Influence of insulin treatment and feed restriction on
follicular development in cyclic gilts
H. Quesnel
∗, A. Pasquier, N. Jan, A. Prunier
Unité Mixte de Recherches sur le Veau et le Porc, Institut National de la Recherche Agronomique, 35590 Saint-Gilles, France
Received 7 January 2000; received in revised form 5 July 2000; accepted 18 July 2000
Abstract
Crossbred gilts were used to investigate whether exogenous insulin can restore normal follicular growth in feed-restricted gilts. After an 18-day altrenogest treatment, the first day of oestrous behaviour was designed as day 0. From day 0 to 13, all gilts received the same amount of feed, calculated to meet 200% of the energy requirements for maintenance. On day 14, luteolysis was induced by injection of an analogue of prostaglandin F2a. All gilts were slaughtered on day 19 and their ovaries removed. In Experiment 1, gilts received a high (240% of maintenance) or low (80%) level of feeding (n=10/group) from day 14 to 18. The number of large follicles (≥5 mm) on day 19 was reduced in feed-restricted gilts (16.9 versus 20.6,P <0.05). The same protocol of feed restriction was used in Experiment 2 (240% versus 80% of maintenance from day 14 to 18), and some gilts received daily injections of insulin (0.6 IU live weight kg−1). The three experimental
groups were H: 240% and no insulin (n=8); H-I: 240%+insulin (n=8) and L-I: 80%+insulin (n=7). On day 18, 4 h after insulin injection, plasma insulin was higher in insulin-treated than in untreated gilts and glucose concentrations were reduced more dramatically in L-I than in H-I gilts (P <0.05). Concentrations of IGF-I were lower in L-I than in other gilts (P <0.05) and plasma IGFBPs were not significantly affected by treatments. On day 19, the number of large follicles (≥5 mm) was not significantly influenced by treatments (19.4, 17.6 and 15.3 for H, H-I and L-I gilts, respectively). Insulin, IGF-I and IGFBP-2 levels in follicular fluids from large follicles did not differ between females whereas IGFBP-3 levels were lower in L-I than in H gilts (P <0.05) and intermediate in H-I gilts. Intrafollicular levels of glucose were higher in feed-restricted than in well-fed gilts (P <0.05). These results suggest that exogenous insulin does not restore final follicular growth impaired by acute undernutrition. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Pig-feeding and nutrition; Food restriction; Follicular growth; Insulin; IGF-I; Steroids
∗Corresponding author. Tel.:+33-223-48-56-49; fax:+33-223-48-50-80.
E-mail address: [email protected] (H. Quesnel).
1. Introduction
In cyclic gilts which are chronically underfed, a transient increase in feed intake (flush-ing) increases ovulation rate (Cox et al., 1987; Flowers et al., 1989; Beltranena et al., 1991). These effects are likely to be mediated by alterations in gonadotrophin secretions (Cox et al., 1987; Flowers et al., 1989) and/or alterations in ovarian responsiveness to gonadotrophin action, both related to nutritionally-induced changes in circulating metabolic hormones (Cox et al., 1987; Beltranena et al., 1991). Indeed, exogenous insulin increases ovulation rate (Cox et al., 1987) and decreases follicular atresia (Matamoros et al., 1991), when ad-ministered during preovulatory follicular maturation, and stimulates follicular growth in nutritionally-restricted gilts (Britt et al., 1988). Insulin also increases intrafollicular con-centrations of insulin-like growth factor I (IGF-I) in diabetic gilts (Edwards et al., 1996) and in prepuberal gilts (Matamoros et al., 1991). Therefore, the stimulatory action of insulin on final follicular maturation in gilts submitted to a nutritional flushing may be mediated by IGF-I stimulatory effects on folliculogenesis. In adult sows which are in energy deficit dur-ing lactation, supplementation with insulin either before or after weandur-ing has no clear effect on ovulation rate, steroidogenesis or follicular IGF-I system (review: Prunier and Quesnel, 2000). Mechanisms underlying the effects of nutrition on the ovaries may thus differ in young and adult sows or may depend on the pattern of feeding applied, feed restriction after high feed intake or flushing after feed restriction. The aim of the main experiment described here was to investigate the effect of insulin supplementation on final follicular development in gilts submitted to feed restriction. Because we previously observed that the luteal phase was not a critical period with respect to the influence of feed restriction on ovulation rate (Quesnel et al., 1999), a preliminary experiment was carried out to determine whether a feed restriction starting at luteolysis is able to alter follicular growth.
