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Regular Article |
c Division of Integrative Biology, Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, United Kingdom
d Department of Applied Physiology, Scottish Agricultural College, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, United Kingdom
e University of Nottingham, Division of Agriculture and Horticulture, School of Biosciences, Loughborough, Leicestershire LE12 5RD, United Kingdom
ABSTRACT
Heifers were assigned either low or high (HE) levels of energy intake and low or high concentrations of dietary crude protein. The effect of these diets on the plasma concentrations of insulin, insulin-like growth factor (IGF)-I, and urea on follicular growth and early embryo development is described. We propose that the observed dietary-induced changes in the ovarian IGF system increase bioavailability of intrafollicular IGF, thus increasing the sensitivity of follicles to FSH. These changes, in combination with increased peripheral concentrations of insulin and IGF-I in heifers offered the HE diet, contribute to the observed increase in growth rate of the dominant follicle. In contrast to follicular growth, increased nutrient supply decreased oocyte quality, due in part to increased plasma urea concentrations. Clearly a number of mechanisms are involved in mediating the effects of dietary energy and protein on ovarian function, and the formulation of diets designed to optimize cattle fertility must consider the divergent effects of nutrient supply on follicular growth and oocyte quality.
fertilization, follicle, follicular development, gene regulation, granulosa cells, growth factors, IGF receptor, insulin, IVF/ART, oocyte development, ovary, ovum, theca cells
INTRODUCTION
Nutritional status is a major factor controlling fertility in cattle [1–3]. Poor nutrition results in delayed puberty, aberrant estrous cycles, lowered conception rates, and reduced birth weight. Specific effects of undernutrition on ovarian follicular growth have also been reported. For example the diameters of preovulatory follicles have been negatively correlated to weight loss in Bos indicus [4], and in postpartum dairy cattle the extent of the energy balance deficit influences follicular growth [5]. Details of mechanisms involved in these changes remain to be characterized fully.
Recently, short-term changes in the plane of nutrition have been shown to affect ovarian follicular dynamics in cattle without any changes in the circulating concentrations of gonadotropin [6]. This is in direct contrast to the situation in monogastric species, such as primates, that show significant changes in LH pulse frequency in response to short-term changes in nutrient intake [7]. In the case of ruminants, it is hypothesized that nutritionally induced changes in circulating metabolic hormones act directly on the ovary to bring about the observed changes in patterns of follicular growth [6]. Supporting this hypothesis is the observation that insulin can influence ovulation rate in energy-deprived beef heifers [8] and ewes [9]. Moreover, treatment of lactating cows with growth hormone (GH) increases the number of small and medium-sized follicles [10, 11], an effect most likely due to GH-induced changes in the circulating concentrations of insulin-like growth factor (IGF)-I and/or insulin. The observations that insulin and IGF-I have potent effects on cultured granulosa cells from cattle [12] and sheep [13, 14] provide further evidence supporting this hypothesis.
Endocrine and metabolic signals that regulate follicular growth also are expected to influence oocyte development either through changes in hormone/growth factor concentrations in follicular fluid or via granulosa-oocyte interaction [15]. For example, as well as regulating follicular growth [6], short-term changes in dietary energy intake influence both oocyte morphology and developmental potential [16, 17]. In addition, high levels of highly degradable protein, as well as increasing plasma ammonia concentrations, increase the concentration of ammonia in bovine follicular fluid [18]. This has been associated with altered follicular growth patterns and a reduction in both the number of ova that cleave following insemination and the proportion that develop to the blastocyst stage. Clearly there are a number of mechanisms through which nutrition can act to influence both follicle dynamics and the developmental competence of oocytes. Although the precise mechanisms are unknown, the observations that the intraovarian IGF system can regulate the response of granulosa and theca cells to gonadotropin [19–21] implicates this system as a candidate for mediating the effects of metabolic hormones on ovarian function.
In this study these mechanisms were analyzed by manipulating the intake of dietary energy and crude protein to produce defined changes in circulating concentrations of metabolic hormones and urea. The resultant changes in the growth of antral follicles and the developmental competence of their oocytes were analyzed. To assess the involvement of the intrafollicular IGF system in this process, dietary-induced changes in the steady-state concentrations of mRNA encoding components of the IGF system in bovine antral follicles was measured and correlated with the observed changes in follicle dynamics and oocyte quality.
MATERIALS AND METHODS
Experimental Design and In Vivo Procedures
The experiments described in this paper were approved by the Animal Experiments Committee of the Scottish Agricultural College and were conducted under the auspices and in accordance with the requirements of the Home Office Animals (Scientific Procedures) Act 1986.
