Abstract
The aim of our study was to test whether a reduction in dietary intake could improve in vitro embryo production in superovulated overfed dairy heifers. Cumulus–oocyte complexes of 16 Prim’ Holstein heifers (14 ± 1 months old) were collected by ovum pick-up (OPU), every 2 weeks following superovulation treatment with 250 μg FSH, before being matured and fertilized in vitro. Embryos were cultured in Synthetic Oviduct Fluid medium for 7 days. Heifers were fed with hay, soybean meal, barley, minerals and vitamins. From OPU 1 to 4 (period 1), all heifers received individually for 8 weeks a diet formulated for a 1000 g/day live-weight gain. From OPU 5 to 8 (period 2), the heifers were allocated to one of two diets (1000 or 600 g/day) for 8 weeks. Heifers’ growth rates were monitored and plasma concentrations of metabolites, metabolic and reproductive hormones were measured each week. Mean live-weight gain observed during period 1 was 950 ± 80 g/day (n = 16). In period 2 it was 730 ± 70 (n = 8) and 1300 ± 70 g/day (n = 8) for restricted and overfed groups respectively. When comparing period 1 and period 2 within groups, significant differences were found. In the restricted group, a higher blastocyst rate, greater proportions of grade 1–3 and grade 1 embryos, associated with higher estradiol at OPU and lower glucose and β-hydroxybutyrate, were observed in period 2 compared with period 1. Moreover, after 6 weeks of dietary restriction (OPU 7), numbers of day 7 total embryos, blastocysts and grade 1–3 embryos had significantly increased. On the contrary, in the overfed group, we observed more <8 mm follicles 2 days before superovulation treatment, higher insulin and IGF-I and lower nonesterified fatty acids in period 2 compared with period 1 (no significant difference between periods for embryo production). After 6 weeks of 1300 g/day live-weight gain (OPU 7), embryo production began to decrease. Whatever the group, oocyte collection did not differ between period 1 and 2. These data suggest that following a period of overfeeding, a short-term dietary intake restriction (6 weeks in our study) may improve blastocyst production and embryo quality when they are low. However, nutritional recommendations aiming to optimize both follicular growth and embryonic development may be different.
Introduction
Nutrition and metabolic status have several effects on reproduction and fertility in cattle, which have been largely reviewed (Robinson 1996, O’Callaghan & Boland 1999). Dietary intake can influence ovarian activity via effects at various levels of the hypothalamus–pituitary–ovarian axis. Changes in the plane of nutrition can affect follicular growth (Gutierrez et al. 1997, Gong et al. 2002, Diskin et al. 2003, Mihm & Bleach 2003), by inducing changes in plasma metabolites and metabolic hormones, such as insulin and insulin-like growth factor-I (IGF-I; Armstrong et al. 2001, Ferguson et al. 2003) and/or in hormones and growth factors in follicular fluid (Landau et al. 2000).
Dietary intake can also affect oocyte morphology (O’Callaghan et al. 2000), oocyte developmental capacity and embryo production: overfeeding has been shown to be detrimental to oocyte quality and embryo development in vivo (Mantovani et al. 1993, McEvoy et al. 1995, Negrao et al. 1997) and in vitro (Papadopoulos et al. 2001), whereas dietary restriction could have a positive effect on oocyte quality (Lozano et al. 2003) and blastocyst production in vitro (McEvoy et al. 1997, Nolan et al. 1998a, Armstrong et al. 2001). The way nutrition influences embryo production remains to be fully characterized. It may affect oocyte development before fertilization, early embryo development or uterine environment. This is why nutritional management of donor cattle in programs of superovulation, embryo transfer and ovum pick-up–in vitro fertilization (OPU-IVF) need to be adapted to optimize the production of oocytes and embryos (Scaramuzzi & Murray 1994).
In France, the age at first calving is on average 24 months for two-thirds of Prim’ Holstein dairy heifers (Gaboriau 2002). As a consequence, growth rates must be high enough to reach a sufficient weight at the first artificial insemination, when heifers are 15 months old (380–400 kg). Recommended live-weight (LW) gains are around 800–900 g/day before 6 months of age and around 700 g/day between 6 and 15 months of age. Growth rates are generally higher than 800 g/day in farms with high genetic potential, where high LW at first artificial insemination is required. French embryo-transfer teams have noticed that embryo production is lower in overfed heifers: a study by Negrao et al.(1997) on superovulated dairy heifers showed a decrease in the number of transferable embryos as the net energy intake per day increased.
Our study was conducted on superovulated dairy heifers to fit with field conditions. The first objective was to study the potentially detrimental effect of several weeks of overfeeding and high growth rate (> 1000 g/day) on in vitro embryo production. The second objective was to test whether in vitro embryo production in the overfed heifers could be improved by a short-term reduction in dietary intake, leading to lower growth rates (around 600 g/day). Heifers were not underfed but only restricted: they grew slowly with all their maintenance and growth needs covered. The duration of the balanced dietary restriction needed to be applicable in field OPU-IVF programs.
By using the OPU-IVF technique we could study the effects of dietary manipulation on oocyte quality – that is to say oocyte developmental capacity – evaluated by in vitro embryo production. By monitoring heifers’ growth rates and metabolic status, and repeating OPU-IVF sessions every 2 weeks throughout the experiment, we wanted to evaluate the minimal duration of diet manipulation needed to modify oocyte collection and embryo production.
