 88, 95 +/- SE 6.0 pg/mL for diets 1 to 4, respectively). For the low-MP treatments, the insulin-to-glucagon ratio was greater with high Leu; for the high-MP treatments, the insulin-to-glucagon ratio was greater with low Leu (means: 4.28, 5.42, 5.16, 4.22 +/- SE 0.456 for diets 1 to 4, respectively). There was no effect of MP or Leu on ovarian follicle numbers or reproductive hormones. Based on hormonal and ovarian responses, we conclude that altering metabolic hormones through manipulation of amino acid supply and balance is unlikely to have a significant effect on ovarian function in dairy cows.

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on bovine granulosa cell steroidogenesis in vitro.

Our previous studies have demonstrated that physiological concentrations of metabolic hormones, including recombinant bovine somatotrophin (BST), insulin-like growth factor-I (IGF-I) and insulin, can significantly stimulate the proliferation of bovine granulosa cells cultured under serum-free conditions. In this study we investigated the effects of these factors on bovine granulosa cell steroidogenesis using the same culture system. Bovine granulosa cells were obtained from antral follicles classified into three size classes: small, < 5 mm; medium-sized, 5-10 mm and large, > 10 mm in diameter. Whilst not affecting steroidogenesis by granulosa cells from small and medium-sized follicles, BST (10-1000 ng/ml) stimulated the secretion of both oestradiol and progesterone by granulosa cells from large follicles in a dose-dependent manner. Insulin (1-1000 ng/ml) and IGF-I (10-1000 ng/ml) stimulated the secretion of oestradiol and progesterone by granulosa cells from all three size categories of follicles in a dose-dependent manner. FSH (200 ng/ml) alone increased progesterone secretion by granulosa cells from all three size classes of follicles, but had no effect on oestradiol secretion by granulosa cells. Both IGF-I (200 ng/ml) and insulin (30 ng/ml) acted in synergy with FSH (200 ng/ml) to stimulate steroidogenesis by granulosa cells from all three size categories of follicles, but no such interaction was observed between BST (50 ng/ml) and FSH (200 ng/ml). In conclusion, BST, IGF-I and insulin significantly influence the steroidogenic activity of bovine granulosa cells cultured under serum-free conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of recombinant bovine somatotrophin, insulin-like growth factor-I and insulin on the proliferation of bovine granulosa cells in vitro.

Treatment of heifers with recombinant bovine somatotrophin (BST) significantly increases the population of small ovarian follicles and peripheral concentrations of somatotrophin, insulin-like growth factor-I (IGF-I) and insulin. To investigate the possible mechanism(s) involved in the action of BST on ovarian follicles, the effects of BST, IGF-I and insulin, given alone or in combination with either FSH or LH, on the proliferation of bovine granulosa cells in vitro were examined using a serum-free culture system. Bovine granulosa cells were obtained from antral follicles classified into three size categories according to diameter: small < 5 mm; medium-sized 5-10 mm and large > 10 mm. The proliferation of granulosa cells was assessed by the incorporation of [3H]thymidine into the cultured cells. Both FSH and LH (1-1000 ng/ml) inhibited the proliferation of bovine granulosa cells obtained from all three size classes of follicles in a dose-dependent manner. BST, at doses ranging from 1 to 1000 ng/ml, had no effect on the proliferation of granulosa cells from small and medium-sized follicles, but inhibited the division of granulosa cells from large follicles in a dose-dependent manner. Treatment with either IGF-I (10-3000 ng/ml) or insulin (0.5-1000 ng/ml) stimulated, in a dose-dependent manner, the proliferation of granulosa cells obtained from all three size categories of follicles. No synergistic interaction between BST (30 ng/ml) and either FSH (50 ng/ml) or LH (5 ng/ml) was observed in granulosa cells from all three size classes of follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultra-structural characteristics of bovine granulosa cells associated with maintenance of oestradiol production in vitro.