Hereford x Friesian heifers (n = 24) weighing 439 ± 19.9 kg (mean ± SD) with a body condition score (scale 1–5) of 2.7 ± 0.14 (mean ± SD) were used in this study. Animals were allocated on the basis of live weight in a 2 x 2 balanced factorial design. They received diets providing daily metabolizable energy (ME) intakes of either 408 (low energy, LE) or 816 (high energy, HE) kJ/kg W0.75 per day (equivalent approximately to 0.8 and 1.6 times maintenance ME requirements, respectively). This was obtained from rations containing dietary crude protein concentrations of either 20 or 27 g/MJ ME (low protein, LP; and high protein, HP, respectively). The energy density of all diets was held constant at 10.4 MJ ME/kg dry matter. Therefore, the two levels of daily ME intake involved offering these diets at two levels of intake on a metabolic live-weight basis consistent with the levels of ME intake detailed above. Protein levels in the diets were adjusted by altering the level of soya bean meal in the pelleted concentrate rations that also consisted of nutritionally improved straw, barley grain, molassed sugar beet pulp, distillers' dark grains, and soya bean meal. Diets were offered daily as two meals at 0800 and 1600 h. The heifers also were offered 2 kg of barley straw daily, as a source of long roughage.
For 4 wk prior to the experiment, all 24 heifers received the LELP diet. Then, during the study (Days 0–32; see Fig. 1), half of the animals received the LE diets and half the HE diets. All animals received LP rations from Days 0 to 11 inclusive, with 12 heifers (6 from each of the two levels of dietary energy intake) switching to the HP diet on Day 12. The delayed introduction of the HP ration was intended to preclude any confounding effects due to possible physiological adaptation by the heifers assigned to this more extreme dietary formulation.
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During the study, the heifers were managed in two groups, 2 days apart, with each group comprising half of the animals on each dietary energy x protein combination. All were accommodated in individual pens on sawdust-bedded floors within the same building. Intravaginal application of a CIDR-B device (controlled internal drug-release; SmithKline Beecham, Hertfordshire, UK), containing 1.9 g progesterone for a 10-day period from Day 0, together with an intramuscular administration of prostaglandin F2
analogue (15 mg Luprostiol; Prosolvin, Intervet, UK) on Day 6, ensured onset of a reference estrus on Day 12 of the experimental program. Fourteen days later, a second injection of the prostaglandin analogue induced a second estrus that occurred on Day 28. All heifers were artificially inseminated on this and the following day (Day 29) using semen from a single Simmental sire (Fig. 1).
Ovarian follicular growth (diameter) was monitored daily from Day 17 of the experimental program (Day 5 after the reference estrus) by transrectal real time B-mode ultrasonography using an Aloka SSD-500v scanner (Aloka Co. Ltd., Tokyo, Japan) equipped with a 5-MHz linear array transducer. Blood samples were collected at CIDR-B insertion and removal, at Day 12, and daily from Day 17 onward by jugular venipuncture into lithium heparin tubes on ice. On each occasion samples were collected at 0800 h, just before the animals were fed.
The heifers were slaughtered at a local abattoir on Day 32 of the experimental program (4 days after the second induced estrus and first insemination), and reproductive tracts were recovered within 25 min postmortem. The ovaries from each animal were examined and the site of the recent ovulation noted before a small section of one ovary (approximately 25% of its mass; alternating left or right ovary from consecutive animals) was removed and immediately processed for in situ hybridization studies. The remaining ovarian tissue was kept warm by prompt placement in an insulated flask (30–35°C) while the reproductive tracts were placed in an insulated, prewarmed (30–35°C) container before transport to the laboratory.
Collection of Tissue for In Situ Hybridization
A portion of the ovary was collected, as described in the previous section, taking care not to rupture any exposed follicles. It was then oriented on a flat surface to expose the maximum number of follicles. A map of the ovarian surface was drawn, showing the location of all follicles. Follicle diameters were recorded before coating with optimal cutting temperature (OCT) compound (Merck, Poole, Dorset, UK) and frozen above liquid nitrogen before immersion in liquid nitrogen. The ovarian blocks were stored at -70°C until required for subsequent in situ hybridization.
After cutting ovarian sections (14 µm), follicles were classified, morphologically, as healthy, atretic, or grossly atretic. Healthy follicles had an intact basement membrane and a healthy granulosa cell layer with only a few pycnotic nuclei. Atretic follicles had fewer granulosa cells with local disruption in the basement membrane and cells with pycnotic nuclei were identified within the granulosa layer. Follicles with a more extensively disrupted basement membrane, and with a significant reduction in the number of granulosa cells and increase in the number of pycnotic nuclei were classified as grossly atretic [22].
