Interaction of bovine granulosa and theca cells in a novel serum-free co-culture system

The objective of this study was to develop a defined culture system in which bovine follicular and granulosa cells are grown in close contact with each other and with the extracellular matrix (ECM) component laminin. Granulosa and theca cells from follicles 4-6 mm in diameter were cultured on either side of laminin-coated BioCoat cell culture inserts in a serum-free medium containing 10 ng insulin ml(-1) at plating densities of 10(5) and 3 x 10(5) cells per membrane side. The cells adopted a clumped arrangement, maintained steroidogenic activity for at least 7 days and demonstrated paracrine communication by increased steroidogenesis and enhanced cell survival compared with cells in mono-culture. Co-cultured theca cells secreted significantly more androstenedione compared with cells in mono-culture. Granulosa cell viability was doubled by co-culture with theca cells. Co-cultures at both cell plating densities were responsive to treatment with physiological combinations of either FSH, LH and LR3 insulin-like growth factor I (IGF-I) (treatment A) or FSH, LR3 IGF-I and androstenedione (treatment B). Significantly more androstenedione was secreted in the presence of treatment A compared with controls. In contrast, oestradiol secretion was increased only by treatment B. Progesterone secretion was unaffected by treatment and did not increase during culture. Co-cultures at the higher plating density demonstrated higher theca cell survival and better maintenance of the follicular cell phenotype. In conclusion, this novel co-culture system provides a unique model for the study of paracrine communication between ovarian somatic cells and cell-ECM interactions during follicle growth.     

Comparison of somatotropin and growth hormone-releasing factor on milk yield, serum hormones, and energy status.

Holstein cows received 12 mg/d of growth hormone-releasing factor (continuous i.v. infusion, n = 5), 14 mg/d of bST (single daily i.m. injection, n = 8), or no treatment (controls, n = 8) for 60 d. Compared with controls (31.6 kg/d), bST and growth hormone-releasing factor increased milk yield to 34.2 and 37.0 kg/d, respectively. The increase in milk yield induced by the growth hormone-releasing factor was greater than that for bST. Milk yield was not different among groups following cessation of treatment. Milk energy output was 24.2 Mcal/d in controls, and growth hormone-releasing factor increased milk energy output to 28.5 Mcal/d. Milk energy output of cows receiving bST was 26.1 Mcal/d. Growth hormone-releasing factor increased DMI (23.2 kg/d) over that of controls (21.1 kg/d), whereas bST (21.5 kg/d) did not. Relative to controls, bST increased averaged daily serum somatotropin from 1.3 to 7.6 ng/ml and insulin-like growth factor-I from 67.5 to 116.0 ng/ml. Relative to bST, growth hormone-releasing factor increased serum somatotropin to 16.3 ng/ml and insulin-like growth factor-I to 202.6 ng/ml. Relative to control (115.8 meq/dl) and bST (158.1 meq/dl), growth hormone-releasing factor increased plasma NEFA (230.3 meq/dl). During treatment, calculated energy balance was negative for cows receiving growth hormone-releasing factor but positive for bST and control cows. Milk composition, body condition score, BW, and apparent digestibility of DM were not different among treatments. We conclude that i.v. infusion of 12 mg/d mg of growth hormone-releasing factor has greater galactopoietic activity than i.m. injections of 14 mg/d of bST.(ABSTRACT TRUNCATED AT 250 WORDS)     

Growth hormone-releasing factor effects on pituitary function, growth, and lactation