We investigated whether the maintenance of oestradiol production by bovine granulosa cells (GC) in vitro was related to GC ultra-structure, and studied the effects of inclusion of serum as a cell attachment factor on oestradiol secretion, cell morphology and ultra-structure. Bovine granulosa cells from medium-sized follicles (4-8 mm diameter), in a serum-free (SF) culture system, maintained oestradiol production for 6 days, whereas oestradiol secretion by cells cultured in serum-coated (SC) wells declined rapidly with time, in culture. SF cells formed clumps consisting of two types of cells. Cells within clumps presented a phenotype similar to GC in vivo, being spherical, tightly joined by extensive gap junctions and interdigitated pseudopodia/microvilli, had abundant rough and smooth endoplasmic reticulum (ER) and mitochondria with trabecular cristae. In contrast, cells cultured in either SC wells or in the flattened base of cell clumps from SF cultures were enlarged, containing less rough ER, had fewer mitochondria (which tended to be round) and contained endosome-like structures, morphological characteristics suggestive of early luteinisation.

Influence of metabolic hormones and nutrition on ovarian follicle development in cattle: practical implications.

Nutrition has long been known to have a profound influence on reproductive performance of female cattle, but the underlying mechanism remains poorly understood. Whilst early investigations focused on the modulation of nutrition on hypothalamic-pituitary axis, more recent studies have tested the hypothesis that metabolic hormones as nutritional signals exert a direct effect at the ovarian level. In cattle, treatment with recombinant bovine somatotrophin (rGH) significantly increases the population of small ovarian follicles. This is associated with increases in circulating concentrations of insulin and insulin-like growth factor-I (IGF-I). Subsequent studies, both in vitro and in vivo, have highlighted the importance of IGF-I and/or insulin acting in synergy with FSH and LH. More recently, we demonstrated that feeding heifers with 200% maintenance requirements for a short period significantly increases circulating insulin concentrations and population of small ovarian follicles. Based on these findings, our recent work has aimed at addressing some practical problems in cattle production. Firstly, we showed that both rGH pretreatment and increased dietary intake significantly enhance the response to standard superovulatory regimes. Secondly, we have demonstrated that feeding a diet to increase circulating insulin concentrations during the early lactation can advance the first ovulation postpartum and increase conception rate to the first service in dairy cows. In summary, nutrition influences ovarian follicle development in cattle possibly through changes in metabolic hormones. These interactions can be manipulated to improve reproductive performance.

Pretreatment with recombinant bovine somatotropin enhances the superovulatory response to FSH in heifers.

One of the primary limiting factors to superovulation and embryo transfer in cattle has been the large variability in response, both between and within animals. It appears that the primary source of this problem is the variability in the population of gonadotropin-responsive follicles present in ovaries at the time of stimulation. We have shown that treatment of heifers with recombinant bovine somatotropin (rbGH) increases the number of small antral follicles (2 to 5 mm) and, therefore, enhances the subsequent superovulatory response to eCG. To investigate further the potential of using this approach to improve superovulatory regimens in cattle, the effect of rbGH pretreatment on the response to pituitary FSH was studied. The estrous cycles of 16 heifers were synchronized using PGF2alpha. On Day 7 of the synchronized cycle, half of the animals were injected with 320 mg sustained-release formulated rbGH, while the other half received 10 ml saline. Five days later, all heifers were given a decreasing-dose regimen of twice daily injections of oFSH for 4 d, incorporating an injection of PGF2alpha with the fifth FSH treatment, to induce superovulation. All animals were artificially inseminated twice with semen from the same bull during estrus. Ova/embryos were recovered nonsurgically on Days 6 to 8 of the following estrous cycle, and the ovulation rate assessed on Day 9 by laparoscopy. Using the same animals as described above, the experiment was repeated twice, 3 and 6 mo later, with no laparoscopy in the third experiment. The animals were randomized both between experiments and for the day of ova/embryo collection. Pretreatment of heifers with rbGH significantly (P < 0.01) increased the number of ovulations, total number of ova/embryos recovered and the number of transferable embryos. The percentage of transferable embryos was significantly (P < 0.05) increased by rbGH pretreatment. In addition, the incidence (2/16) of follicular cysts with a poor ovulatory response (< 6 ovulations) for the rbGH-pretreated heifers was significantly lower (P < 0.05) when compared with the incidence (7/16) in the control animals. It is concluded that pretreatment with rbGH may provide a useful approach for improving superovulatory response in cattle.

The effect of increased dietary intake on superovulatory response to FSH in heifers.