Riboprobes
Riboprobes for IGF binding protein (BP)-2 and IGFBP-4 [23] and IGF-II, and type 1 IGF receptor [24] were labeled with 35S-UTP according to the method described by Armstrong et al. [23]. The insulin receptor probe was obtained by reverse transcription-polymerase chain reaction using RNA isolated from bovine luteal tissue. The forward and reverse primers were: 5'-aactcttcttccactataaccc-3' and 5'-gcaatgtcgtttctctcc-3', respectively, and amplified a 100-base pair cDNA corresponding to positions 1493–1592 of the human insulin receptor cDNA [25].
In Situ Hybridization
Frozen sections (14 µm) were dehydrated, fixed, and probed with 35S-labeled riboprobes according to the method described by Xu et al. [22]. After the final high stringency wash the sections were dipped in autoradiographic K2 photographic emulsion (Ilford Limited, Mobberley, Cheshire, UK) and exposed for 3 wk at 4°C. Sections were then developed (Kodak D-19; H.A. West Watson Cres., Edinburgh, UK) and fixed using Hypam fixer (Ilford Limited) before staining in hematoxylin and eosin. The sections were finally mounted in DPX (R.A. Lamb, London, UK) before microscopic examination using both light- and dark-field illumination.
Image Analysis
The intensity of the in situ hybridization signal was analyzed using an NIH-Image analysis system (NIH, Bethesda, MD) as described previously [23]. Briefly, the number of graphic pixels occupied by silver grains (identified by a set gray threshold) within a defined area of the tissue section was counted and presented as a percentage of the total pixel number within the defined area. The hybridization intensity is therefore presented as the percentage of occupied pixels to total pixels within a defined area of the tissue. Background hybridization intensity, measured with the sense RNA probes, was subtracted from the measurements obtained with the antisense probes to give the final hybridization signal. Within each follicle three separate fields were analyzed for each probe. There was no significant difference (P > 0.05) in hybridization intensity obtained with antisense and sense RNA probes within a nonexpressing region of a tissue section. Under the conditions described here the hybridization signal was proportional to the length of time the slides were exposed to photographic emulsion for up to 3 wk.
In Vivo Zygote Collection and Evaluation
On arrival at the laboratory, and not more than 2 h postmortem, the uterine horn and oviduct ipsilateral to the corpus luteum were each flushed with 10 ml phosphate-buffered fluid containing 0.4% w/v BSA (Ovum Culture Medium; Imperial Laboratories, Andover, Hampshire, UK) to retrieve recently ovulated and fertilized eggs for evaluation of their development in vivo.
Oocyte Retrieval and Embryo Production In Vitro
The collection of ovaries was timed to coincide with presumptive emergence of the first follicular wave and to be in advance of establishment of follicular dominance. The status of all ovaries at slaughter was determined with respect to the numbers of follicles in 1- to 4-mm, 4- to 8-mm, and >8-mm diameter categories. Follicles in the 1- to 4-mm and 4- to 8-mm categories were aspirated separately, and all retrieved oocyte-cumulus complexes (OCC) classified as competent, on the basis of conventional qualitative evaluation criteria [26] were retained for in vitro oocyte maturation (within-category). The OCCs were matured on 4-day-old bovine granulosa cell layers in 50-µl droplets of Medium 199 with Earles salts (Life Technologies, Paisley, UK) supplemented with 10% v/v heat-inactivated steer serum (Globepharm, Esher, UK). The maturation medium was overlaid with mineral oil. After 24 h maturation at 38.5°C (5% CO2 in air), oocytes were fertilized in vitro, using swim-up-derived frozen-thawed spermatozoa from the same Simmental sire (20 h) as used for artificial insemination in modified Tyrode, albumin, lactate, pyruvate medium containing 0.6% w/v fatty acid-free BSA (pH = 7.8; 290–310 mOsm). They were then transferred (Day 1) for further culture, until Day 8, in a modified version of synthetic oviductal fluid [27] containing 0.99 mmol L-1 sodium pyruvate and 9.90 mmol L-1 sodium lactate and supplemented with 10% v/v heat-inactivated steer serum. Postfertilization culture was carried out in 50-µl droplets under mineral oil (5% CO2, 5% O2, 90% N2; 38.5°C), and eggs were transferred to fresh droplets at 48-h intervals. Incidence of cleavage (Day 3) and blastocyst development (Days 7 and 8) were determined by visual evaluation (up to 400x magnification). Embryos at the appropriate stage of development for the day of culture and of good morphological quality, as determined by the criteria of Lindner and Wright [28] were classified as viable. All oocyte and embryo evaluations were carried out by operators who were unaware of the treatments from which the eggs were derived.
Metabolite and Hormone Assays
Urea Plasma samples were analyzed for urea on a BMD/Hitachi 705 autoanalyzer using a commercially available kit (Boehringer Mannheim [Diagnostics and Biochemicals], Lewes, East Sussex, UK).
