78 H. Quesnel et al. Animal Reproduction Science 64 2000 77–87
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 andor 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 weaning 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 12 Piétrain, 14 Landrace, 14 Large
White were given an altrenogest treatment Regumate®, Roussel-Uclaf, Romainville, France; 20 mgday 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. Throughout the experiment, gilts received twice daily, at 09.00 and 13.30 h, a diet con-
taining 3200 kcal of digestible energykg, 17.1 crude protein and 0.95 lysine. From day 0 to 13, feed allowance for all gilts was calculated to meet 200 of the energy requirements
for maintenance. Maintenance requirements were calculated for individual gilts on the basis of their metabolic body weight 240 kcal ME
∗
body weight kg
0.60
Noblet et al., 1999. On day 14, all gilts received an injection of an analogue of prostaglandin F2a Alfabédyl®,
Distrivet, Paris, France; 2 mggilt i.m. to induce luteolysis. Gilts averaged 266 ± 6 days and 188 ± 13 kg body weight mean ± S.D.. From day 14 to 18, feed allowance represented
either 240 H, n = 10 or 80 L, n = 10 of energy requirements for maintenance. Feed
H. Quesnel et al. Animal Reproduction Science 64 2000 77–87 79
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
− 1
and 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 12 Piétrain, 14 Landrace, 14 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 weightkg 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 insulin-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 follpool.
Left ovaries were transferred into minimum essential medium MEM, Sigma, La Ver- pillière, France and all follicles ≥5 mm were carefully dissected and measured. These
follicles were incubated individually in 24-well tissue culture plates in 1 ml of MEM for 2 h, at 37
◦
C in 95 air and 5 CO
2
. Medium was collected and stored at −20
◦
C until as- sayed for oestradiol-17b and testosterone. Both production and release from follicular fluid
80 H. Quesnel et al. Animal Reproduction Science 64 2000 77–87
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 = 5gilt and as pool from two follicles insulin, n = 2gilt. 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 59 mIU ml
− 1
for insulin and 6.3 at 433 ng ml
− 1
for IGF-I. Average sensitivity, estimated as 90 of total binding was 3 mIU ml
− 1
for 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-17b and 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
− 1
and 4.9 at 18.1 ng ml
− 1
, and assay sensitivity was 25 pg ml
− 1
and 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; 2 ml were subjected to SDSpolyacrylamide gel 12.5 electrophoresis under non-reducing conditions. The proteins were then transferred to a nitrocellulose mem-
brane BA85, 0.45 mm; Schleicher and Schuell, Dassel, Germany. The membrane sheets were treated and incubated with 90,000 cpm ml
− 1
of mixture of
125
I-IGF-I and
125
I-IGF-II 5050 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 All data number and diameter of follicles and hormonal levels were analysed by analysis
of variance using the GLM procedure of SAS SAS, 1996. All models included the effect of treatment. For intra-follicular concentrations, a split-plot design was used with sow within
treatment as the error term for the treatment effect. An additional analysis was performed with diameter of follicles introduced as a covariate. In Experiment 2, when the effect of
group was significant P 0.05, differences between group means were calculated using the LSMEANS procedure of SAS with the PDIFF option.
H. Quesnel et al. Animal Reproduction Science 64 2000 77–87 81
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