We have previously shown that the number of ovarian follicles <4 mm in diameter can be increased by enhanced dietary intake in heifers. This study investigated the effect of the same dietary treatment on superovulatory response. The estrous cycles of 24 mature Hereford x Friesian heifers were synchronized by a standard progesterone plus prostaglandin protocol. The animals were fed with either 100% (group M, n = 12) or 200% (group 2M, n = 12) maintenance requirements for a 3-week period. Starting from day 4 of the synchronized estrous cycle, all the animals were superovulated using a standard 4-day FSH regime followed by an injection of GnRH analogue (GnRHa) to induce ovulation. Rectal ultrasound scanning was carried out to assess ovarian follicular populations at the start of FSH treatment and on the day of GnRHa injection, and to determine the number of corpora lutea 5 days after GnRHa injection. The body weight (BW) and body condition score (BCS) were recorded weekly and plasma samples were collected throughout the experimental period. There were no differences in either BW or BCS between two groups at the start of the experiment. The BW and BCS were maintained during the experiment in the group M, whilst animals in the group 2M showed a non-significant (P > 0.05) increase in BW and BCS. Circulating concentrations of insulin were significantly (P < 0.01) higher in heifers from the group 2M throughout the controlled feeding period. The group 2M had significantly (P < 0.05) more follicles 2-4 mm in diameter at the start of FSH treatment and more (P < 0.01) follicles >9 mm in diameter on the day of GnRHa injection, when compared with the group M. Similarly, 5 days after GnRHa injection there were significantly (P < 0.01) more corpora lutea in the group 2M (18.1+/-2.2) than in the group M (10.6+/-3.0). In addition, plasma progesterone concentrations following GnRHa injection were significantly (P < 0.01) higher in heifers from the group 2M. In conclusion, these results confirm that increased dietary intake can enhance the recruitment of ovarian follicles in heifers. This treatment may provide a valuable approach to improving superovulatory response in cattle.

The recruitment of ovarian follicles is enhanced by increased dietary intake in heifers.

The objective of this study was to determine whether dietary-induced changes in growth hormone (GH), insulin, and IGF-I alter the pattern of ovarian follicular development in heifers. Twenty-eight 2- to 3-yr-old Hereford-Friesian heifers were equally allocated (n = 7) to one of four dietary treatments: 1) Control (C) fed a maintenance diet as two meals per day; 2) twice maintenance (2M2) given as two meals per day; 3) twice maintenance (2M6) given as six meals per day; 4) feed-deprived (F) fed maintenance requirements as for C, changed to straw ad libitum for 3 d from a synchronized estrus. On d 4 (estrus = d 0) heifers were transferred to grass silage (ad libitum access) until the end of the experiment. Blood samples were collected hourly for 10 h on d 1 and 3 and daily for an additional 14 d. Follicular development was monitored daily by ultrasonography until d 14. The number of small follicles (< 4 mm) was increased (P < .05) by 37% on d 1 and 2 in 2M2 and 2M6 heifers, with no carryover effect of nutrition to the second follicular wave. Number of medium-sized (4 to 8 mm) and large (> 8 mm) follicles did not vary (P > .05) among treatments. The FSH concentrations were not different (P > .05) among treatments. Insulin concentrations were higher (P < .05) in 2M2 and 2M6 heifers than in C or F heifers, with no carryover after the diet was changed. Increase in number of small follicles was independent of changes in FSH and IGF-I, negatively associated with GH, but positively associated with circulating insulin. These results indicate that a short-term increase in nutritional plane can affect follicular recruitment in cycling heifers.

The effect of recombinant bovine somatotropin on ovarian function in heifers: follicular populations and peripheral hormones.

The objective of this study was to investigate the possible effect of recombinant bovine somatotropin (BST) on ovarian folliculogenesis and ovulation rate. Twelve Hereford x Friesian heifers received daily injections of either 25 mg BST (6 heifers) or vehicle (6 heifers) for a period of two estrous cycles until slaughter. Blood samples were collected three times a week for measurements of peripheral growth hormone (GH), insulin-like growth factor I (IGF-I), FSH, LH, estradiol, and progesterone. Serial blood samples were also taken every 10 min for 8 h on Days 12 and 19 of the second estrous cycle to monitor GH, IGF-I, FSH, and LH profiles. At the end of treatment (Day 7 of the third estrous cycle), the heifers were killed and their ovaries were collected. Ovulation rate was determined by counting the number of fresh corpora lutea (CL). All antral follicles greater than or equal to 2 mm in diameter were dissected to assess antral follicle populations. Granulosa and thecal cells from the three largest follicles and CL from each heifer were collected for FSH and LH binding measurements. All heifers had a single ovulation. The treated heifers had significantly more antral follicles (60.2 +/- 6.7) than did the animals in the control group (33.2 +/- 3.2) (p less than 0.001). When follicles were grouped according to diameter, the mean numbers of follicles greater than 10 mm, 5-10 mm, and 2-5 mm in diameter were 0.8 +/- 0.2, 6.8 +/- 1.4, and 52.5 +/- 6.5 for the treated group, and 0.8 +/- 0.2, 6.5 +/- 1.0, and 25.8 +/- 2.7 for controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of recombinant bovine somatotrophin on ovarian follicular growth and development in heifers.

