S. Landau et al. Animal Reproduction Science 64 2000 181–197 183
oestrus post-partum Moallem et al., 1997. Increased supply of dietary protein is associated with increased circulating insulin levels in ruminants in many studies Bassett et al., 1971.
Infusions of branched chain amino-acids such as leucine Kuhara et al., 1991; Downing et al., 1995 or the inclusion of leucine-rich components in the diet Landau et al., 1996;
Blache et al., 1996 increase plasma insulin. Protein in excess affects folliculogenesis in sheep in as few as 5 days Landau et al., 1996.
Schrick et al. 1992 have shown in beef cows that insulin concentration follow an oestrus-like rhythm, with a peak on the day of oestrus but no information relating insulin
to the oestrus cycle in dairy cows has been published to date. The objectives of the present study in high-yielding dairy cows were to assess: 1 whether
peripheral insulin concentration is affected by phase of the oestrus cycle; 2 whether insulin and glucose concentration in peripheral blood and in FF are affected by nutrition and; 3
possible relationships between insulin and preovulatory follicular status.
2. Material and methods
2.1. Experiment 1: circulating insulin during the luteal and the follicular phases 2.1.1. Animals and diets
Twelve Israeli Holstein multiparous dairy cows 60–80 days post-partum were fed a total mixed ration formulated according to NRC 1989. Ingredients were of dry matter
DM: corn silage 19.8, wheat silage 7.2, vetch hay 5.7, whole cotton seeds 13.9, ground corn grain 11.1, ground barley grain 9.1, ground sorghum grain
2.3, citrus pulp 6.2, sugarbeet molasses 6.7, corn gluten feed 5.9, soyabean meal 3.6, rapeseed meal 3.2, sunflower meal 2.3, fish meal 0.7, and a mineral
and vitamins premix. The feed was distributed at 11.00 h. Average body condition score BCS, in a 0–5 scale from thin to obese was 2.22. All BCS values were in the range
1.75–2.50. Milk yield averaged 37.2 kg. Animals were cared for according to the Israel Council on Animal Care Guidelines 1994.
2.1.2. Oestrus synchronisation The oestrus synchronisation procedure was as follows: an intravaginal progesterone re-
leasing device CIDR-B; Eazi-breed, AHI Plastic Co., New Zealand containing 1.9 g of progesterone was vaginally inserted for 9 days. An intra-muscular injection of PGF
2
a
Lu- talyse, Upjohn Co., Kalamazoo, MI; 2.5 ml per cow was administered 48 h before CIDR
removal. Oestrus behaviour was observed three times daily from day of CIDR removal. A second PGF
2
a
injection was administered at 06.00 h on day 16 of the first synchronised oestrus cycle. Cows were inseminated at the second oestrus and subsequent pregnancy was
assessed 50 days after insemination by trans-rectal palpation. 2.1.3. Blood sampling
Blood was sampled daily at 08.00 h from the caudal vein for four consecutive days of the luteal phase before the second injection of PGF
2
a
and every 8 h, commencing 24 h after the second PGF
2
a
injection, and finishing 24 h after standing oestrus behaviour was
184 S. Landau et al. Animal Reproduction Science 64 2000 181–197
observed. Centrifugation was carried out immediately and plasma samples n = 132 for 12 cows were stored at −20
◦
C until thawed for analyses. 2.2. Experiment 2: short-term dietary effect on plasma insulin during the luteal phase
2.2.1. Animals and diets The experiment was carried out with six cows, using a Latin-Square design with two
replicates and three treatments. Cows were kept as a group in a roofed building on a dirt floor, and were equipped with a passive chip identifying system, which allowed each cow
access to one feed trough, and provided individual feed intake data. Oestrus synchronisation procedures and oestrus detection scheme were as in experiment 1 and only cyclic animals
with a functional CL were selected.