Materials and Methods
Animals and housing
All experimental work was performed in an experimental farm of the French Union Nationale des Coopératives d’Elevage et d’Insémination Animale (UNCEIA), in Isère, in the south-east of France. Sixteen Prim’ Holstein dairy heifers of 14 ± 1 months old, an average LW of 340 ± 25 kg and a body condition score (BCS; on a scale from 0 = thin to 5 = fat; Bazin 1984) of 1.9 ± 0.2 were used for the study. Heifers were confirmed to be cyclic by rectal palpation before being introduced into the experimental farm, where they were housed together indoors and fed a maintenance diet composed of natural prairie hay and a commercial concentrate. During the experiment they were housed separately and fed individually the experimental diets. Water was available ad libitum. Straw was used as bedding. All experimental procedures were carried out in accordance with French recommendations.
Experimental protocol
The experiment was carried out using two batches of eight heifers, from December 2000 to April 2001 and from December 2001 to April 2002. The experimental protocol was the same for both periods (Fig. 1). The same technician worked on embryo production in both batches.
Before the start of the experiment, estrous cycles were synchronized with subcutaneous 3 mg Norgestomet implants (Crestar; Intervet, Angers, France) inserted under the convex surface of the ear for 10 days. On the day of implant insertion, heifers received an i.m. injection of 3 mg norgestomet and 3.8 mg estradiol valerate (Crestar). Two days before implant removal, they received 500 μg cloprostenol i.m. (Estrumate; Schering-Plough Vétérinaire, Levallois-Perret, France). On day 6 of the synchronized estrous cycle (day 0 was the day of estrus), large follicles (diameter ≤8 mm) were aspirated and small follicles (2 mm < diameter < 8 mm) were counted. Two days later, heifers were superovulated with a total dose of 250 μg follicle-stimulating hormone (FSH; Stimufol; Mérial, Lyon, France) divided into 5 i.m. injections 12 h apart, at decreasing doses. Cumulus–oocyte complexes (COCs) were collected by ovum pick-up (OPU) 12 h after the last FSH injection, during the luteal phase of the cycle. All follicles were aspirated and counted as small (2 mm < diameter < 8 mm) or large (diameter ≥ 8 mm) follicles. The day after, heifers received an additional Norgestomet implant for 10 days to induce estrus 2 days after implant removal. On day 9 of the second cycle, large follicles were aspirated, heifers were superovulated 2 days later and OPU performed again during the luteal phase. As shown in Fig. 1, OPU 2 took place 4 weeks after OPU 1.
Subsequent OPU sessions (OPU 3-8) were performed every 2 weeks; in order to prevent return to estrous between OPUs, heifers received Norgestomet implants every 10 days, from the day after OPU 2 until the end of the experiment. As described previously, large follicles were aspirated and small follicles (2 mm < diameter < 8 mm) counted 2 days before the beginning of the superovulation treatment.
Period 1 (P1) is defined as the time between OPU 1 and 4: it includes OPUs 1, 2, 3 and 4. Period 2 (P2) is defined as the time between OPU 4 and 8: it includes OPUs 5, 6, 7 and 8. COCs collected from OPU 2 and 8 were not used for embryo production but were frozen for later molecular analyses (data not shown).
Experimental diets and growth measurements
Experimental diets were composed of natural prairie hay, soybean meal, barley and a mineral and vitamin mix. We tested a global variation in energy and protein supply, and worked in terms of the growth objective. Diets differed in the quantities of hay and barley offered, in order to adjust the diet to the growth objective (Jarrige 1988).
After a dietary transition of 3 weeks, all heifers (n = 16) received individually for 8 weeks (period 1 was the control period) a diet designed to produce a LW gain of about 1000 g/day. After OPU 4, heifers were then allocated to one of two diets according to LW gain and oocyte production observed during period 1. One group of heifers (n = 8) continued to receive the same diet (overfed group) whereas the other group of heifers (n = 8) received a diet designed to limit growth to about 600 g/day (restricted group), until the end of the experiment. Diets were offered twice daily at 0800 and 1600 h. Heifers were weighed each week to measure their LW gain and body condition was scored every 4 weeks.
Ultrasound-guided transvaginal follicular aspiration (OPU)
Follicular aspiration was performed as described by Bols et al.(1995). We used an ultrasound scanner Starvet 3 (Pie medical 200; Hospimedi, Valdampierre, France) with a 5 MHz annular array probe. Heifers received an epidural injection of 4 ml lidocaine (Lurocaine®; Vétoquinol, Lure, France). A disposable-needle guidance system was used and disposable 18-G 1/2 needles were changed between each animal. Tubing and needles were rinsed with collection medium (phosphate buffer with 2 g/l BSA, 100 IU/ml penicillin and 50 μg/ml streptomycin; IMV Technologies, L’Aigle, France), supplemented with 100 IU/ml heparin (Leo, Saint Quentin en Yvelines, France). Follicular fluid was collected in 50 ml sterile tubes. Tube contents were then filtered over a 100 μm steel filter and rinsed with collection medium over a Petri dish. Tubes, dishes, collection medium and filters were maintained at 38 °C throughout the procedure.