Progesterone Plasma progesterone concentrations were measured without prior extraction, using an 125I-labeled progesterone double-antibody RIA [29]. The nonextraction assay was modified and validated to enable the use of a rabbit antiprogesterone first antibody, donkey antirabbit IgG, and normal rabbit serum that were obtained as gifts from Diagnostics Scotland, Carluke, Lanarkshire, UK. The sensitivity of the assay at an 80% effective dose (ED80) was 0.51 ng/ml. The inter- and intraassay coefficients of variation for low, medium, and high controls were, 15.7% and 9.5%, 9.3% and 7.8%, and 6.9% and 4.9%, respectively.
Insulin Plasma insulin concentrations were measured using an 125I-labeled insulin double-antibody RIA [30]. The assay was modified to use porcine insulin (I-3505, 24 IU/mg) obtained from Sigma; guinea pig antiporcine insulin, normal guinea pig serum, and sheep antiguinea pig IgG obtained as a gift from Diagnostics Scotland, Law Hospital, Carluke, UK. The sensitivity of the assay at ED80 was 2.50 mIU/L. The inter- and intraassay coefficients of variation for low, medium, and high controls were 12.6% and 10.8%, 7.6% and 7.5%, and 7.1% and 6.6%, respectively.
Insulin-like growth factor-I Plasma IGF-I was measured by RIA after acid gel filtration to remove IGFBPs [31], using a rabbit polyclonal antibody raised against human recombinant IGF-I [32]. The sensitivity of the assay was 22 pg, and the inter- and intraassay coefficients of variation were 13% and 8%, respectively.
Follicle-stimulating hormone Plasma FSH was measured using an 125I-labeled FSH double-antibody RIA [33]. The assay used bovine FSH (USDA-bFSH-1-2, for standards and USDA-b-1-2, for radioiodination), obtained as a gift from Dr. D.J. Bolt through the USDA Animal Hormone Program, Beltsville, MD. Rabbit antiovine FSH-1 antiserum was obtained as a gift from Dr. A.F. Parlow through the program of the National Institute of Diabetes and Digestive and Kidney Diseases, Harbor-UCLA Medical Center, Torrance, CA. Normal rabbit serum and second antibody, donkey antirabbit IgG were obtained as gifts from Diagnostics Scotland, Law Hospital, Carluke, UK. The sensitivity of the assay at ED80 was 69 ng/L. The inter- and intraassay coefficients of variation for low, medium, and high controls were 10.3% and 8.8%, 5.7% and 5.2%, and 6.6% and 6.1%, respectively.
Statistical Analyses
Data relating to plasma insulin, IGF-I, urea, FSH, and follicular growth were analyzed using repeated-measures (split-plot) ANOVA (Genstat 5, Version 3.2; Lawes, Rothamstead, 1993). Level of feeding (energy intake) and protein concentration in the diet (CP/MJ ME) and their associated interactions were used as the between-animal stratum. Sample date and associated interactions with energy intake and dietary protein concentration formed the within-animal stratum.
Live-weight change and growth rate of the dominant follicle of the second follicular wave following the reference estrus were analyzed by regressing heifer live weight and follicle diameter, respectively, to day of experiment for each animal and then analyzing the regression coefficients by a two-way ANOVA.
Oocyte quality and embryo production data, subjected to square root or arcsine transformation as appropriate, were also analyzed using split-plot ANOVA with the blocking structure comprising slaughter date, animal, and follicle size category. Thus, the between-animal stratum examined effects of energy intake and dietary protein concentration and associated interactions. The within-animal stratum examined effects of follicle size category and associated interactions with energy intake and dietary protein concentration. In addition, within each follicle size category, single-factor ANOVA was used to compare embryo production data for animals with contrasting mean plasma urea concentrations (moderate, <6.0 mmol/L; high, 
6.0 mmol/L) over the 15-day period prior to oocyte collection (Days 17–31 inclusive).
The effects of energy intake and dietary protein concentration on the expression of mRNA encoding components of the IGF system were analyzed, after arcsine transformation, using an unbalanced-linear-mixed model according to the method of residual maximum likelihood (REML; Genstat 5, Version 3.2; Lawes, Rothamstead, 1993). The model was constructed using random effects described by the fixed block structure in which replicate observations were nested within follicles that were nested within cows and fixed effects described by the main effects of dietary energy levels, dietary protein concentrations, and follicle size plus their interactions. Individual animals were used as an absorbing factor in the analysis so as to reduce the size of the matrices involved in the calculation.
RESULTS
Growth Rate
During the course of the experiment, heifers offered the HE diets gained weight (1.12 kg/day), whereas those offered the LE diets lost weight (-0.14 kg/day; SED = 0.12; P < 0.001). Growth rate was unaffected by protein level in the diet. Energy intake and dietary protein concentration had no effect on body condition score (P > 0.1).