Cows were, on average, at 76.7 range 67–85 days post-partum when feeding of the experimental diets was initiated. The control diet consisted of a total mixed ration formulated
as in experiment 1, distributed at 11.00 h and fed ad libitum. The two other diets were isoenergetic and isonitrogenous, but differed in the degradability of protein sources Landau
et al., 1996. Diets consisted of mixtures of the total mixed ration with soyabean meal SBM, or with corn gluten meal and ground corn grain CGM, respectively, as described
in Table 1. Supplements were selected to be of medium ruminal degradability and leucine content SBM or of low ruminal degradability and high leucine content CGM according
to Tagari et al. 1994. Each cycle of the Latin-Square lasted 7 days, including 6 days of adaptation to diets and 1 day for blood sampling. As blood sampling was carried out during
the luteal phase days 9 and 16 of the cycle, only two dietary treatments could be given in each synchronised oestrus cycle. Therefore, the oestrus synchronisation procedure had
Table 1 Diet composition of cows in experiments 2 and 3: cows were offered a basic total mixed ration TMR, with or
without soyabean meal SBM, corn gluten meal CGM or corn grain CG Ingredient
Amount kg
Crude protein Net energy for lactation
Neutral detergent fibbre kg
of DM Mcal Mcalkg DM kg
of DM Experiment 2
Control Basic TMR 22.0
3.76 17.1
37.8 1.72
7.7 34.9
SBM Basic TMR
20.0 4.28
19.7 37.9
1.74 7.3
33.3 SBM
1.78 CGM
Basic TMR 20.0
4.28 19.7
37.9 1.74
7.3 33.6
CGM 1.20
CG 0.22
Experiment 3 CG
Basic TMR 22.5
3.92 16.7
40.9 1.74
n.d. n.d.
CG 1.00
SBM Basic TMR
22.0 4.56
19.5 41.3
1.75 n.d.
n.d. SBM
1.65 CGM
Basic TMR 22.0
4.57 19.3
41.1 1.74
n.d. n.d.
CGM 1.20
CG 0.30
S. Landau et al. Animal Reproduction Science 64 2000 181–197 185
to be repeated. Two injections of PGF
2
a
, given in the evening of day 16 18.00 h and the morning of day 17 08.00 h, were used to synchronise the second oestrus. As a result,
over the experiment, each cow received all three diets and was fed on one of the diets for two periods. This allowed evaluation of the repeatability of insulin and glucose during two
consecutive periods on each cow when given the same diet.
2.2.2. Blood sampling On days 9 and 16 of the oestrus cycle for two consecutive synchronised cycles, blood
was sampled from the caudal vein seven times at 2 h intervals between 06.00 and 18.00 h. 2.2.3. Ultrasound procedures
An ultrasound scanner Pie Medical 150 Veterinary Ultrasound, Maastricht, The Nether- lands equipped with a 5.0 MHz annular array probe was used. In experiments 2 and 3, the
presence of a preovulatory follicle, ovulation, and the presence of a corpus luteum were assessed 1, 4, and 8 days following CIDR removal, respectively.
2.3. Experiment 3: short-term dietary effects during the luteal phase on insulin and glucose in the follicular fluid
2.3.1. Animals and diets The experiment was carried out using 16 Israeli Holstein multiparous dairy cows eight
cows in January–February and eight cows in May, commencing 60–80 days post-partum. During days 0–9 of the oestrus cycle, the cows were fed the basic total mixed ration of
experiments 1 and 2. Cows were then stratified to three experimental groups according to dry matter intake and milk yield. Groups differed in the ration distributed during days
10–17 of the oestrus cycle. All diets were isoenergetic. Cows in group CG n = 5 had access to a total mixed ration supplemented with corn grain, while the other groups received
isonitrogenous diets, e.g. SBM n = 5 or CGM n = 6, similar to those in experiment 2 Table 1. The oestrus synchronisation procedures and scheme were as in experiment 2, but
oestrus detection was also confirmed by using computerised pedometric activity Afikim, Israel. Two injections of PGF
2
a
given the evening of day 16 18.00 h and the morning of day 17 08.00 h were used to synchronise the second oestrus.
2.3.2. Blood sampling The effect of diets on insulin was assessed during the luteal phase, e.g. on days 9 and 16
of the oestrus cycle for two consecutive cycles. On these days, blood was sampled from the caudal vein seven times at 2 h intervals from 06.00 to 18.00 h.
2.3.3. Ultrasound procedure On day 18 of the synchronised cycle, ovaries were viewed by ultrasonography, 40 h
after of the first PGF
2
a
administration. Follicular fluid was aspirated from the largest and second largest visible follicles, using the ovum pick-up procedure Kruip et al., 1994;
Bols et al., 1995 and an ultrasound scanner Scanner 200; Pie Medical, Maastricht, The Netherlands. Cows were given local anaesthesia with 5 ml of 2 lidocaine HCl injected
186 S. Landau et al. Animal Reproduction Science 64 2000 181–197
epidurally between the last sacral and first caudal vertebrae. Needles were changed between cows. All follicles were aspirated until empty, as assessed on the scanner LCD screen, into
a single calibrated tube where the volume of follicle fluid was determined. Aspirated FF was yellowish in colour and was not contaminated with blood. The fluid was centrifuged
and stored at −18
◦
C until assayed. Twenty six follicles from 16 cows were successfully aspirated in the experiment.