In vitro maturation, fertilization and culture
COCs from a single heifer were treated separately from those of other heifers. COCs were isolated from Petri dishes and washed three times in collection medium. Their quality was graded according to classical morphological criteria as described by Marquant-Le Guienne (1998). Briefly, grade 1 COCs had intact cumulus, compact multi-layered cumulus cells and homogeneous ooplasm. Grade 2 COCs had incomplete compact cumulus with at least five layers of cumulus cells and/or less homogeneous ooplasm. Grade 3 COCs had incomplete cumulus which could be partially absent around the oocyte, at least three layers of compact cumulus cells and/or non homogeneous ooplasm. Grade 4 COCs had incomplete, expanded or absent cumulus and/or very heterogeneous ooplasm. In vitro maturation was performed in M199 medium (M4530; Sigma, St Louis, MO, USA) supplemented with 10 μg/ml FSH (Stimufol®; Mérial, Lyon, France), 50 μg/ml gentamycin (G1264; Sigma), 1 μg/ml 17β-estradiol (E2257; Sigma) and 5 ng/ml epidermal growth factor (E6135; Sigma). Quality 1, 2 and 3 COCs were washed once in maturation medium before being incubated for 24 h at 38.5 °C in an humidified atmosphere of 5% CO2 in air. In vitro fertilization (IVF) was performed on matured oocytes as described by Guyader-Joly et al.(1999) with some modifications, using frozen-thawed semen from the single ejaculate of a Prim’ Holstein bull. The capacitation medium was a modified Tyrode’s calcium-free medium (sp-TALP, pH 7.2) supplemented with 6 mg/ml BSA (A7906; Sigma), 1 mM pyruvic acid sodium (P2256, Sigma) and 50 μg/ml gentamycin (G1264). The fertilization medium was a modified Tyrode’s solution (fert-TALP, pH 7.6) containing 6 mg/ml fatty acid-free BSA (A6003), 0.2 mM pyruvic acid sodium (P2256), 100 IU/ml penicillin (P3032), 50 μg/ml streptomycin (S9137), 10 μg/ml heparin-sodium salt (H3125), 20 μM penicillamine (P4875), 10 μM hypotaurine (H1384) and 1 μM epinephrine (E4250; all reagents were from Sigma). Oocytes and spermatozoa were incubated for 18 h at 38.5 °C in an humidified atmosphere of 5% CO2 in air. The day of IVF was defined as day 0.
After IVF, zygotes were washed once in fert-TALP and once in culture medium, which was Synthetic Oviduct Fluid medium (UNCEIA, Maisons-Alfort, France) supplemented with 3 mg/ml BSA (A9647), 100 IU/ml penicillin (P3032), 50 μg/ml streptomycin (S9137), 50 μg/ml gentamycin (G1264) and 20 μg/ml nystatin (N4014; Sigma). All zygotes from one heifer were cultured in one microdrop of 45 μl Synthetic Oviduct Fluid medium, covered with mineral oil (M3516; Sigma) at 38.5 °C in an humidified atmosphere of 5% CO2 and 5% O2 in air. Five microlitres of foetal calf serum (Hyclone, Logan, UT, USA) were added 48 h after beginning in vitro culture. Zygotes were observed on day 2 to count the number of cleaved embryos. After 7 days of in vitro culture, the embryo stage was evaluated (morula, early blastocyst, blastocyst and expanded blastocyst) and the quality was graded according to classical morphological criteria (grade 1, excellent; grade 2, good; grade 3, medium; grade 4, poor) as previously described by INRA-UNCEIA (1990).
Blood sampling
Blood samples were collected weekly by caudal venipuncture, before morning feeding, to be assayed for glucose, β-hydroxybutyrate (BHB), nonesterified fatty acids (NEFAs), urea, insulin and IGF-I. Blood samples were also collected at the time of follicular puncture (dominant follicle puncture and OPU) to be assayed for progesterone and estradiol. Blood samples were collected into heparinized tubes and centrifuged immediately at 1350 g for 10 min. Plasma aliquots were stored at −20 °C until required for analysis.
Assays
Weekly blood samples were analysed by photometric methods for glucose (Glucose-RTU; BioMérieux, Lyon, France), NEFAs (NEFA C; Wako Chemicals, Neuss, Germany), urea (Urea-kit S; BioMérieux, Lyon, France), BHB (method adapted from Barnouin et al. (1986)). Insulin and IGF-I were analysed by radioimmunoassays respectively based on porcine insulin (Insulin-CT; CIS Bio International, Gif-sur-Yvette, France) and human recombinant IGF-I (IGF-I-RIACT; CIS Bio International). IGF-binding proteins were removed by treating each plasma sample with an acid solution to separate the IGF-I from the binding proteins. The acidified sample was then neutralized and the binding proteins saturated by the addition of IGF-II, therefore leaving all the IGF-I free to be measured. The standards only contained IGF-I and the antibodies used were specific for IGF-I.