Growth and lactation are complex processes controlled by several metabolic hormones such as insulin, glucagon, and thyroid hormones but most notably growth hormone. Growth hormone secretion is regulated by two hypothalamic hormones, somatostatin, an inhibitory regulatory factor, and growth hormone-releasing factor. The quantity and pattern of growth hormone secretion is ultimately regulated in concert by the secretion of both regulatory factors from the hypothalamus through the hypothalamo-hypophyseal portal system, where they exert their actions at unique pituitary receptors. Because the potential use of exogenous growth hormone administration for the stimulation of growth efficiency and lactation has been demonstrated, recent efforts have been directed toward the enhancement of production through manipulation of endogenous growth hormone secretion via its releasing factor. Thus far, releasing factor-stimulated growth and lactation has not been achieved to the same extent as that of exogenous growth hormone. Growth hormone-releasing factor has stimulated growth in two growth hormone-deficient children, as well as female, but not male, rats. Although all food-producing species tested to date respond to growth hormone-releasing factor with the appropriate growth hormone response, continuous or pulsatile administration of releasing factor has not resulted in increased growth rate in sheep, chicks, or hogs. Despite levels of circulating growth hormone that would be expected to produce a 30% increase in milk production if given exogenously to dairy cows, releasing factor-stimulated growth hormone secretion has resulted in only a 3 to 9% increase. It is clear from these studies that further developments are necessary to demonstrate the practical application of growth hormone-releasing factor.     

Pregnancy and bovine somatotropin in nonlactating dairy cows: I. Ovarian, conceptus, and insulin-like growth factor system responses

Nonlactating dairy cows were used to examine effects of bovine somatotropin (bST) on components of the insulin-like growth factor (IGF) system. Estrus was synchronized in cows with a Presynch + Ovsynch protocol and timed AI (TAI; n = 55) or not TAI (cycling, C; n = 23) on d 0 (time of synchronized ovulation). On d 0 and 11, cows received bST (500 mg) or no bST, and were sacrificed on d 17. Pregnancy rates were less in bST cows (27.2%, 9 of 33) than in controls (63.6%; 14 of 22). In contrast, conceptuses were larger in bST-treated cows (39.2 +/- 4.8 cm) than in controls (20 +/- 4.3 cm). Total interferon-tau in uterine luminal flushings (ULF) was greater in bST-treated cows (7.15 > 2.36 microg). Number of class 2 follicles (6 to 9 mm) was less in bST-C cows on d 7 and 16. On d 17, corpus luteum (CL) weight tended to be greater in bST-treated cows. Concentrations of progesterone were greater after d 10 in C than in pregnant (P) cows. In the ULF, IGF-binding protein-3 was greater in bST-P cows than in pregnant cows. A tendency for an increase in IGF-I hormone concentrations in the ULF was detected on d 17 in bST-treated and cyclic cows. Endometrial mRNA for IGF-I, IGF-II, IGFBP-2, and IGFBP-3 increased in bST-C, but not in bST-P cows. Treatment with bST increased plasma concentrations of insulin, IGF-I, and growth hormone (GH). In conclusion, bST may have hyperstimulated plasma IGF-I and insulin to cause asynchrony between conceptus and uterus that was detrimental to pregnancy.     

Steroidogenesis in bovine granulosa cells: the effect of short-term changes in dietary intake

The nutritional status of a cow is a key factor in the regulation of both follicle growth and oocyte quality. In this study, the effect of diets designed to increase circulating insulin and insulin-like growth factor I (IGF-I) concentrations on steroid production by granulosa cells in vitro was examined to analyse the mechanisms through which these changes occur. Hereford x Friesian heifers (n = 24) were offered maintenance or twice maintenance diets during the experimental period (17 days). Circulating concentrations of FSH did not differ between the two dietary groups, whereas insulin and IGF-I concentrations showed significant diet x day of oestrous cycle interactions. Ovaries were collected on day 3 of the first follicle wave after synchronization of oestrus. Granulosa cells were isolated from small (1-4 mm) and medium-sized (4-8 mm) follicles and cultured in the presence of long R3-IGF-I or bFSH or both. After 4 days in culture, granulosa cells isolated from small follicles, but not medium-sized follicles, collected from cattle offered the twice maintenance diet secreted significantly higher (P < 0.05) amounts of oestradiol compared with granulosa cells collected from cattle offered the maintenance diet. The effect was apparent in either the presence or absence of FSH and long R3-IGF-I. This nutritional effect on aromatase activity in granulosa cells was not apparent after day 6 of culture. There was no effect of diet on progesterone production by granulosa cells after 4 or 6 days of culture. These results support the hypothesis that dietary-induced changes in circulating insulin and IGF-I concentrations have a direct effect on the steroidogenic potential of bovine granulosa cells from small follicles. The dietary-induced increases in aromatase activity in small follicles combined with the increased concentration of metabolic hormones are possible mechanisms through which short-term changes in nutrition may affect follicle dynamics.     