2. Materials and methods
2.1. Experiment 1
After their second or third oestrus, 20 crossbred gilts (1/2 Piétrain, 1/4 Landrace, 1/4 Large White) were given an altrenogest treatment (Regumate®, Roussel-Uclaf, Romainville, France; 20 mg/day) for 18 days. They were checked for oestrus twice daily in the pres-ence of a mature boar. The first day of oestrous behaviour was designed as day 0.
refusals were not observed in the group L and seldom occurred in the group H, but were not measured. All gilts were slaughtered between 08.30 and 10.00 h on day 19.
Blood samples were collected at 08.45 h on days 8 and 15 using heparinised vacuutainers. They were immediately placed on ice and centrifuged for removal of plasma (for 10 min at 2000 g). Plasma samples were stored at−20◦C until assayed. Progesterone concentrations were determined by a validated RIA, after extraction in solvent (Saumande et al., 1985). Assay sensitivity was 0.5 ng ml−1and intraassay CV was 14.9% at 44.5 ng ml−1.
Ovaries were collected at slaughter. They were weighed and surface diameter of the largest follicles was determined using a calliper rule. Large follicles (≥5 mm) were counted in both ovaries.
2.2. Experiment 2
Twenty-three crossbred gilts (1/2 Piétrain, 1/4 Landrace, 1/4 Large White) received an 18-day Regumate® treatment after their second or third oestrus. The first day of oestrous behaviour, observed in the presence of a mature boar was designed as day 0 and considered as the beginning of the experiment.
During the whole experiment, gilts received twice daily, at 09.00 and 13.30 h, the same diet as in Experiment 1. From day 0 to 13, feed allowance for all gilts represented 200% of the energy requirements for maintenance. On day 14, luteolysis was induced in all gilts as in Experiment 1. Gilts averaged 258±14 days and 160±9 kg body weight (mean±S.D.). From day 14 to 18, feed allowance represented 240% of energy requirements for 16 well-fed gilts (H) and 80% for 7 restricted gilts (L). From day 14 to 18, half the well-fed gilts and all the restricted gilts received subcutaneous injections of an intermediate-acting human insulin (Insulatard®, Novo Nordisk, Boulogne Billancourt, France), once daily, at a dose of 0.6 IU live weight/kg (groups H-I and L-I). We previously described that a single injection of this formulation of insulin (0.5 IU kg−1) maintained for 10 h high levels of plasma insulin compared with control sows (Quesnel and Prunier, 1998). To attenuate insulin-induced hypoglycaemia, especially in feed-restricted gilts, injections were performed during the morning meal, and the afternoon meal was distributed 4.30 h after the injection when in-sulin levels were supposed to peak in inin-sulin-treated gilts (Quesnel and Prunier, 1998). No hypoglycaemic shock was observed. Gilts of the control group (H) were not injected.
Blood samples were collected using heparinised vacuutainers on day 18 at 13.15 h, before the afternoon meal. Plasma was harvested after centrifugation and stored at−20◦C until assayed. Ovaries were collected on day 19 at slaughter for control gilts and by laparotomy for insulin-treated gilts. Ovaries were immediately placed in sterile ice-cold 0.9% saline. Large follicles (with diameter≥5 mm as estimated with callipers) were counted. Fluid from the five largest follicles and from two to five medium-sized (3.0–4.9 mm) follicles of right ovaries was aspirated and stored at−20◦C, individually (large follicles) or pooled (medium follicles, two to three foll/pool).
contribute to the amounts of steroids measured in medium (Driancourt and Terqui, 1996). After incubation, three or four follicles of each gilt were coated with Tissue-Tek embed-ding medium, frozen in liquid nitrogen and stored at−70◦C, before assessing their quality (healthy or atretic) by histological procedures previously described (Quesnel et al., 1998).
2.3. Glucose and hormone assays
Follicular fluids from large follicles of each gilt were analysed individually (glucose, IGF-I,n =5/gilt) and as pool from two follicles (insulin,n=2/gilt). Pools of follicular fluids from medium-sized follicles were analysed for glucose only. Plasma was analysed individually for glucose, IGF-I and insulin. Determinations were performed in duplicate.