Plasma Insulin and IGF-I Concentrations
Mean plasma insulin and IGF-I concentrations (Fig. 2) were higher for heifers offered the HE compared to the LE diets (P < 0.001) but were unaffected by the protein concentration in the diet (P > 0.05). Both hormones showed similar changes in concentrations during the estrous cycle with maximum concentrations on the day of ovulation. The ovulatory increases in plasma insulin and IGF-I concentrations were more pronounced in cattle offered the HE diet.
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Plasma FSH and Progesterone Concentrations
Plasma FSH concentrations did not differ significantly (P > 0.05) among dietary treatments. Mean values ranged from 0.14 ng/ml on the day of the reference estrus up to maximum values of 0.22 ng/ml during the first and second waves of follicle growth following the reference estrus. Mean plasma progesterone concentrations (Fig. 3a) between Days 17 and 26 of the experiment (Days 5 and 14 of the estrous cycle) were significantly higher in heifers offered the HE than the LE diets (6.2 vs. 4.8 ng/ml; SED = 0.6; P < 0.05). However, there was a significant interaction (P < 0.05) between dietary energy level and protein concentration with day of cycle for plasma progesterone levels during this period. As Day 14 of the estrous cycle approached, progesterone levels in heifers offered the LEHP and HEHP diets converged, whereas progesterone levels in heifers offered the LELP and HELP diets diverged. Mean plasma progesterone levels between Days 5 and 14 of the estrous cycle for heifers offered the LELP, LEHP, HELP, and HEHP diets were 4.3, 5.3, 6.6, and 5.7 ng/ml, respectively (SED = 0.8).
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Follicle Dynamics
The mean diameter of the dominant follicle (DF) of the first follicular wave after the induced reference estrus on Day 12 of the experimental program (Fig. 3b) was greater for heifers offered HE rather than LE diets (11.0 vs. 8.1 mm; SED = 0.73; P < 0.01). There was a significant interaction (P < 0.001) between dietary protein concentration and day of cycle on the diameter of the DF of the first wave. This indicated that maximum size and subsequent initiation of atresia of the DF occurred 1–2 days earlier in heifers offered HP rather than LP diets. Growth rate of the DF of the second follicular wave was significantly greater in heifers offered the HE than the LE diets (1.78 vs. 1.22 mm/day; SED = 0.20; P < 0.05). The diameter of this follicle at the point of induced ovulation was also greater in heifers offered HE rather than LE diets (15.6 vs. 12.1 mm; SED = 0.8; P < 0.001). Dietary protein concentration had no effect on the growth rate of the DF of the second follicular wave. Energy intake and dietary protein concentration had no effect on the number of 1- to 4-mm diameter follicles (P = 0.57) or 4- to 8-mm diameter follicles (P = 0.61) on the day of slaughter.
Plasma Urea Concentrations
Plasma urea levels increased between Days 0 and 12 of the experimental program in heifers offered the HE but not the LE diets (Fig. 4). Plasma urea concentrations increased further between Days 12 and 17 of the program in heifers offered the HP but not the LP diets. Mean plasma urea levels for heifers offered the LELP, LEHP, HELP, and HEHP diets between Days 17 and 31 of the program were 4.3, 6.3, 6.1, and 7.4 mmol/L, respectively (SED = 0.4; P < 0.001).
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Fertilization In Vivo
Examination of ovaries postmortem indicated that ovarian follicle populations were similar for all diets and that all 24 heifers had ovulated recently, one (HELP diet) with twin ovulations. Native zygotes, fertilized in vivo and collected by flushing the reproductive tracts postmortem, were retrieved from 19 heifers. Among these, 4 LELP (67%), 3 LEHP (60%), 2 HELP (50%), and 2 HEHP (50%) donor-derived ova, respectively, had reached the 8-cell stage of development at collection. The remainder were less advanced.