Pregnancy, bovine somatotropin, and dietary n-3 fatty acids in lactating dairy cows: I. Ovarian, conceptus, and growth hormone-insulin-like growth factor system responses

The objective was to examine effects of bovine somatotropin (bST), pregnancy, and dietary fatty acids on reproductive responses in lactating dairy cows. Beginning at approximately 17 d in milk (DIM), a comparison was made of isoenergetic diets comprising supplementary lipids of whole cottonseed vs. calcium salts of fish oil enriched lipid (FO). Ovulation was synchronized in cows with a presynchronization plus Ovsynch protocol, and cows were inseminated artificially by appointment or not inseminated (d 0 = time of synchronized ovulation; 77 ± 12 DIM). On d 0 and 11, cows received bST (500 mg) or no bST. All cows were slaughtered on d 17. Number of cows in each group was as follows: control diet had 5 bST-treated cyclic (bST-C), 5 non-bST-treated cyclic (no bST-C), 4 bST-treated pregnant (bST-P), and 5 non-bST-treated pregnant (no bST-P) cows; and cyclic cows fed FO diet had 4 bST-treated (bST-FO) and 5 non-bST-treated cyclic (no bST-FO-C) cows. Feeding FO increased milk production, number of class 1 follicles (2 to 5 mm), and decreased insulin during the period before d 0 compared with control-fed cows. The bST increased milk production, pregnancy rate [83% (5/6) vs. 40% (4/10)], conceptus length (45 vs. 34 cm), and interferon-τ in the uterine luminal flushings (9.4 vs. 5.3 μg) with no effect on interferon-τ mRNA concentration in the conceptus. Treatment with bST increased plasma growth hormone (GH) and insulin-like growth factor (IGF)-I. Among control-fed cows (cyclic and pregnant), bST decreased progesterone concentration in plasma. Cows fed FO had less plasma insulin than control-fed cyclic cows, and FO altered the plasma GH (bST-FO > bST-C) and IGF-I (bST-C > bST-FO-C) responses to bST injections. Endometrial IGF-I mRNA was reduced in pregnant cows and tended to decrease in those fed FO. The IGF-II mRNA was increased in the endometrium of pregnant and bST-treated cows fed the control diet. Cows fed FO had increased concentrations of IGF-II mRNA, when bST was not injected. The insulin-like growth factor binding protein-2 (IGFBP-2) mRNA was increased in bST-P cows, whereas bST decreased the IGFBP-2 mRNA in all cyclic cows. In summary, bST and FO seemed to modulate reproductive responses that may be beneficial to the developing conceptus and pregnancy rate.     

Role of transforming growth factor-beta1 in gene expression and activity of estradiol and progesterone-generating enzymes in FSH-stimulated bovine granulosa cells

Survival and inhibitory factors regulate steroidogenesis and determine the fate of developing follicles. The objective of this study was to determine the role of transforming growth factor-beta1 (TGFB1) in the regulation of estradiol-17beta (E(2)) and progesterone (P(4)) secretion in FSH-stimulated bovine granulosa cells. Granulosa cells were obtained from 2 to 5 mm follicles and cultured in serum-free medium. FSH dose (1 and 10 ng/ml for 6 days) and time in culture (2, 4, and 6 days with 1 ng/ml FSH) increased E(2) secretion and mRNA expression of E(2)-related enzymes cytochrome P450 aromatase (CYP19A1) and 17beta-hydroxysteroid dehydrogenase type 1 (HSD17B1), but not HSD17B7. TGFB1 in the presence of FSH (1 ng/ml) inhibited E(2) secretion, and decreased mRNA expression of FSH receptor (FSHR), CYP19A1, and HSD17B1, but not HSD17B7. FSH dose did not affect P(4) secretion and mRNA expression of 3beta-hydroxysteroid dehydrogenase (HSD3B) and alpha-glutathione S-transferase (GSTA), but inhibited the amount of steroidogenic acute regulatory protein (STAR) mRNA. Conversely, P(4) and mRNA expression of STAR, cytochrome P450 side-chain cleavage (CYP11A1), HSD3B, and GSTA increased with time in culture. TGFB1 inhibited P(4) secretion and decreased mRNA expression of STAR, CYP11A1, HSD3B, and GSTA. TGFB1 modified the formation of granulosa cell clumps and reduced total cell protein. Finally, TGFB1 decreased conversion of androgens to E(2), but did not decrease the conversion of estrone (E(1)) to E(2) and pregnenolone to P(4). Overall, these results indicate that TGFB1 counteracts stimulation of E(2) and P(4) synthesis in granulosa cells by inhibiting key enzymes involved in the conversion of androgens to E(2) and cholesterol to P(4) without shutting down HSD17B reducing activity and HSD3B activity.     