The effects of recombinant bovine somatotrophin (BST) on the dynamics of ovarian follicular growth and development and peripheral insulin concentrations were investigated. Initially, studies were carried out in a population of Hereford x Friesian heifers to validate the ultrasound technique. In the first experiment, 12 heifers were injected daily with either 25 mg BST or vehicle for two oestrous cycles, and the effects on follicular dynamics and peripheral insulin were determined. In a second experiment, 12 heifers were given a single injection of 10 ml saline or 320 mg BST in a sustained-release formulation to examine the temporal relationships between growth hormone (GH), insulin-like growth factor-1 (IGF-1), insulin and the number of follicles. The validation studies demonstrated that small follicles (< 5 mm in diameter) could be clearly detected by real-time ultrasound, and that 75% (9 of 12) of heifers showed three waves of dominant follicle development during the oestrous cycle, whereas the remainder had only two waves. The changes in the numbers of follicles of the three size categories (< 5 mm, small; 5-10 mm, medium-sized and > 10 mm, large) also displayed a wave pattern similar to that of the dominant follicle, with a marked reduction in the number of subordinate follicles as the dominant follicle grew and reached its maximum size. In Expt 1, BST treatment increased the number of small follicles and caused a rise in peripheral insulin concentrations (P < 0.01) throughout the treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions between nutrition and ovarian activity in cattle: physiological, cellular and molecular mechanisms.

The effects of acute changes in dietary intake on ovarian activity can be correlated with changes in circulating concentrations of metabolic hormones including insulin, insulin-like growth factor I (IGF-I), growth hormone and leptin. There is no corresponding change in circulating gonadotrophin concentrations and it is proposed that the dietary induced changes in ovarian activity, resulting from acute changes in dietary intake, are a result of direct actions of these metabolic hormones on the ovary. Changes in the peripheral concentrations of insulin, IGF-I and leptin were also associated with the initiation of a synchronized wave of follicle growth and it is hypothesized that oestrogen secreted by the developing follicle is involved in regulating the secretion of these metabolic hormones. At the cellular level, physiological concentrations of insulin and IGF-I interact to stimulate oestradiol production by granulosa cells. In contrast, leptin inhibits FSH-stimulated oestradiol production by granulosa cells and LH-stimulated androstenedione production by theca cells. At the molecular level, dietary energy intake affects the expression of mRNA encoding components of the ovarian IGF system and these changes can directly influence the bioavailability of intrafollicular IGF. This, in turn, can increase the sensitivity or response of follicles to FSH and is one mechanism through which nutrition can directly affect follicle recruitment. Dietary induced increases in intrafollicular IGF bioavailability also have a negative effect on oocyte quality, and diets that are optimal for follicle growth may not necessarily be optimal for oocyte maturation.

Control of ovarian function in cattle.