Insulin and IGF-I concentrations were measured by double antibody RIAs (Prunier et al., 1993; Louveau and Bonneau, 1996), after an acid-ethanol extraction for IGF-I as-say. The intraassay CV was 6.3% at 59mIU ml−1for insulin and 6.3% at 433 ng ml−1for IGF-I. Average sensitivity, estimated as 90% of total binding was 3mIU ml−1for insulin and 0.08 ng ml−1 for IGF-I. Concentrations of glucose were measured by automatic en-zymatic methods with a Cobas Mira (Hoffman Laroche, Basel, Switzerland) apparatus. Oestradiol-17band testosterone concentrations in incubation medium were determined by validated RIAs without extraction (Thibier and Saumande, 1975; Bonneau et al., 1987). Intraassay CVs were 8.3% at 2.2 ng ml−1and 4.9% at 18.1 ng ml−1, and assay sensitivity was 25 pg ml−1and 0.2 ng ml−1, for oestradiol and testosterone, respectively.
2.4. Analysis of IGFBPs
Western ligand blotting was performed according to the method of Hossenlopp et al. (1986) modified as follows. Samples (individual plasma and pools of the five largest folli-cles from each gilt; 2ml) were subjected to SDS/polyacrylamide gel (12.5%) electrophoresis under non-reducing conditions. The proteins were then transferred to a nitrocellulose mem-brane (BA85, 0.45mm; Schleicher and Schuell, Dassel, Germany). The membrane sheets were treated and incubated with 90,000 cpm ml−1of mixture of125I-IGF-I and125I-IGF-II (50/50) for 2 h at 21◦C. The membranes were exposed to a Kodak X-Omat AR film with two intensifying screens for 3–6 days at−70◦C. The relative level of IGFBPs was analysed by densitometric scanning using phosphorImager (STORM, Molecular Dynamics) and IM-AGEQUANT software. To prevent gel to gel variation in IGFBP evaluation, the different treatments were represented on each gel.
2.5. Statistical analysis
Table 1
Influence of feeding level on ovarian characteristics on day 19 of the oestrous cycle in gilts (mean±SEM, Experiment 1)
Variable Feeding group Significance
H L
Number of gilts 10 10
Ovarian weight (g) 17.3±0.7 13.5±0.7 P=0.001
Number of follicles (≥5 mm) 20.6±1.1 16.9±0.8 P=0.016 Maximum follicular diameter (mm) 8.2±0.4 7.3±0.3 P=0.100
3. Results
3.1. Experiment 1
The weight of the ovaries and the number of follicles≥5 mm on day 19 of the cycle were decreased by feed restriction (P <0.001 andP <0.05, respectively, Table 1) whereas the maximum diameter of follicles was not significantly affected (P =0.1, Table 1).
Concentrations of progesterone measured on days 8 and 15 (i.e. in the mid-luteal phase and 24 h after luteolysis induction) were not significantly different between treatment groups (56.5±3.6 and 36.4±4.4 ng ml−1on days 8 and 15, respectively).
3.2. Experiment 2
3.2.1. Plasma insulin, glucose and IGF-I system in response to treatments
As expected, plasma insulin levels measured 4 h after insulin administration on day 18 were significantly higher in insulin-treated than in not-treated gilts (Table 2). This was
Table 2
Influence of exogenous insulin (0.5 IU live weight/kg, once daily) and feeding level on concentrations of insulin, glucose, IGF-I and IGFBPs in plasma 4 h after the injection on day 18 of the oestrous cycle in gilts (mean±SEM, Experiment 2)a
Variable Group
H H-I L-I
Number of gilts 8 8 7
Plasma insulin (mIU ml−1) 37±8 a 77±6 b 73±8 b
Plasma glucose (mg l−1) 933±45 a 697±70 b 266±38 c
Plasma IGF-I (ng ml−1) 316±23 a 290±47 a 136±17 b
Plasma IGFBPs (arbitrary units)
43–39 kDa 214±32 207±20 169±29
34 kDa 15±3 14±2 21±4
29 kDa 8±2 7±1 9±2
24 kDa 2.4±0.6 1.7±0.4 1.4±0.3
Table 3
Influence of exogenous insulin (0.5 IU live weight/kg, once daily) and feeding level on the number and diameter of follicles in gilt ovaries removed on day 19 of the oestrous cycle (mean±SEM, Experiment 2)a
Variable Group
H H-I L-I
Number of follicles (≥5 mm) 19.4±2.0 17.6±0.7 15.3±1.4 Maximum follicular diameter (mm) 8.7±0.3 x 8.4±0.2 x 7.6±0.4 y Mean diameter of the largest 10 follicles (mm) 7.7±0.3 x 7.5±0.2 x 6.7±0.3 y
aMeans within row with different letters (x, y) tend to differ (P <0.1).