In Vitro Fertilization and Blastocyst Production
Yields of oocytes and the proportions classified as suitable for in vitro embryo production, together with subsequent postfertilization development data, are presented in Table 1. Oocyte yields from animals on the contrasting diets were not significantly different, although follicle size category influenced the proportions subsequently classified as suitable for in vitro embryo production (P < 0.005), with proportionately more OCCs from medium-sized follicles (4–8 mm) selected. All 24 donors yielded a minimum of two suitable oocytes each from their 4- to 8-mm follicle cohorts, whereas three animals, all receiving the HP diet (1 = LEHP; 2 = HEHP), failed to yield suitable oocytes from small (1- to 4-mm) follicles. Zygote cleavage data, recorded on Day 3 (48 h post-in vitro fertilization) and reflecting presumptive fertilization incidence among selected oocytes, were unaffected by either donor diet or follicle size category. In contrast, blastocyst production data, expressed as proportions of zygotes cleaved by Day 3 and classified in terms of their viability (Table 1), were influenced by dietary energy (LE > HE; P = 0.032). Moreover, within-animal data analysis indicated that cleaved ova derived from 1- to 4-mm follicles were less capable of development to blastocysts in vitro than those derived from 4- to 8-mm follicles (P = 0.023). For animals yielding 1- to 4-mm follicle-derived ova that cleaved following fertilization in vitro, blastocysts were generated from five of six LELP (83%), four of five LEHP (80%), four of six HELP (67%), and one of three HEHP (33%) donors, respectively. The proportions of blastocysts classified as viable were higher (P = 0.056) among ova from the medium-size follicle size category, while dietary protein concentration tended to influence the proportions of viable blastocysts that were graded as excellent or good (grades 1 + 2/viable: LP > HP, P = 0.062).
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A retrospective analysis of the developmental competence of blastocysts derived from donors exhibiting moderate or high plasma urea levels in the 14 days prior to oocyte collection was performed. In respect of blastocysts produced from selected oocytes derived from small follicles, proportionately more viable blastocysts (0.31 ± 0.081 vs. 0.14 ± 0.051; P < 0.08) were produced from donors with moderate (<6 mmol/L) plasma urea concentrations (n = 10) than from those exhibiting high (>6 mmol/L) plasma urea concentrations (n = 11).
Ovarian IGF System
The spatial distribution of mRNA encoding components of the IGF system within bovine antral follicles is shown in Figure 5. The IGF-II and IGFBP-4 mRNA were confined to thecal tissue; in contrast, mRNA encoding IGFBP-2 was located in granulosa cells. Both type 1 IGF receptor mRNA and insulin receptor mRNA were detected in both granulosa and thecal tissue with expression in granulosa tissue greater than in thecal tissue. In addition to granulosa and thecal tissue, mRNA encoding type 1 IGF receptor was also detected in oocytes from preantral and antral follicles. The IGF-I mRNA was not detected in bovine ovarian follicular tissue. The HE diet reduced the steady-state concentrations of mRNA encoding all the components of the IGF system in healthy follicles. The reduction was significant (P < 0.05) in the case of IGFBP-2, IGFBP-4, and insulin receptor mRNA. No effect of diet was observed in atretic or grossly atretic follicles (data not shown). All subsequent statistical analyses were carried out using data from healthy follicles alone. The interaction between follicle size and dietary energy and protein is shown in Figure 6. Significant (P < 0.05) reductions in the steady-state concentrations of mRNA encoding IGFBP-2 and -4 and insulin receptor in small (<4-mm) follicles were induced by the HE diet. There was no direct effect of dietary protein on the expression of mRNA encoding components of the IGF system.
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DISCUSSION
The overall aim of this work was to analyze mechanisms through which dietary energy and protein influence follicular dynamics and the developmental competence of oocytes in cattle. It is the first study to demonstrate a direct effect of dietary intake on the expression of mRNA encoding components of the ovarian IGF system and supports the hypothesis that the nutritional regulation of follicular growth is mediated, at least in part, by the action of circulating metabolic hormones on the ovarian IGF system.
The high level of energy intake described in this study significantly increased plasma insulin and IGF-I concentrations relative to the LE diet. In contrast, dietary protein concentration had no effect on these metabolic hormones. The absence of any significant changes in FSH concentrations supports the results of other studies that failed to show any effects of short-term changes in the plane of nutrition on FSH concentrations in ruminants [2]. The ovulatory increase in plasma IGF-I concentrations supports results of related studies in sheep [34] and is probably a consequence of increased plasma estradiol stimulating hepatic IGF-I production because treatment of ovariectomized cattle with estradiol was shown to increase serum IGF-I concentrations significantly [35].
Although there is no direct evidence of an endocrine role for insulin and IGF-I in the control of follicular growth, increasing indirect evidence indicates that such a role exists. For example, plasma insulin and IGF-I concentrations have been positively correlated with follicular growth in postpartum dairy cattle [5, 36], and twinning rate in cattle is also associated with elevated plasma IGF-I concentration [37]. The stimulation of antral follicle growth following recombinant GH treatment of nonlactating heifers was similarly associated with increased circulating concentrations of insulin and IGF-I [38, 39]. Collectively, the available evidence suggests that the increased size of the dominant follicle in cattle fed the HE diet in the present study was due to endocrine actions of both these metabolic hormones on follicular growth.
Luteal progesterone production during the induced estrous cycle was significantly increased in cattle offered the HE diet and is probably a reflection of increased follicle size prior to ovulation. This has relevance to embryo survival in the pregnant cow as increases in luteal progesterone production as early as Day 5 of pregnancy have been shown to significantly increase embryo growth and associated interferon-
production [40].