In cattle, ovarian function is controlled by complex local and systemic feedback mechanisms involving gonadotrophins from the pituitary gland and steroids and proteins from the ovaries. This control system ensures that in more than 96% of females, only one follicle will ovulate per oestrous cycle. Follicular growth and development in cattle occurs in a wave-like pattern, with two or three waves of follicles growing and regressing per oestrous cycle. Each wave is characterized by the emergence, from a pool of growing follicles, of a large dominant follicle which reaches a mature stage of development and may be induced to ovulate and form a functional corpus luteum with a single treatment of either human chorionic gonadotrophin (hCG) or gonadotrophin-releasing hormone (GnRH). The growth of this dominant follicle is associated with a marked reduction in both the number and growth of subordinate follicles in both ipsi- and contralateral ovaries, suggesting a systemic exertion of 'dominance'. The dominance concept is further supported by the observation that the subsequent wave of follicular growth cannot be detected until after the start of the regression of the previous dominant follicle. Furthermore, the dominance mechanism does not involve an action of inhibin, as previously proposed. In addition, in cattle, inhibin may not have a primary role in the control of follicle-stimulating hormone (FSH) release, unlike its role in other species such as sheep. Gonadotrophins provide the primary endocrine drive for the growth of follicles > 2 mm in diameter, but it is now becoming evident that other systemic and locally produced factors profoundly influence ovarian function. For example, short-term treatment of cattle with recombinant bovine somatotropin (BST) can double the number of small (< 5 mm) antral follicles without altering the pattern of circulating gonadotrophin concentrations. These follicles appear to be fully functional since they can be stimulated to develop further and ovulate with superovulatory treatment regimens. Dominance also appears to be exerted via a different pathway from that involved in the BST-induced recruitment of small follicles. The identification of the factor responsible for dominance, together with the elucidation of the mechanisms controlling follicular recruitment and growth should ensure that the full benefits ensuing from the precise control of ovarian function in cattle are achieved.

Effects of chronic treatment with a gonadotrophin-releasing hormone agonist on peripheral concentrations of FSH and LH, and ovarian function in heifers.

The effect of chronic treatment with a gonadotrophin-releasing hormone agonist (GnRHa) on ovarian function in cattle was investigated by injecting heifers i.m. twice a day with saline, 5 micrograms GnRHa (Buserelin) or 10 micrograms GnRHa (n = 7) for 21 days. Blood samples were taken twice a day during the treatment period, and then three times a day for 7 days and once daily for a further 4 days. Frequent samples were also collected on day 1, day 10 and day 21 of treatment. The ovaries of all heifers were examined daily using real-time ultrasonography throughout the experimental period. No significant differences in the response were observed between two doses of GnRHa. The first GnRHa injection produced a large LH and FSH surge and this acute response was still present by day 21 of treatment, but both the magnitude and duration of response were significantly attenuated (P < 0.01). After an initial increase, LH returned to the basal concentration, which was maintained until the termination of treatment, when concentrations increased significantly, with a preovulatory surge occurring approximately 6 days later. Peripheral FSH concentrations during the oestrous cycle in control animals displayed a pattern of three waves, each of which closely preceded a wave of follicular development. Concentrations of FSH in GnRHa-treated heifers showed a normal pattern for the first wave after the start of treatment. During the next wave, concentrations increased and remained at the peak values until about 4 days after the end of treatment. An additional ovulation was induced in 11 of 14 GnRHa-treated heifers within 2-3 days of the start of treatment, and a significant (P < 0.05) increase in serum progesterone concentrations was detected 2 days later. All GnRHa-treated heifers then showed a normal follicular wave, with the development and regression of a dominant follicle. The dominant follicles from the next wave grew to only 7-9 mm in diameter and remained at this size until the end of treatment, when they resumed growth, ovulated approximately 7 days later and formed corpora lutea. We conclude that chronic treatment of heifers with GnRHa for 3 weeks suppresses pulsatile secretion of LH and blocks the development of dominant follicles beyond 9 mm in diameter, preventing the preovulatory LH surge and ovulation. However, GnRHa did not suppress the secretion of FSH within the 3 week treatment period. The maintenance of the dominant follicles for an extended period should provide an ideal model to study the control of follicular atresia in cattle in vivo.

Enhancement of ovarian follicle development in heifers by treatment with recombinant bovine somatotrophin: a dose-response study.