accompanied by hypoglycaemia which was more pronounced in L-I than in H-I gilts (Ta-ble 2). Concentrations of IGF-I were significantly lower in L-I than in H-I and H gilts. Autoradiography of IGFBPs in plasma revealed five bands (data not shown), likely cor-responding to IGFBP-3 (43–39 kDa) and BP-2 (34 kDa), and to unidentified IGFBPs (29 and 24 kDa). Their levels did not significantly differ between groups (Table 2). The ratio of IGF-I:43–39 kDa IGFBP (1.8; 1.4 and 0.9 in H, H-I and L-I gilts, respectively) tended to be lower in L-I than in H females (P <0.07) and was intermediate in H-I gilts.
3.2.2. Number and diameter of follicles
On day 19, the number of follicles≥5 mm in diameter did not significantly differ between groups of gilts (Table 3). The maximal diameter and the mean diameter of the 10 largest follicles tended to be lower in L-I than in H-I and H females (Table 3).
3.2.3. Steroid hormones
Although the majority of follicles had an oestradiol concentration in incubation medium in excess of 1.5 ng ml−12 h−1, some follicles produced low level of oestradiol, suggesting that they were atretic. Health assessment, by histology of follicles chosen at random confirmed that atretic follicles had an oestradiol concentration in the incubation medium<1 ng ml−1 2 h−1whereas healthy follicles produced >1.5 ng ml−12 h−1. Using this oestradiol threshold of 1 ng ml−12 h−1to discriminate healthy and atretic follicles, 6.5% of large follicles were identified as atretic. This percentage was not influenced by treatments (P >0.1).
Oestradiol and testosterone concentrations released in the incubation medium by healthy follicles did not significantly differ between groups of females (6.8 ±0.6 and 12.8± 1.1 ng ml−12 h−1for oestradiol and testosterone, respectively). They were highly correlated with follicular diameter (P <0.01) and correlated with each other (P <0.001, Fig. 1). The ratio oestradiol:testosterone was not significantly influenced by treatments (0.64±0.05).
3.2.4. Follicular insulin, glucose and IGF-I system on day 19
Fig. 1. Relationship between oestradiol and testosterone concentrations in culture medium (ng ml−12 h−1) for large follicles (≥5 mm) from H, H-I and L-I gilts; H: high level of feeding; H-I: high level of feeding+insulin; L-I: low level of feeding+insulin.
Fig. 2. Glucose, IGF-I and IGFBPs (43–39 and 34 kDa) in the follicular fluid of large follicles (≥5 mm) removed on day 19 of the oestrous cycle of H, H-I and L-I gilts; H: high level of feeding; H-I: high level of feeding+insulin; L-I: low level of feeding+insulin.abMeans with different superscripts differ (P <0.05).
influenced by treatments (Fig. 2). Glucose concentrations in large follicles differed between groups of females (P <0.05) and are significantly higher in L-I than in H females (Fig. 2). For most gilts, intrafollicular glucose levels varied little between the five follicular fluids collected. Levels of glucose in medium-sized follicles were higher in L-I than in H and H-I females (P <0.05; 843±9; 819±74 and 1074±80 in H, H-I and L-I, respectively). They were highly correlated to glucose levels in large follicles (r=0.68, P <0.004).
4. Discussion
on day 19 of the oestrous cycle. As nearly 94% of these follicles were healthy (data from Experiment 2), this number of large follicles is likely to reflect the subsequent ovulation rate. Conversely, nutritional flushing of limited-fed gilts during the follicular phase increases the ovulation rate (Dailey et al., 1972). In contrast, feed restriction during the late luteal phase has no effect on the ovulation rate (Quesnel et al., 1999). This suggests that the most sensitive period to the level of feeding with respect of ovulation rate is the last 4–6 days of the cycle in agreement with previous findings (Clark et al., 1972; Dailey et al., 1972).
In prepuberal ovariectomised gilts, the level of feeding was shown to alter metabolic clearance of steroids, through changes in hepatic portal blood flow (Prime and Symonds, 1993). We previously reported that the decline in progesterone at prostaglandin-induced luteolysis was lengthened in feed-restricted gilts compared with well-fed gilts (80% ver-sus 300%, Prunier et al., 1999), which may impair folliculogenesis through endocrine or paracrine action. However, results from Experiment 1 do not show a significant influence of feed restriction on progesterone levels measured 24 h after luteolysis induction. Such a difference between studies may be related to the age or the body weight of the females. We can also suggest that, because of the great variability in progesterone levels 24 h after luteolysis induction, a single measurement is not sufficient to observe nutritional effects. In the present experiment, a strong negative correlation was found between plasma pro-gesterone level at day 15 and the diameter of the large follicles (r = −0.59, P =0.007), supporting the hypothesis of a detrimental effect of high levels of progesterone at luteolysis on preovulatory growth.