The spatial patterns of expression of mRNA encoding components of the ovarian IGF system in antral follicles support previous observations [24, 26, 41, 42]. The results also clearly demonstrate the presence of mRNA encoding type 1 IGF receptor in oocytes from preantral and antral follicles, confirming that IGF-I can regulate oocyte maturation directly through binding to its own receptor.
Although nutritional status has been shown to regulate the expression of mRNA encoding components of the hepatic IGF system [43], the results presented here show, for the first time, a direct effect of dietary intake on the ovarian IGF system. Specifically, increased dietary energy significantly decreased the steady-state concentration of mRNA encoding IGFBP-2 and -4 and insulin receptor in small antral follicles. Previous studies [23] have shown that during the development of dominance there is a significant decrease in the steady-state concentration of mRNA encoding IGFBP-2 in granulosa cells from the dominant follicle. A reduction in the local level of IGFBP-2 would increase IGF bioactivity in the dominant follicle that, in turn, would be expected to increase the sensitivity/response of the dominant follicle to FSH, thus allowing its continued growth in an environment of decreasing systemic FSH concentrations.
Similarly, in heifers offered the HE compared to the LE diet in the present study, the reduction in the steady-state concentration of mRNA encoding IGFBP-2 and -4 in small antral follicles is expected to increase the bioavailability of intrafollicular IGF (both locally produced IGF-II and systemically derived IGF-I) in these follicles. The consequent increase in the sensitivity/response toward FSH would be expected to result in an increased rate of follicular growth.
The factors involved in regulating IGFBP-2 and -4 mRNA expression in the bovine ovary are not understood. Although we have shown that FSH decreases IGFBP-2 mRNA expression in cultured granulosa cells [23], the roles of insulin and IGF-I, the most likely candidates for mediating the effects of dietary energy on the ovarian IGF system have not been examined. The decrease in insulin receptor mRNA expression in cattle offered the HE diet is most probably due to insulin-mediated receptor downregulation. The functional significance of this latter observation, however, is not known.
Oocyte quality in small follicles was negatively correlated with plasma urea concentrations (Table 1 and Fig. 4). Exposure of follicle-enclosed oocytes to high levels of ammonia and/or urea has previously been observed to compromise their capacity to develop to the blastocyst stage following a period of in vitro culture [18]. In that study, however, the detrimental effect of ammonia and/or urea on oocyte quality was greater for oocytes from medium than from small follicles. The mechanisms by which oocyte competence from specific follicle size categories is compromised by these nitrogen moieties are not understood, and the most vulnerable follicle size category may be linked to the energy status. In the present experiment the highest levels of urea reflected the combined effects of feeding a high protein diet at a high level (HEHP). It is therefore not possible, with the present experimental design, to ascertain whether energy intake per se may have modified the known detrimental effect of high plasma urea on the oocyte.
The ovarian IGF system also has the potential to interact directly with the oocyte through the type 1 IGF receptor. Small follicles from heifers offered the HEHP diet in the present study had significantly reduced levels of mRNA encoding IGFBP-2 and -4, and as discussed, we expect this to increase the bioactivity of IGF in these follicles, which is probably a critical factor controlling oocyte developmental capacity. Indeed our results indicate that overstimulation by IGF may be detrimental to oocyte development.
In conclusion we have shown that dietary energy and protein can directly affect the expression of mRNA encoding components of the ovarian IGF system. We hypothesize that the resultant changes in the ovarian IGF system increase the sensitivity of follicles toward FSH and, in combination with dietary-induced increases in the concentration of circulating insulin and IGF-I, contribute to the observed increase in growth rate of the dominant follicle. Dietary protein concentration also influenced oocyte quality, with developmental competence being negatively correlated with plasma urea levels. In addition, we hypothesize that nutritionally induced changes in the ovarian IGF system may play a key role in regulating oocyte quality. Finally, the data presented here indicate that future studies concerned with the formulation of diets designed to optimize cattle fertility must take into account the possibility of divergent actions of nutrient supply on follicular growth and oocyte quality.
FOOTNOTES
First decision: 25 August 2000.
1 Ministry of Agriculture Fisheries and Food (contract DSO206) and Biotechnology and Biological Sciences Research Council supported this study. Scottish Agricultural Colleges receives funding from the Scottish Executive Rural Affairs Department. The Medical Research Council funded K.J.W. 
2 Correspondence: FAX: 0131 440 0434; david.armstrong{at}bbsrc.ac.uk
Accepted: January 16, 2001.
Received: July 19, 2000.