Inter-assay coefficients of variation were 4.3% at 3.47 mM, 12.65% at 0.31 mM, 8.3% at 2.99 mM, 6.5% at 0.82 mM, 11.3% at 153.8 pM and 3.5% at 57.45 ng/ml for glucose, NEFAs, urea, BHB, insulin and IGF-I respectively. Corresponding intra-assay coefficients of variation were 1.3% at 3.47 mM, 2.4% at 0.31 mM, 3.4% at 2.99 mM, 1.8% at 0.82 mM, 4.8% at 153.8 pM and 3.8% at 57.45 ng/ml.
Blood samples collected at the moment of follicular puncture were analysed by enzyme immunoassay for progesterone (Ovucheck plasma; Vétoquinol) and by radioimmunoassay for estradiol as previously described by Grimard et al.(1995). Intra-assays coefficients of variation were 17.4% at 6.6 ng/ml and 6.7% at 60 pg/ml for progesterone and estradiol respectively. Corresponding inter-assays coefficients of variation were 5.6% at 6.6 ng/ml and 8.8% at 60 pg/ml.
Statistical analyses
Statistical analysis was performed using a linear mixed model, with the MIXED procedure of the SAS software (SAS Institute 2000) for repeated measures, including a random female effect in all models. The year effect was tested in all models and removed when nonsignificant.
Effects of the number of OPU sessions, experimental group and their interaction where tested on LW, BCS, follicular characteristics, COCs and embryo production (Least square means (Ls means) of the interaction were subsequently compared with the contrast statement of the mixed procedure). Effects of period, experimental group and their interaction where tested on LW gain, metabolites, metabolic and reproductive hormone plasma concentrations, follicular characteristics, COCs and embryo production (lsmeans of the interaction were subsequently compared using Scheffe’s test for multiple comparisons). Results are presented as lsmeans ± standard error of the mean (s.e.m.).
Percentages of large and small aspirated follicles, grade 1–3 and grade 1 collected oocytes, maturation, cleavage, development, blastocysts, grade 1–3 and grade 1 embryos were compared by a X2 test. A P ≤ 0.05 was significant while 0.05 < P < 0.10 was considered to be a trend.
Results
Growth rate, body weight and BCS
During the 3-week dietary transition, the mean LW gain was 490 ± 100 g/day (lsmean ± s.e.m.). The LW gain observed in period 1 for the 16 heifers was 950 ± 80 g/day (for an objective of 1000 g/day), with a LW of 380 ± 7 kg and a BCS of 2.3 ± 0.1 at the end of period 1. In period 2, LW gains significantly differed (P ≤ 0.01) and were 730 ± 70 and 1300 ± 70 g/day for restricted and overfed groups respectively. LW and BCS began to differ significantly between the two groups respectively from OPU session 7 (Fig. 2) and OPU session 6 (Fig. 2), and remained different until the end of the experiment.
Metabolite and metabolic hormone plasma concentrations
Lsmeans ± s.e.m. of plasma concentrations of metabolites and metabolic and reproductive hormones for the interaction between period and group are presented in Table 1. A year effect was observed for NEFAs and BHB and was taken into account in all models. As expected, when comparing the two groups of heifers during period 1 (Table 1), no differences were observed for plasma glucose, insulin, IGF-I, NEFAs, BHB and urea. During period 2, plasma insulin and BHB were significantly reduced in the restricted compared with the overfed group of heifers (Table 1). No differences were observed for the other metabolites.
The restricted group had reduced plasma glucose and BHB in period 2 compared with period 1 (P ≤ 0.05) while insulin, IGF-I and NEFA concentrations did not differ (Table 1). On the contrary, the overfed group had increased plasma insulin and IGF-I and reduced plasma NEFAs in period 2 compared with period 1 (P ≤ 0.05) while there were no differences for glucose and BHB. Weekly insulin plasma concentrations are illustrated in Fig. 3: during the last 2 weeks of experiment, insulinaemia was very significantly higher in the overfed group. Urea was increased in period 2 compared with period 1 for both groups (Table 1).
Reproductive hormone plasma concentrations
A year effect was observed for estradiol. No differences were observed for plasma progesterone and estradiol 2 days before superovulation treatment (time of ≥ 8 mm follicle aspiration) or at the time of OPU when comparing the two groups of heifers during period 1 (Table 1). When comparing the two groups of heifers during period 2, progesterone and estradiol concentrations did not differ.
Progesterone (measured 2 days before superovulation and at OPU) was reduced in period 2 compared with period 1 for both groups of heifers. Estradiol concentrations measured 2 days before FSH treatment did not differ between periods, but estradiol concentrations measured at the time of OPU were increased in period 2 compared with period 1, and this was significant in the restricted group (Table 1).
Follicular characteristics, oocyte collection and embryo production
Lsmeans ± s.e.m. of follicular characteristics, oocyte collection and embryo production for the interaction between period and group are presented in Table 2. A year effect was observed for large follicles 2 days before the beginning of superovulation treatment and at the time of OPU, day 7 total embryos, blastocysts, grade 1–3 embryos and grade 1 embryos: it was taken into account in all models. In period 1, lsmeans did not differ between the two groups of heifers for all variables concerning follicular characteristics, oocyte collection and embryo production. However, the percentages of development, blastocysts, grade 1–3 embryos and grade 1 embryos were lower in the restricted group. No difference was observed between groups in period 2 (Table 2). The evolution of numbers of total embryos, blastocysts and grade 1–3 embryos at day 7 of in vitro culture, according to OPU sessions, is shown in Fig. 4 for each group of heifers. No significant difference was found between groups compared at each OPU session.