Therefore, a severe level of feed restriction starting at luteolysis is sufficient to impair the terminal follicular growth in gilts. This protocol of feed restriction was then used in Exper-iment 2 to determine whether insulin supplementation is able to restore normal follicular growth. Insulin treatment doubled plasma insulin concentrations 4 h after the injection in re-stricted and in well-fed gilts. Simultaneously, glucose concentrations were decreased, more severely in feed-restricted than in well-fed gilts. However, insulin treatment did not increase the number of large follicles in gilts fed the high dietary level (2.4 times maintenance). This contrasts with data from Cox et al. (1987), showing that insulin increases ovulation rate of cyclic gilts during their first pubertal oestrous cycle and that the amplitude of increase is greater for gilts fed a high dietary energy (2.3 times maintenance). However, the pattern of feeding of the animals differs between these experiments. Insulin treatment was associated to acute feed restriction in our experiment whereas it was associated to chronic limited feeding or to nutritional flushing in previous experiments (Cox et al., 1987).
the ratio of IGF-I:IGFBP-3 was not significantly altered (data not shown). Follicles may have produced locally IGF-I to protect themselves against plasma IGF-I reduction and risk of atresia. The decrease in IGFBP-3 levels in large follicles but not in plasma is somewhat surprising, as intrafollicular IGFBP-3 is likely to reflect plasma levels (Samaras et al., 1993; Besnard et al., 1997). However, IGFBP-3 level was measured in plasma 4 h after the morn-ing meal whereas it was measured in follicular fluids 19–21 h after the last meal. Therefore, variations in plasma IGFBP-3 at slaughter as well as changes in vascular permeability may explain the difference in follicular IGFBP-3 between groups.
In the present study, the ability of large follicles to produce steroids on day 19 of the oestrous cycle, was mainly related to the follicular size, but was not significantly altered by insulin treatment and/or feeding level. In contrast, both stimulatory and inhibitory influ-ences of exogenous insulin on steroidogenesis in large follicles were reported by Whitley et al. (1998a, b). The lack of alteration in steroid production and in IGFBP-2, the IGFBP as-sociated with atresia suggests that at day 19, preovulatory follicles presented similar degree of maturity and health in the three groups of gilts.
As mentioned above, most large follicles (≥5 mm) were healthy, whereas medium-sized follicles collected at day 19 of the oestrous cycle were likely to be all atretic (Guthrie et al., 1995; Driancourt and Terqui, 1996). Glucose concentrations in large healthy follicles are similar to those previously described (Cabrera et al., 1985). They are similar to and strongly correlated with levels in atretic follicles, suggesting that glucose reaches follicular fluid from the circulation by passive diffusion through the follicular wall. Therefore, the higher levels of glucose in large and medium-sized follicles observed at slaughter in L-I gilts compared with well-fed gilts may reflect higher plasma levels at slaughter (after a 19 h fasting), due to an enhanced neoglucogenesis during the night in feed-restricted gilts.
Insulin concentrations in large follicles (∼13mIU ml−1, i.e.∼0.5 ng ml−1) were similar to concentrations measured in pooled porcine follicular fluid (Hammond et al., 1983), but less variable than follicular fluid levels reported in women (from <2–65mIU ml−1, Diamond et al., 1985). They are similar to porcine preprandial plasma concentrations. These concentrations are 500–1000 times lower than those of IGF-I in plasma as in follicles, suggesting that in vivo insulin action on folliculogenesis is more likely mediated through insulin receptor than through type I (IGF-I) receptor in swine as in human (Poretsky et al., 1999).
The main conclusions of these two experiments are that (1) feed restriction starting at luteolysis, hence occurring during recruitment and selection of the preovulatory follicles may impair final follicular growth; (2) exogenous insulin does not clearly alleviate the negative effects of acute undernutrition; (3) feed restriction and insulin administration may alter the levels of IGF-I and IGFBP-3 in plasma and follicular fluids. These findings do not provide clear evidence that insulin or IGF-I (or both) deficiency is involved in impaired folliculogenesis occurring in feed restricted gilts.
Acknowledgements
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