Treatment with recombinant bovine somatotrophin (bST) can enhance the development of ovarian antral follicles in cattle. The underlying mechanism was examined further by performing a dose-response study to investigate the effects of bST on peripheral concentrations of somatotrophin, insulin-like growth factor I (IGF-I), insulin, FSH and LH, and ovarian follicle development. Twenty mature heifers were randomly divided into five groups and injected s.c. at 6 h intervals for 7 days with 25% of one of the following daily doses of bST: 0, 3.13, 6.25, 12.5 or 25.0 mg. Ovarian follicular dynamics were monitored by real-time ultrasonography. Blood samples were collected daily during the experiment, and every 15 min for 8 h on days 1 and 5 of bST treatment. Treatment with bST increased (P < 0.01) peripheral concentrations of somatotrophin in a dose-dependent manner. Serum concentrations of both IGF-I and insulin were significantly (P < 0.01) increased in all heifers given 12.5 or 25.0 mg bST per day. Peripheral concentrations of IGF-I and insulin in all animals in the group given 3.13 mg bST and two heifers in the group given 6.25 mg bST were not different from those in the control group, while concentrations in the other two heifers given 6.25 mg bST were significantly (P < 0.01) higher. The number of ovarian follicles < 5 mm in diameter was increased (P < 0.05) in response to bST, but only in heifers (n = 10) with significantly increased serum concentrations of IGF-I and insulin. There were no effects of treatment on peripheral concentrations of FSH, LH and progesterone, and on the numbers of follicles > 5 mm in diameter. In conclusion, this study has demonstrated in vivo that the effect of treatment with bST on ovarian follicle development appears to be mediated through an increase in circulating IGF-I or insulin concentrations, rather than via an alteration in the secretion of pituitary gonadotrophins or a direct effect of bST on ovarian follicles.

Effect of dietary-induced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows.

Although it has become increasingly clear that fertility in modern dairy cattle is declining in association with increased milk yields, the underlying mechanism is poorly understood. The first ovulation post partum is delayed in dairy cows undergoing selection for genetic merit for milk yield in association with lower circulating insulin concentrations. The aim of this study was to investigate whether feeding a diet to increase circulating insulin concentrations can overcome this delay in the first ovulation post partum. The experiment was a 2 x 2 factorial design (n = 10 per group) involving diet and genetic merit for milk yield. The dietary treatment started on the day of calving and lasted for 50 days. Plasma samples were collected each day and ovarian ultra-sonography was performed three times a week during the experimental feeding period. Milk yield was recorded each day, and body weight and body condition score were determined each week. Milk samples were collected three times a week from day 50 to day 105 post partum, and reproductive performance data were recorded for all the cows as part of the routine farm practice. The dietary treatment induced significant differences in plasma insulin concentrations in both high and low genetic merit cows. Although high genetic merit cows produced more milk, lost more body weight and had lower body condition scores during the experiment, no significant effect of diet was observed on these measurements. The high insulin inducing diet increased the proportion of cows ovulating within 50 days of calving and reduced the intervals from calving to first ovulation, and tended to reduce the intervals from calving to first service and to conception. These fertility parameters were also more favourable in low than in high genetic merit cows, but no interaction between diet and genetic merit was observed for any of these parameters. Genetic merit, but not diet, also affected the number of services required per conception and the conception rate. In conclusion, these results have confirmed that genetic selection for high milk yield is associated with a decrease in reproductive performance in dairy cows. More importantly, this study has demonstrated that it is possible to alleviate this problem by nutritional manipulation.

Effect of recombinant bovine somatotropin on the superovulatory response to pregnant mare serum gonadotropin in heifers.

Treatment of mature heifers with recombinant bovine somatotropin (BST) increases the number of ovarian follicles of 2-5 mm in diameter. This study was carried out to investigate whether the increase in the number of small follicles induced by BST treatment can enhance the superovulatory response. In the first experiment, 24 heifers were assigned, in a randomized block design, to four treatment groups: 1) control; 2) BST; 3) pregnant mare serum gonadotropin (PMSG); 4) BST+PMSG. On Day 7 of the estrous cycle, animals in groups 2 and 4 received injections of 320 mg BST (in a sustained release formulation), while heifers in groups 1 and 3 received 10 ml saline. Five days later heifers in groups 3 and 4 were treated with a single dose of 2000 IU PMSG to induce superovulation. Embryos were recovered nonsurgically on Days 6-8 of the following cycle, and all heifers underwent laparoscopy on Day 9 to assess ovulation rate. In a second experiment, 24 heifers were assigned randomly to four treatment groups: 1) PMSG (1000 IU); 2) BST (320 mg) + PMSG (1000 IU); 3) PMSG (2000 IU); 4) BST (320 mg) + PMSG (2000 IU), and then treated as for experiment I. In both experiments, all heifers were scanned daily using real-time ultrasound from the day before BST injection until the day of ovulation to monitor dynamics of ovarian follicular growth and development, and blood samples were collected daily throughout the experimental period for measurement of peripheral progesterone, estradiol-17 beta, growth hormone (GH), and insulin-like growth factor-I (IGF-I).(ABSTRACT TRUNCATED AT 250 WORDS)

Control of follicular growth: local interactions and nutritional influences.