When comparing follicular characteristics, oocyte collection and embryo production between OPU sessions 1 and 4, after 8 weeks of overfeeding for all heifers, evolution was only observed for follicles with a significant increase in the number of small follicles 2 days before the first FSH injection at OPU session 4. The total number of follicles aspirated at OPU and oocyte collected did not vary. No change was observed from OPU 1 to OPU 4 for embryo production data (Fig. 5a).
Two days before the beginning of superovulation treatment, the number of small follicles was significantly increased in period 2 compared with period 1 for the overfed group whereas there was no difference in the restricted group. The number of large follicles was equivalent in the two periods for the two groups. On the day of OPU, the number of small follicles increased in period 2 for all heifers (period effect P = 0.01) but no difference between periods was observed group by group and the number of large follicles did not vary (Table 2). For both groups, the numbers of collected, grade 1–3 and grade 1 oocytes were not different between periods (Table 2). No effect on morphological quality of oocytes was observed. Recovery rate (collected oocytes/aspirated follicles) did not differ between the two groups in period 1 (66% for the restricted group and 62% for the overfed group) and in period 2 (65% for the restricted group and 60% for the overfed group), as well as grade 1–3 and grade 1 oocyte rates (Table 2).
The number of fertilized oocytes and cleaved embryos increased in period 2 for all heifers (period effect P < 0.001) and group by group for fertilized oocytes as illustrated in Table 2. Maturation rate (fertilized oocytes/ grade 1–3 oocytes used for in vitro maturation) increased significantly in period 2 for the restricted group (82 versus 97%) and the overfed group (86 versus 94%). Cleavage rate did not differ between periods 1 and 2. The numbers of day 7 total embryos, blastocysts, grade 1–3 embryos (transferable embryos) and grade 1 embryos (freezable embryos) were increased in period 2 for the two groups (period effect P < 0.01). Nevertheless, the increase was higher and significant for blastocysts and grade 1 embryos in the restricted group, as shown in Table 2. In this group, rates of blastocysts, grade 1–3 and grade 1 embryos (as percentages of fertilized oocytes) were significantly increased in period 2 (Table 2).
Follicular characteristics, oocyte collection and embryo production were compared between OPU sessions 4 and 7 for both groups of heifers. At OPU session 7, effects were mainly observed on embryo production in the restricted group (Fig. 5b): the numbers of cleaved embryos, day 7 total embryos, blastocysts and grade 1–3 embryos significantly increased from OPU 4 to 7. The effect of dietary treatment was observed after at least 6 weeks (the time between OPU 4 and 7). When comparing OPU sessions 5 and 6 to OPU 4, no such effect was observed (data not shown).
From OPU 6 to 7 the numbers of total embryos, blastocysts and grade 1–3 embryos continued to increase in the restricted group whereas they decreased in the overfed one.
Discussion
The objective of this study was to test whether a reduction in dietary intake, leading to a lower growth rate and associated metabolic changes, could improve in vitro embryo production in superovulated overfed dairy heifers.
After an 8-week period of high dietary intake (period 1 with a LW gain of around 950 g/day), heifers either continued to receive the same high intake or were restricted (period 2). At the beginning of period 2, embryo production improved in the same way for the two groups of heifers. Nevertheless, restricted heifers (with a mean LW gain of 730 g/day) showed a higher blastocyst rate and higher proportions of grade 1–3 and grade 1 embryos during period 2 in comparison with the overfeeding period. Moreover, after 6 weeks of dietary restriction (OPU 7), numbers of day 7 total embryos, blastocysts and grade 1–3 embryos had significantly increased in that group. On the other hand, heifers kept on a high plane of nutrition throughout the experiment (with a mean LW gain of 1300 g/day in period 2) showed a higher number of follicles <8 mm 2 days before FSH treatment during the second period. However, no significant difference between periods was observed for oocyte collection and embryo production in this group. After 6 weeks of 1300 g/day LW gain (OPU 7), embryo production began to decrease. We conclude that a period of at least 6 weeks of dietary restriction could improve embryo production in overfed dairy heifers.
Growth rate, body weight and BCS
As expected with the experimental scheme, two periods could be differentiated: in period 1, all heifers showed a high growth with an average LW gain around 950 g/day. In period 2, significantly different LW gain were achieved in the two groups of heifers: 730 g/day for the restricted group versus 1300 g/day for the overfed group. Such LW gain is close to that observed in French farms (Troccon 1993, Gaboriau 2002) as well as in other studies on heifers (Bergfeld et al. 1994, Saumande et al. 1998, Carson et al. 2000, Armstrong et al. 2001, Chelikani et al. 2003). The increase from 950 g/day in period 1 to 1300 g/day in period 2 for the overfed group could be due to an improvement of nutritional efficiency during the 4 months of experiment. Indeed, even the restricted group had a LW gain which was higher than expected. As LW gain increased in the same way for the 2 years of the experiment, we believe that it was not due to a difference in hay quality (soybean meal and barley quality was standard). During period 2, BCS and LW began to differ significantly respectively after 4 weeks (OPU 6) and 6 weeks (OPU 7).