2.3.4. Classification of follicles Two populations were identified, using oestradiol and the ratio of oestradiol to proges-
terone Badinga et al., 1992 in FF as the discriminating factor. Oestradiol concentration ranged between 2.8 and 8.9 in follicles deemed subordinate, and 691.2 and 1023.7 ngml
in follicles deemed preovulatory P 0.0001. The progesterone:oestradiol ratio ranged between 0.001 and 1.17 in follicles deemed subordinate, and 11.7–30.7 in follicles deemed
preovulatory P 0.0001.
The statistical analyses included data of 14 preovulatory and 12 subordinate follicles from five CG-fed cows three preovulatory, five subordinate follicles, six CGM-fed cows
four preovulatory, three subordinate follicles and four SBM-fed cows seven preovulatory and four subordinate follicles.
2.3.5. Assays Glucose was assessed in 20 ml samples of plasma by the Trinder method, using a colori-
metric glucose oxidase reaction Sigma, Rehovot, Israel read at 505 nm using an Elx808 microplate reader Bio-Tek Instruments, Winooski, VT, USA.
Insulin was assayed in duplicates of 200 ml plasma by radio-immuno assay, using the Coat-A-Count kit DPC, Los Angeles, CA, USA, designed for human insulin analyses and
validated for cattle by Reimers et al. 1982. The sensitivity of assay was 0.09 mgml. Intra- and inter-assay CV for 3 plasma pools across 2 assays were 5.7 and 8, respectively. The
cross reaction with IGF-I was also checked, by adding 30 ml of IGF-I supplied by Diagnostic System Laboratories Webster, TX, USA at concentrations of 0, 60 and 600 ngml to 5
insulin standards of 200 ml 0.27, 0.79, 2.4, 4.4 and 8.3 ngml. When graphs of Logit bound were plotted against insulin concentrations, all slopes and intersects of the
standard curves were strictly identical, showing no cross reactivity of IGF-I on the assay of insulin.
Concentration of 17b-estradiol in the follicle fluid was determined by previously de- scribed and validated RIA Wolfenson et al., 1995. Assay sensitivity was 3.9 pg per tube.
Intra- and inter-assay coefficients of variation were 13.3 and 13.9, respectively. Proges- terone was assayed, using the Coat-A-Count kit DPC, Los Angeles, CA, USA. Assay
sensitivity was 0.02 ngml. Intra- and inter-assay coefficients of variation were 7.3 and 16.6, respectively. The total insulin or glucose content was calculated for each follicle as
the product of volume as aspirated and insulin or glucose concentration in FF.
2.4. Statistical analyses The effects of oestrus phase and follicular wave experiment 1 and the effects of dietary
treatments, sampling time, and their interactions on the plasma concentrations of insulin
S. Landau et al. Animal Reproduction Science 64 2000 181–197 187
experiments 1, 2, 3 and glucose experiments 2, 3 were tested by analysis of variances using a General Linear Model GLM of the Statistical Analysis Systems Institute 1988.
Analysis for experiment 2 was according to a Latin-Square design including dietary treat- ment, cow and feeding cycle as main effects. As plasma glucose and insulin may be
affected by DMI Richards et al., 1989, which is itself related with days post-partum and milk production Hart et al., 1978, variancecovariance analyses were carried out on mean
plasma insulin and glucose values, using DMI, milk production and days post-partum as covariates.
A repeated measure procedure with cow treatment as the term of error in the GLM was used in experiment 3 to analyse dietary effects on circulating insulin and glucose. The
status preovulatory versus subordinate of individual follicles was established, relying on estradiol concentration in FF, the status being a main effect in the GLM. In order to assess
the effects of diet, preovulatory follicular status and their interaction on insulin and glucose, the statistical significance of differences between group means was evaluated using Tukey’s
studentised range tests.
The effects of diet and preovulatory follicular status on insulin and glucose were re- evaluated in cows from which FF had been aspirated from pairs of one preovulatory and
one subordinate follicle by using paired T-test.
3. Results