REFERENCES
production and uterine hormone receptors during early pregnancy. J Reprod Fertil 1999; 54(suppl):33–48This article has been cited by other articles:
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L. M. Chagas, J. J. Bass, D. Blache, C. R. Burke, J. K. Kay, D. R. Lindsay, M. C. Lucy, G. B. Martin, S. Meier, F. M. Rhodes, et al. Invited Review: New Perspectives on the Roles of Nutrition and Metabolic Priorities in the Subfertility of High-Producing Dairy Cows J Dairy Sci, September 1, 2007; 90(9): 4022 - 4032. [Abstract] [Full Text] [PDF]
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E. Borowczyk, J. S. Caton, D. A. Redmer, J. J. Bilski, R. M. Weigl, K. A. Vonnahme, P. P. Borowicz, J. D. Kirsch, K. C. Kraft, L. P. Reynolds, et al. Effects of plane of nutrition on in vitro fertilization and early embryonic development in sheep J Anim Sci, June 1, 2006; 84(6): 1593 - 1599. [Abstract] [Full Text] [PDF]
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S Freret, B Grimard, A A Ponter, C Joly, C Ponsart, and P Humblot Reduction of body-weight gain enhances in vitro embryo production in overfed superovulated dairy heifers. Reproduction, April 1, 2006; 131(4): 783 - 794. [Abstract] [Full Text] [PDF]
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A A Fouladi-Nashta and K H S Campbell Dissociation of oocyte nuclear and cytoplasmic maturation by the addition of insulin in cultured bovine antral follicles. Reproduction, March 1, 2006; 131(3): 449 - 460. [Abstract] [Full Text] [PDF]
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S J Adamiak, K Powell, J A Rooke, R Webb, and K D Sinclair Body composition, dietary carbohydrates and fatty acids determine post-fertilisation development of bovine oocytes in vitro Reproduction, February 1, 2006; 131(2): 247 - 258. [Abstract] [Full Text] [PDF]
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B. W. Hess, S. L. Lake, E. J. Scholljegerdes, T. R. Weston, V. Nayigihugu, J. D. C. Molle, and G. E. Moss Nutritional controls of beef cow reproduction J Anim Sci, June 1, 2005; 83(13_suppl): E90 - 106. [Abstract] [Full Text] [PDF]
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Z. Roth and P.J. Hansen Sphingosine 1-Phosphate Protects Bovine Oocytes from Heat Shock During Maturation Biol Reprod, December 1, 2004; 71(6): 2072 - 2078. [Abstract] [Full Text] [PDF]
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T. R. Bilby, A. Guzeloglu, S. Kamimura, S. M. Pancarci, F. Michel, H. H. Head, and W. W. Thatcher Pregnancy and Bovine Somatotropin in Nonlactating Dairy Cows: I. Ovarian, Conceptus, and Insulin-Like Growth Factor System Responses J Dairy Sci, October 1, 2004; 87(10): 3256 - 3267. [Abstract] [Full Text] [PDF]
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T. P. Fleming, W. Y. Kwong, R. Porter, E. Ursell, I. Fesenko, A. Wilkins, D. J. Miller, A. J. Watkins, and J. J. Eckert The Embryo and Its Future Biol Reprod, October 1, 2004; 71(4): 1046 - 1054. [Abstract] [Full Text] [PDF]
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R. Webb, P. C. Garnsworthy, J.-G. Gong, and D. G. Armstrong Control of follicular growth: Local interactions and nutritional influences J Anim Sci, January 1, 2004; 82(13_suppl): E63 - 74. [Abstract] [Full Text] [PDF]
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N. H. Ciccioli, R. P. Wettemann, L. J. Spicer, C. A. Lents, F. J. White, and D. H. Keisler Influence of body condition at calving and postpartum nutrition on endocrine function and reproductive performance of primiparous beef cows J Anim Sci, December 1, 2003; 81(12): 3107 - 3120. [Abstract] [Full Text] [PDF]
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G. Ptak, M. Tischner, N. Bernabo, and P. Loi Donor-Dependent Developmental Competence of Oocytes from Lambs Subjected to Repeated Hormonal Stimulation Biol Reprod, July 1, 2003; 69(1): 278 - 285. [Abstract] [Full Text] [PDF]
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R. P. Wettemann, C. A. Lents, N. H. Ciccioli, F. J. White, and I. Rubio Nutritional- and suckling-mediated anovulation in beef cows J Anim Sci, February 1, 2003; 81(14_suppl_2): E48 - 59. [Abstract] [Full Text] [PDF]
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), LEHP (
), HELP (
), and HEHP (
) diets and during the second follicle wave for heifers offered the HE (
) or LE (•) diets (averaged over both dietary protein concentrations). A prostaglandin injection was given on Day 26 of the cycle. The bar represents a pooled standard error of the difference (SED)