Metabolite and metabolic hormone plasma concentrations
Changes in dietary intake, LW gain and BCS were associated with changes in plasma metabolites and metabolic hormones. In period 2 compared with period 1, plasma glucose and BHB decreased in the restricted group whereas plasma insulin and IGF-I increased and NEFAs decreased in the overfed group. Those results are consistent with those from other studies in growing heifers: decreased plasma glucose, insulin and IGF-I and increased plasma NEFAs are associated with dietary restriction (Yambayamba et al. 1996, Chelikani et al. 2004); increased glucose, insulin and IGF-I and decreased NEFAs are associated with high energy intake (Burgwald-Balstad et al. 1995, Yelich et al. 1996, Lalman et al. 2000). Increased plasma BHB in the overfed group is probably due to higher feed intake and a ruminal rather than a hepatic origin of BHB (Gutierrez et al. 1997). Plasma urea increased from period 1 to period 2 in the two groups but values were in a normal range.
Reproductive hormones
Higher plasma progesterone in period 1 compared with period 2 was a consequence of the experimental scheme. OPU 1 and 2 were performed during the luteal phase, whereas after OPU 2 and until the end of the experiment, heifers were under the effect of norgestomet implants (Fig. 1), and secretion of progesterone decreased to very low levels, as already described (Kojima et al. 1992, Stegner et al. 2004). Plasma progesterone remained very low for the two groups of heifers during period 2.
Plasma estradiol measured on the day of OPU was increased in period 2 compared with period 1 (global period effect): it could be due to the global increase in the number of aspirated follicles <8 mm. Surprisingly, the increase in plasma estradiol in period 2 was significant in the restricted group, whereas there was no increase in the number of aspirated follicles with a diameter ≥ 8 mm. An increase in the diameter of follicles ≥ 8 mm, leading to more estradiol being secreted, cannot be excluded. Moreover, the rate of estradiol clearance could be related to feed intake: in the restricted group, a lower hepatic clearance could lead to a higher estradiol concentration in plasma. Jorritsma et al.(2003) found higher estradiol plasma concentrations in fasted heifers compared with control heifers.
Follicular characteristics, oocyte collection and embryo production
At the end of period 1, heifers were allocated according to LW gain and oocyte production of OPU 1-4. When comparing period 1 lsmeans for the two groups of heifers, no difference was found for numbers of follicles (2 days before superovulation treatment and at the time of OPU) and for numbers of collected oocytes, showing that experimental groups were well paired. This is important for an experiment with a small number of animals, and where variability between animals is a difficulty. Nevertheless, percentages of day 7 embryos and day 7 blastocysts were lower in the group that would later be restricted. Heifers were allocated to the overfed or restricted group according to mean LW gain and oocyte production during period 1, not according to embryo production. Individual variability between heifers for both oocyte and embryo production was high: a classification of heifers according to mean overall oocyte production would be different from a classification according to overall day 7 embryo production. It would have been very difficult to allocate heifers to the dietary treatments using three criteria at the same time. Therefore, we compared periods within group. We wanted to observe the evolution of oocyte and embryo production from period 1, the control period for each group, to period 2, according to the evolution of LW gain in each group.
A positive effect of the short-term increase of dietary intake was found on the growth of small follicles: the number of follicles <8 mm (2 days before the first FSH injection) was increased in the overfed group of heifers from period 1 (950 g/day LW gain) to period 2 (1300 g/day LW gain). When comparing follicular characteristics between OPU sessions 1 and 4 after 8 weeks of overfeeding for all heifers, a significant increase in the number of these small follicles was observed at OPU session 4. Such an effect of a short-term increase of the plane of nutrition on small follicles has already been reported in studies with heifers (Gutierrez et al. 1997, Armstrong et al. 2002). However, it was not associated with a higher number of follicles aspirated at OPU, and did not enhance the recruitment of small follicles by the superovulatory treatment, whereas this could have been expected. In heifers, a short-term increase in dietary intake could be associated with an increase in the number of small follicles (2–4 mm) before a superovulation treatment, and large follicles (> 9 mm) after the treatment (Gong et al. 2002). The increase in the number of small follicles before superovulation treatment was associated with higher plasma insulin and IGF-I.