The effects of genetic and phenotypic production potential on response to recombinant bovine somatotropin.

Evidence was sought for an interaction between both phenotypic and genetic production potential and response of milk production to administration of bST in three trials of 38, 43, and 35 cows. In each trial, bST was administered in doses of 0, 10.3, 20.6, and, in trial 1 only, 41.2 mg/d for 38 wk from wk 4 of lactation. Data were analyzed for each experiment separately and combined across experiments. Analyses included separate regressions for treated and untreated animals for milk production during the production period on pretreatment production and estimated breeding value for milk production. Breeding value was estimated as the sire's estimated transmitting ability plus one-half of the maternal grandsire's estimated transmitting ability. With the exception of regression on estimated breeding value in trial 1 and in combined data, differences between treated and untreated animals in the regression of milk production on pretreatment milk production or on estimates of breeding value were not statistically significant. However, regressions on pretreatment production were substantially lower for treated than for untreated animals in each of the three trials. Regressions on breeding value estimated from sire and maternal grandsire estimated transmitting abilities were substantially, but not significantly, lower for untreated than for treated animals in all three trials. The results suggest that cows with high production potential for nongenetic reasons may show diminished response to bST, whereas cows with genetically high production potential show enhanced response. However, borderline statistical significance argues for considerable further examination before drawing firm conclusions.     

Effect of long-term infusion with recombinant growth hormone-releasing factor and recombinant bovine somatotropin on development and function of dominant follicles and corpora lutea in Holstein cows.

The objective of this study was to evaluate the effects of recombinant bovine growth hormone-releasing factor (rGRF) or recombinant bovine somatotropin (rbST) on growth and function of the first-wave dominant follicle and corpus luteum. Primiparous Holstein cows (117 d postpartum) were infused with 12 mg/d of rGRF (n = 10) or 29 mg/d of rbST (n = 10) for 63 d, and non-infused cows (n = 10) were controls. At slaughter on d 5 of an estrous cycle, blood and ovaries were collected and data from cows with a corpus luteum were analyzed (control, n = 8; rGRF, n = 5; rbST, n = 6). Treatment with rGRF or rbST increased somatotropin (ST) and IGF-I in serum similarly compared with controls. In contrast, rbST-treated cows had higher concentrations of ST in follicular fluid (FF) compared with rGRF-treated and control cows. In addition, rbST, but not rGRF, increased the number and decreased the size of estrogen-active follicles (EA; estradiol > progesterone concentrations in FF), increased the abundance of IGF binding proteins-2, -3, and -4 in FF from EA follicles, and increased the number but decreased the size of corpora lutea and decreased concentration of progesterone in serum compared with controls. Based on these results, we concluded that long-term infusion of rbST alters growth and function of the first-wave dominant follicle and the corpus luteum in cattle.     