Regulation of ovarian activity is an integrated process encompassing both extraovarian signals and intrafollicular factors. Initiation of primordial follicle growth and the early stages of folliculogenesis can occur without gonadotropins, but FSH may affect the rate of preantral follicle growth. Antral follicle development from 1 to 4 mm in sheep and cattle is completely gonadotropin dependent. These recruited follicles express a range of mRNA encoding steroidogenic enzymes, gonadotropin receptors, and local regulatory factors and their receptors. As follicles continue to mature, there is a transfer of dependency from FSH to LH, which may be part of the mechanism involved in selection of follicles for continued growth. Locally produced growth factors, such as the IGF and members of the transforming growth factor-beta superfamily, work in concert with gonadotropins throughout the follicular growth continuum and can have significant effects on follicle selection. Environmental influences, such as changes in nutrition, also have an effect on ovarian activity. This can occur without significant variation in circulating gonadotropin concentrations and can be correlated with changes in circulating concentrations of metabolic hormones, including insulin, IGF-I, GH, and leptin. Nutrition can also affect the expression of mRNA encoding components of the ovarian IGF system to regulate the sensitivity/response of follicles toward gonadotropins. Hence, the roles of growth factors in follicular development and survival depend on gonadotropin status and differentiation state of the follicle. In conclusion, it is the integration of these extraovarian signals and intrafollicular factors that determine whether a follicle will continue to develop or be diverted into atretic pathways.

Molecular mechanisms regulating follicular recruitment and selection.

Ovarian follicular growth and development is an integrated process encompassing both extraovarian signals, such as gonadotrophins and metabolic hormones, and intraovarian factors. Follicular development has been classified into gonadotrophin-independent and -dependent phases. In the latter, FSH provides the primary drive for follicular recruitment and LH is required for continued development of follicles to the preovulatory stage. A transient increase in circulating FSH precedes the recruitment of a group of follicles, and these recruited follicles are characterized by expression of mRNAs encoding P450scc and P450arom in granulosal cells. As follicles mature, there is a transfer of dependency from FSH to LH, which may be part of the mechanism(s) involved in selection of follicles for continued growth. Indeed, changes in the pattern of expression of mRNA for gonadotrophin receptors and steroid enzymes within follicular cells appear to be closely linked to changes in peripheral concentrations of gonadotrophins. The mechanism of selection of dominant follicles still requires clarification, but seems to be linked to the timing of mRNA expression encoding LHr and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in granulosal cells. Additional intraovarian systems, including the ovarian IGF and activin/inhibin systems, also exert a role. For example, it appears that the development of follicular dominance in cows is associated with the FSH-dependent inhibition of the expression of mRNA encoding insulin-like growth factor binding protein 2 (IGFBP-2) in granulosal cells. In conclusion, the integration of these endocrine signals and intraovarian factors within follicles determines whether follicles continue to develop and become dominant or are diverted into apoptotic pathways leading to atresia.

The tyrosine kinase c-Abl regulates p73 in apoptotic response to cisplatin-induced DNA damage.

Cancer chemotherapeutic agents such as cisplatin exert their cytotoxic effect by inducing DNA damage and activating programmed cell death (apoptosis). The tumour-suppressor protein p53 is an important activator of apoptosis. Although p53-deficient cancer cells are less responsive to chemotherapy, their resistance is not complete, which suggests that other apoptotic pathways may exist. A p53-related gene, p73, which encodes several proteins as a result of alternative splicing, can also induce apoptosis. Here we show that the amount of p73 protein in the cell is increased by cisplatin. This induction of p73 is not seen in cells unable to carry out mismatch repair and in which the nuclear enzyme c-Abl tyrosine kinase is not activated by cisplatin. The half-life of p73 is prolonged by cisplatin and by co-expression with c-Abl tyrosine kinase; the apoptosis-inducing function of p73 is also enhanced by the c-Abl kinase. Mouse embryo fibroblasts deficient in mismatch repair or in c-Abl do not upregulate p73 and are more resistant to killing by cisplatin. Our results indicate that c-Abl and p73 are components of a mismatch-repair-dependent apoptosis pathway which contributes to cisplatin-induced cytotoxicity.