The total number of collected oocytes and the morphological quality of the oocytes did not differ in period 2 compared with period 1: no effect of dietary intake on oocyte production could be observed. Overfeeding had no positive effect on oocyte collection and restriction did not alter it. However, the number of fertilized oocytes was higher in period 2 compared with period 1 for both groups of heifers, because of an increase in maturation rate. A global improvement in embryo production was observed for all heifers during period 2, as illustrated by numbers of cleaved and day 7 embryos. It showed that the quality of collected oocytes, defined as their ability to be fertilized and to develop during in vitro culture, was better in period 2, even if there was no difference in morphological grades. This overall effect cannot be due to the level of dietary intake. An hypothesis of an improvement in oocyte quality with the age of the heifers, from 14 to 18 months old, could be made (Marquant-Le Guienne 2003). Moreover, the improvement in embryo production from period 1 to period 2 was higher in the restricted group compared with the overfed group, with a significant increase for the number of day 7 blastocysts and for the morphological quality of day 7 embryos. This favourable effect can be attributed to dietary restriction and to the reduction in LW gain in period 2. Indeed, others studies in heifers showed an enhancement of in vitro embryo production after a reduction of dietary intake (McEvoy et al. 1997, Nolan et al. 1998b, Armstrong et al. 2001). Comparison between OPU sessions showed that, in our study, the positive effect of dietary restriction occurred after 6 weeks. A detrimental effect of high LW gain and dietary intake could have been expected on embryo production, according to results in vivo in sheep (McEvoy et al. 1993, Creed et al. 1994, McEvoy et al. 1995) and in cattle (Mantovani et al. 1993, Negrao et al. 1997, Yaakub et al. 1999), or in vitro in cattle with impaired embryo development associated with high plasma concentrations of both insulin and IGF-I (Armstrong et al. 2001). However, no such effect was observed in our study (comparisons between OPU sessions 1 and 4 for all heifers, then between periods 1 and 2/OPU sessions 4 and 7 for the overfed group). Perhaps a longer period of overfeeding should have been necessary, as embryo production began to decrease only after 6 weeks in the continuously overfed group (when heifers had reached a BCS of 3, on a scale from 0 to 5). Indeed, Adamiak et al.(2005) found a detrimental effect of high feeding level on oocytes from animals with moderately high body condition (associated with hyperinsulinaemia for a significant proportion of animals) but not from low body condition ones. In our study, insulinaemia was very significantly higher after 7 weeks in the overfed group compared with the restricted one.
The effects of manipulating dietary intake on follicular concentrations of steroids and metabolites and on the expression of ovarian growth factors need to be more widely investigated, and might be of physiological importance, as it could affect the quality of both oocyte and granulosa cells.
In farms, it is important to take into account that variations in dietary intake can have possible divergent effects on follicle growth, oocyte maturation and embryo quality. Our results suggest that a recommendation of a short-term dietary restriction (at least 6 weeks) before the beginning of an in vitro or in vivo embryo production program should be applied when heifers are overfed (high LW gain or BCS).
Growth rate, plasma concentrations of metabolites, metabolic and reproductive homones in period 1 (OPU 1–4) and period 2 (OPU 5–8), for restricted and overfed groups (lsmeans ± s.e.m.).
| Group 950 → 730 g/day (n = 8) | Group 950 → 1300 g/day (n = 8) | |||||||
|---|---|---|---|---|---|---|---|---|
| Period 1 | Period 2 | Period 1 | Period 2 | |||||
| Plasma concentration | Lsmean | s.e.m | Lsmean | s.e.m. | Lsmean | s.e.m. | Lsmean | s.e.m. |
| * a versus b, P ≤ 0.05; a versus c, P ≤ 0.01 between periods; x versus y, P ≤ 0.05 between groups. | ||||||||
| Weekly | ||||||||
| Glucose (mM) | 4.9a | 0.1 | 4.7c | 0.1 | 5.0 | 0.1 | 4.9 | 0.1 |
| Insulin (pM) | 127.1 | 12.2 | 135.3x | 12.6 | 148.3a | 12.2 | 199.6cy | 12.6 |
| IGF-I (ng/ml) | 220.5 | 22.2 | 236.7 | 22.5 | 267.0a | 22.2 | 294.8b | 22.5 |
| NEFA (mM) | 0.11 | 0.01 | 0.12 | 0.01 | 0.13a | 0.01 | 0.10b | 0.01 |
| BHB (mM) | 0.74a | 0.03 | 0.67bx | 0.03 | 0.78 | 0.03 | 0.85y | 0.03 |
| Urea (mM) | 2.6a | 0.1 | 3.1c | 0.1 | 2.7a | 0.1 | 3.1c | 0.1 |
| Two days before FSH | ||||||||
| Progesterone (ng/ml) | 4.9a | 0.7 | 0.5c | 0.7 | 5.4a | 0.7 | 0.4c | 0.7 |
| Estradiol (pg/ml) | 5.6 | 0.6 | 6.4 | 0.6 | 7.5 | 0.6 | 6.3 | 0.6 |
| At the time of OPU | ||||||||
| Progesterone (ng/ml) | 4.7a | 0.7 | 0.5c | 0.7 | 5.2a | 0.7 | 0.5c | 0.7 |
| Estradiol (pg/ml) | 8.3a | 1.3 | 12.2b | 1.3 | 7.2 | 1.3 | 9.8 | 1.3 |
Follicular characteristics, oocyte collection and in vitro embryo production in period 1 (OPU 1–4) and period 2 (OPU 5–8) for restricted and overfed groups (lsmeans ± s.e.m.). Percentages are calculated on aspirated follicles at OPU, collected oocytes and fertilized oocytes respectively for follicular characteristics at OPU, oocyte collection and embryo production data.