Effects of recombinant bovine somatotropin on milk production, body composition, and physiological parameters

Twenty-eight multiparous Holstein cows were utilized to determine effects of long-term administration of recombinant bovine somatotropin on lactational performance, body condition, body composition, hormones, blood constituents, and physiological parameters. Treatments were 0 (control), 10.3, 20.6, and 41.2 mg recombinant bovine somatotropin administered daily as subcutaneous injections beginning 4 to 5 wk postpartum and continuing for 38 wk. A total mixed diet of 28% corn silage, 22% alfalfa hay cubes, and 50% corn-soybean meal-based grain mixture was fed. Fat-corrected milk yields were increased 12 to 25% for cows treated with somatotropin as compared with controls (29.9 kg/d). Milk composition was similar among treatments. Cows receiving somatotropin consumed 4 to 10% more feed and were 11 to 17% more efficient than controls for conversion of feed to milk. Body weight changes were not significantly different among treatments, but cows receiving somatotropin gained 4 to 10% less weight than controls. Body fat (kg) of cows receiving 20.6 and 41.2 mg/d was less than that of cows receiving 10.3 mg/d or no somatotropin. Estimated weights of body protein and mineral, most blood constituents, respiratory rates, and body temperature were not affected by somatotropin administration. Plasma fatty acids were elevated and hematocrit values were reduced. Plasma insulin, serum somatotropin, and heart rate increased concurrently with somatotropin administration.     

Effect of bovine somatotropin and rumen-undegradable protein on mammary growth of prepubertal dairy heifers and subsequent milk production

Rapid body growth during the prepubertal period may be associated with reductions in mammary parenchymal growth and subsequent milk yield. The objective of this study was to test effects of dietary rumen-undegradable protein (RUP) and administration of recombinant bovine somatotropin (bST) during the prepubertal period on mammary growth and milk yield of dairy heifers. Seventy-two Holstein heifers were used in the experiment. At 90 d of age, 8 heifers were slaughtered before initiation of treatment. Remaining heifers were assigned randomly to 1 of 4 treatments. Treatments consisted of a control diet (5.9% RUP, 14.9% CP, DM basis) or RUP-supplemented diet (control diet plus 2% added RUP) with or without 0.1 mg of bST/kg of BW per day applied in a 2 x 2 factorial design. A total of 6 heifers per treatment (3 each at 5 and 10 mo of age) were slaughtered for mammary tissue analysis. Remaining heifers were bred to evaluate impact of treatment on subsequent milk yield and composition. Mammary parenchymal growth was not affected by RUP or bST treatment. Total parenchymal mass increased from 16 to 364 g, and parenchymal DNA from 58 to 1022 mg from 3 to 10 mo of age, respectively. Furthermore, number of mammary epithelial cells likely was not affected by diet or bST because the epithelial cell proliferation index, assessed by Ki-67 labeling, was not affected by treatment, nor was total parenchymal DNA and lipid content. Neither deleterious effects of increased rates of gain nor positive effects of bST were evident in prepubertal mammary growth. Subsequent milk production and composition was not different among treatments.     

Compensatory milk production within the bovine udder: effects of short-term non-milking of single quarters

Daily quarter-milk yields of eight high-yielding cows (24-28 kg/d) and eight heifers (14-24 kg/d) were measured to examined to examined compensatory changes in milk production between quarters within an udder. Either one, two or three quarters per cow were left unmilked for 12 d, in early or late lactation, and then all quarters of all cows were milked normally for a further period of 12 d. Concentrate feeding levels were constant throughout the experiment. The mean daily yield per cow fell to 26, 59, and 75% during the period of milking either one, two or three quarters respectively. Twelve days after resumption of normal milking the total daily yield per cow was the same for cows continuously milked in two, three or all four quarters. Daily yield in cows with only one quarter milked continuously recovered to only 78% of the level of the pretreatment period. During the treatment period, the mean daily yield of the continuously milked quarters increased by almost 14% for cows milked in one quarter only, by more than 10% for two quarters milked, and by 4% per quarter if three quarters were milked. Milk yields of these quarters remained above their pretreatment levels when milking was resumed in the adjacent quarters. There was no difference in the compensatory effect between cows and heifers in early lactation, but the compensatory effects were lower in the lower in late lactation heifers.     

Effects of insulin,recombinant bovine somatotropin,and their interaction on insulin-like growth factor-I secretion and milk protein production in dairy cows

This trial was designed to test the effects of insulin, recombinant bovine somatotropin (rbST), and their interaction on milk protein and selected blood parameters in dairy cows. Eight Holstein cows (86 +/- 10 d in milk) were divided in two groups and used in two replicates of a Latin square design with four animals, four periods, and four treatments: 1) intravenous infusion of saline, 2) infusion of saline and subcutaneous administration of 40 mg of rbST per day, 3) intravenous infusion of 12 mg of insulin per day coupled with glucose infusion, and 4) rbST administration combined with insulin and glucose infusion. The glucose infusion rate was adjusted to maintain euglycemia. Each experimental period lasted 14 d: treatments were administered during the first 6 d, and no treatment was administered during the following 8-d resting phase. The average daily amount of glucose infusion needed to avoid hypoglycemia was 2.8 kg/cow when only insulin was infused as opposed to 2.2 kg/cow when both insulin and rbST were administered, indicating that either rbST causes a peripheral resistance to insulin or rbST increased liver gluconeogenesis or both. Data from the last 3 d of infusion were analyzed by using the SAS system for mixed models. Percent protein of milk tended to be lower (2.84 vs. 2.79%) and milk urea content was lower (16.6 vs. 14.8 mg/dl) during rbST administration, regardless of insulin infusion. Insulin infusion increased percent protein (2.78 vs. 2.85%) and percent casein (2.36 vs. 2.46%) and decreased milk urea content (17.1 vs. 14.3 mg/dl) regardless of rbST administration. For milk yield, protein yield, casein yield, lactose percent, and lactose yield, there were significant interactions between insulin and rbST administration. For example, casein yield averaged 1.17, 1.12, 1.20, and 1.28 kg/d for saline, insulin, rbST, and insulin combined with rbST, respectively. Similarly, there was a significant interaction between insulin and rbST on IGF-I levels, which were 122.5, 181.3, 342.3, and 492.2 ng/ml for saline, insulin, rbST, and insulin combined with rbST, respectively. In conclusion, these results clearly demonstrated that insulin interacts with bST in early lactation to improve milk protein synthesis and yield in dairy cows. These effects are probably mediated through a combination of bST nutrient mobilization, bST-induced gluconeogenesis, bST-induced insulin peripheral resistance, and bST/insulin synergism on insulin-like growth factor-I secretion and on mammary epithelial tissue.     

Expression of lysyl oxidase and effect of copper chloride and ammonium tetrathiomolybdate on bovine ovarian follicle granulosa cells cultured in serum-free media

Subfertility that will respond to appropriate copper supplementation is a widespread problem in the UK dairy herd and, although characterized by reduced or absent oestrus and reduced conception rates, the exact cause remains unknown. The aim of this study was to investigate the expression of mRNA for the copper-dependent enzyme, lysyl oxidase, and the effect of copper and/or copper chelating thiomolybdates on FSH-induced differentiation of bovine granulosa cells cultured in serum-free media. Expression of lysyl oxidase mRNA was investigated using bovine specific primers and RT-PCR on cell lysates obtained from bovine granulosa cells cultured under optimum conditions for 0, 16, 24, 48, 96, 144 and 192 h. The effect of thiomolybdates and copper were investigated by supplementing optimized granulosa cell culture media with ammonium tetrathiomolybdate at 0, 0.1, 1, 10, 100 and 1000 micro g ml(-1), copper chloride at equimolar concentrations (0, 0.0516, 0.516, 5.16, 51.6, 516 micro g ml(-1)) or equimolar combinations of both media. Lysyl oxidase mRNA was expressed by the granulosa cells throughout the 192 h of culture. Thiomolybdate depressed oestradiol production in a dose-dependent manner at doses > 1 micro g ml(-1) and prevented the characteristic clumped appearance of granulosa cells in this serum-free system. Although the supplementation of copper alone had no effect at physiological doses, the use of the equimolar copper and thiomolybdate media ameliorated the effect of tetrathiomolybdates on both oestradiol production and cellular morphology. In conclusion, the results of the present study indicate that lysyl oxidase is expressed by granulosa cells, that thiomolybdates can prevent FSH-induced differentiation of bovine granulosa cells in vitro and that these effects can be reversed by copper supplementation. Overall, these data support the hypothesis that copper-responsive subfertility results from perturbation of the normal pattern of ovulatory follicle growth and development, an effect that may be mediated, at least in part, via lysyl oxidase activity.     