| Group 950 → 730 g/day (n = 8) | Group 950 → 1300 g/day (n = 8) | |||||||
|---|---|---|---|---|---|---|---|---|
| Period 1 | Period 2 | Period 1 | Period 2 | |||||
| Lsmean | s.e.m. | Lsmean | s.e.m. | Lsmean | s.e.m. | Lsmean | s.e.m. | |
| * a versus b, P ≤ 0.05, a versus c, P ≤ 0.01 between periods; x versus y, P ≤ 0.05, x versus z, P ≤ 0.01 between groups. | ||||||||
| Follicular characteristics | ||||||||
| Two days before FSH | ||||||||
| Follicles ≥ 8 mm | 2.2 | 0.2 | 1.7 | 0.2 | 2.1 | 0.2 | 1.7 | 0.2 |
| Follicles < 8 mm | 23.8 | 3.1 | 24.1 | 3.1 | 21.6a | 3.1 | 27.3b | 3.1 |
| At the time of OPU | ||||||||
| Follicles ≥ 8 mm | 6.6 | 1.3 | 6.3 | 1.3 | 5.7 | 1.3 | 6.7 | 1.3 |
| Follicles < 8 mm | 11.3 | 2.6 | 14.1 | 2.5 | 13.2 | 2.6 | 16.1 | 2.5 |
| Oocyte collection | ||||||||
| Collected oocytes | 12.2 | 2.5 | 13.3 | 2.5 | 11.7 | 2.5 | 13.8 | 2.5 |
| Grade 1–3 oocytes | 10.2 | 2.1 | 11.1 | 2.1 | 9.6 | 2.1 | 11.5 | 2.1 |
| % | 83.4 | 83.8 | 82.1 | 83.9 | ||||
| Grade 1 oocytes | 3.7 | 0.9 | 3.7 | 0.9 | 2.9 | 0.9 | 3.4 | 0.9 |
| % | 30.4 | 28.0 | 24.8 | 24.5 | ||||
| Embryo production | ||||||||
| Fertilized oocytes | 7.2a | 1.9 | 11.0b | 1.9 | 7.3a | 1.9 | 11.1b | 1.9 |
| Cleaved embryos | 4.6 | 1.5 | 7.4 | 1.5 | 4.4 | 1.5 | 7.3 | 1.5 |
| % | 63.0 | 67.2 | 60.3 | 65.2 | ||||
| Day 7 embryos | 1.4 | 0.8 | 2.9 | 0.8 | 2.2 | 0.8 | 3.8 | 0.8 |
| % | 19.3x | 26.4 | 30.0y | 33.7 | ||||
| Day 7 blastocysts | 0.9a | 0.7 | 2.7b | 0.7 | 1.9 | 0.7 | 3.1 | 0.7 |
| % | 12.2ax | 24.2c | 26.1z | 28.1 | ||||
| Day 7 grade 1–3 embryos | 0.9 | 0.7 | 2.3 | 0.7 | 1.7 | 0.7 | 3.0 | 0.7 |
| % | 12.9ax | 21.1b | 22.8y | 26.6 | ||||
| Day 7 grade 1 embryos | 0.3a | 0.4 | 1.5b | 0.4 | 0.6 | 0.4 | 1.6 | 0.4 |
| % | 3.9ax | 13.2c | 8.8y | 14.6 | ||||
Evolution of LW and BCS for restricted (□, n = 8) and overfed (▪, n = 8) groups. (a) LW from OPU sessions 1–8; (b) body condition scored every 4 weeks, at OPU sessions 1, 2, 4, 6 and 8. Lsmeans ± s.e.m.; a versus b, P ≤ 0.05; a versus c, P ≤ 0.01.
Citation: Reproduction 131, 4; 10.1530/rep.1.00689
Evolution of insulin plasma concentrations measured weekly for restricted (□, n = 8) and overfed (▪, n = 8) groups. Lsmeans ± s.e.m.; a versus b, P ≤ 0.05; a versus c, P ≤ 0.01.
Citation: Reproduction 131, 4; 10.1530/rep.1.00689
Evolution of numbers of total embryos (a), blastocysts (b) and grade 1–3 embryos (c) at day 7 of in vitro culture from OPU 1 to OPU 7 for restricted (□, n = 8) and overfed (▪, n = 8) groups (lsmeans ± s.e.m.).
Citation: Reproduction 131, 4; 10.1530/rep.1.00689
Comparison of follicular characteristics, oocyte and embryo production between OPU sessions 1 (beginning of period 1), 4 (end of period 1) and 7 (end of period 2). Evolution of numbers of follicles 2 days before superovulation treatment and aspirated on the day of OPU, collected and fertilized oocytes, cleaved embryos, total embryos, blastocysts and grade 1–3 embryos at day 7 of in vitro culture (a) from OPU session 1 to 4 (8 weeks) for all heifers (n = 16) and (b) from OPU session 4 to 7 (6 weeks) for restricted (n = 8) and overfed (n = 8) groups. Difference of lsmeans ± s.e.m.; *P ≤ 0.05; ***P ≤ 0.001.
Citation: Reproduction 131, 4; 10.1530/rep.1.00689
We are very grateful to S. Ponchon for in vitro embryo production, N. Tron and M. Durand for precious technical assistance in OPU and management of heifers, C. Ficheux and N. Jeanguyot for help with metabolic and hormonal assays respectively. This work was financially supported by the UNCEIA (Union Nationale des Coopératives d’élevage et d’Insémination Animale), the UMR 1198 INRA/ENVA Biologie du Développement et Reproduction (Institut National de la Recherche Agronomique – Ecole Nationale Vétérinaire d’Alfort) and the ANRT (Association Nationale de la Recherche Technique). The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.
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