Insulin-like growth factor (IGF) system in the oocyte and somatic cells of bovine preantral follicles

Many studies have highlighted the role of the insulin-like growth factor (IGF) system in the control of antral follicular growth. However, much less is known about the involvement of the IGF system in the regulation of preantral follicular development. In an attempt to address this lack of knowledge, the present study describes the spatial and temporal patterns of expression of mRNA encoding components of the IGF system in bovine follicles during preantral stages of development. mRNA was detected by in situ hybridization using frozen sections (14 microm) of bovine ovarian tissue. Serial sections were probed with 35S-labelled bovine riboprobes. Type 1 IGF receptor mRNA was detected in granulosa cells and in the oocyte of preantral follicles; however, in this study, as in previous studies, it was not possible to detect mRNA encoding either IGF-I or -II. IGF binding protein (IGFBP)-2 mRNA was present in granulosa cells and oocytes of preantral follicles, and immunoreactive IGFBP-2 was detected around granulosa cells during this early stage of development. Occasionally, preantral follicles were identified in which there was no expression of IGFBP-2 in granulosa cells or the oocyte. IGFBP-3 mRNA was detected in the oocyte of preantral follicles and in the surrounding stromal tissue. mRNAs encoding IGFBP-2 and -3, and type 1 IGF receptor were first detected in type 2 follicles. In conclusion, although the IGF ligands are not expressed in preantral follicles, mRNAs encoding the type 1 IGF receptor, and IGFBP-2 and -3 were present and showed unique spatial patterns of expression within preantral follicles.     

Effect of methionyl bovine somatotropin in a prolonged-release vehicle on milk production, hormone profiles and health in dairy cows.

Milk production of dairy cows in 14 herds was increased by 3.8-32.1% by the administration of recombinant methionyl bovine somatotropin (bST) in a sustained-release vehicle at 14 d intervals at 40-94 d post partum. A greater response in multiparous than in primiparous cows was found in cows turned out to graze spring pasture. Administration of recombinant bST resulted in elevated plasma bST during the first 9 d after injection. Clinical characteristics such as respiration, heart rate and body temperature were unaffected by bST treatment, as were blood erythrocyte and leucocyte counts, haemoglobin concentrations and haematocrit values. Plasma levels of glucose, free fatty acids, urea and P, and serum alanine aminotransferase and aspartate aminotransferase were not affected by bST treatment, and acetone was not detected. No adverse effects of bST on general health, infection status of mammary glands, mastitis incidence and reproduction were found.     

Interactive effects of granulosa cell apoptosis, follicle size, cumulus-oocyte complex morphology, and cumulus expansion on the developmental competence of goat oocytes: a study using the well-in-drop culture system

Using a well-in-drop (WID) oocyte/embryo culture system that allows identification of follicular origin, we have investigated the effects of granulosa cells (GCs) apoptosis, follicle size, cumulus-oocyte complexes (COCs) morphology, and cumulus expansion on the developmental competence of goat oocytes matured and cultured individually following parthenogenetic activation. The WID system supported oocyte maturation and embryo development to a level similar to the conventional group system. The majority of goat oocytes acquired competence for development up to the 8-16 cell stage in follicles larger than 2 mm, but did not gain the ability to form morula/blastocyst (M/Bs) until follicles larger than 3 mm in diameter. The extent of atresia affected M/Bs formation. This effect varied according to the follicle size. Cumulus expansion increased with follicle size and decreased with increasing incidence of GCs apoptosis. Oocyte developmental potential was also correlated with cumulus expansion. Regardless of the degree of follicle atresia, 73-84% of the floating cells in the follicular fluid (FF) underwent apoptosis. Correlation between floating cell density in FF and oocyte developmental potency suggests the possibility to use the floating cell density as a simple and non-invasive marker for oocyte quality. It is concluded that the developmental potential of an oocyte is determined by multifactor interactions, and multiple factors must be considered together to accurately predict the quality of an oocyte.