M. Kaim

Preovulatory follicular status and diet affect the insulin and glucose content of follicles in high-yielding dairy cows.

Insulin and glucose may be limiting factors for ovarian function in dairy cows genetically selected for high milk yield. The effects of nutrition on the intrafollicular content of insulin and glucose were investigated in Israeli Holstein dairy cattle fed a basic total mixed ration and producing 34-39kg of milk daily. In experiment 1, carried out in 11 oestrus-synchronised cows, little variation in insulin concentration was found in plasma sampled during the luteal phase, but high variation was found in plasma sampled during the follicular phase. Therefore, in order to prevent confounding the effects of diet and of phase in cycle in the following experiments, experimental diets were fed during the luteal phase of synchronised oestrus cycles. In experiment 2, designed as Latin-Square, six cows received sequentially diets containing 17.1 (control) or 19.7% of crude protein, using two sources of supplementary protein, i.e. soyabean meal (SBM) and corn gluten meal (CGM), differing in ruminal degradability and leucine content. When dry matter intake was used as covariant, plasma insulin on day 16 was 29.5 and 26.4% higher in cows fed diets containing SBM and CGM than in the control (P<0.05). In experiment 3, 17 cows were individually fed the basic diet and then switched to isoenergetic diets containing SBM (n=5), CGM (n=6) or corn grain (CG, n=6) given from day 10 to 16 of the synchronised oestrus cycle. On the eve of day 16, and in the morning of day 17, they were administered PGF(2alpha) and the content of 26 largest follicles was aspirated by using the transvaginal ovum pick-up technique. Follicles were sorted into two classes (preovulatory and subordinate) according to oestradiol concentration and the progesterone:oestradiol ratio in follicular fluid (FF). Higher concentrations of insulin (0.282 versus 0.127ng/ml, P<0.0001) and of glucose (0.614 versus 0.386g/l, P<0.002), were found in FF from preovulatory follicles. The insulin concentration in the FF of cows fed the CG diet was 26% higher than in their counterparts fed CGM (P<0.04), SBM being intermediate. Dietary effects did not reach significance in subordinate follicles. The finding that preovulatory follicular status is associated with increased intrafollicular insulin and glucose suggests that insulin is involved in follicular maturation. The nutritional effect on intrafollicular glucose and insulin may have practical implications to optimise feeding in dairy cows during phases of the oestrus cycle.

Seasonal differences in progesterone production by luteinized bovine thecal and granulosa cells

This study examined seasonal differences in progesterone (P4) production by granulosa cells (GC) and thecal cells (TC) that were luteinized in vitro during the winter or the summer; it also compared plasma P4 concentrations of lactating dairy cows in the two seasons. First-wave dominant follicles obtained from Holstein cows were dissected on day 6 of the cycle, GC and TC were separated, enzymatically dispersed, and cultured for 9 days in media containing 1% fetal calf serum, forskolin (10 micromol/mL) and insulin (2 microg/mL), to induce cell luteinization. All experimental procedures were identical and characteristics of the follicles were similar in the two seasons. During 9 days of culture, P4 production by luteinized GC was higher in winter than in summer, but the difference only tended to be significant. In contrast, luteinized TC produced three times as much P4 in winter as in summer (324 versus 100 ng/10(5)cells). In the in vivo experiment, P4 concentrations in plasma collected during entire estrous cycles in winter and summer were compared. The cows were, on average, at 70 days postpartum and yielded similar amounts of milk. Concentrations of progesterone in plasma were significantly higher in winter than in summer; during the mid-luteal phase the difference between the two seasons was 1.5 ng/mL. These results indicate that chronic effects of heat-stress are possibly carried over from an impaired follicle to an impaired corpus luteum (CL), and that luteinized TC are more susceptible to heat-stress than luteinized GC.

Delayed effect of progesterone on endometrial morphology in dairy cows

Two experiments were conducted to investigate the effect of vaginal progesterone (P4) administered during the luteal phase, on endometrial morphology during the subsequent oestrous cycle. In experiment 1, lactating Holstein cows were allotted to three groups: (1) Control group in which cows remained untreated; (2) The CIDR group in which cows were treated with two P4-containing controlled intravaginal-drug releasing devices (CIDR) during days (d) 6-12 of the cycle; and (3) The PG + CIDR group of cows that received two prostaglandin F2 alpha (PGF2 alpha) injections on d 6 and 7 of the oestrous cycle, to regress the corpus luteum (CL), and were treated with CIDRs on d 6-12, like the CIDR group. All cows were slaughtered on d 3 of the subsequent oestrous cycle. In experiment 2, cows were allotted to three groups: (1) Control cows that remained untreated; (2) CIDR cows that were treated with two CIDRs from d 6 to 15; and (3) Early PG cows that received three i.m. injections of PGF2 alpha on d 3 and 4 of the oestrous cycle to reduce plasma P4. All cows were slaughtered on d 15 of the subsequent cycle. In both experiments, blood was collected during the treated and subsequent cycles to determine P4 and oestradiol (E2) concentrations, and tissue samples from the uterine horn ipsilateral to the CL were collected on the day of slaughter to evaluate endometrial morphology. In both experiments, plasma P4 differed between treatments during the treated cycle but no differences in P4 and E2 concentrations were recorded during the subsequent cycle. In experiment 1, the endometrial morphology of the cows from CIDR and PG + CIDR groups differed from that found in the control group: The surface epithelium was medium to high and the glands were round and tortuous, as compared with low epithelium and oblong glands in the control. In addition, the density of blood vessels and the level of edema was higher in the CIDR-treated cows than in the control cows. In experiment 2, the endometrial morphology of the CIDR-treated group differed from that of the control and early PG groups: Low surface epithelium and oblong glands in the former compared with high epithelium and tortuous glands in the latter. In summary, P4 supplementation during the luteal phase had delayed effect on endometrial morphology at different stages of the subsequent oestrous cycle.

The effect of GnRH administered to dairy cows during oestrus on plasma LH and conception in relation to the time of treatment and insemination

The ocstrous cycles of 480 dairy cows were synchronized with prostaglandin F2α and progesterone coils during two consecutive years. Within 20 h of oestrus detection, cows were treated with saline, 100 or 250 μg GnRH, or 10 μg of the GnRH analogue Buserelin. When GnRH was administered before the spontaneous preovulatory LH surge, the height of the LH surge was 25.8 ± 2.1 ng ml−1 compared to 9.7 ± 0.7 ng ml−1 when GnRH was administered after the spontaneous surge and compared to 12.0 ± 0.8 ng ml−1 in saline-treated cows (P < 0.001). During experimental periods when first-insemination conception rates of control primiparous and multiparous cows were 30.0% and 33.3%, respectively, GnRH administered within 3 h of oestrus detection increased conception to 64.3% in primiparous cows (P < 0.005) and 56.1% in multiparous cows (P < 0.05). When GnRH was administered 16–20 h after oestrus detection or during experimental periods when conception of control animals was higher than 50%, treatment with GnRH had no effect on conception. Multiparous cows which required two to four inseminations and were treated with GnRH within 3 h of oestrus detection had a conception rate of 28.6% when inseminated within 3 h of oestrus detection compared to 67.9% when inseminated 4–30 h following oestrus detection (P < 0.05). Primiparous cows had a significantly higher conception rate than multiparous cows among GnRH-treated animals (65.1% vs. 50.4%) but not among control animals (53.0% vs. 46.2%).

Management of reproduction in dairy heifers based on the synchronization of estrous cycles.

Israeli-Holstein breed dairy heifers (n=571), 13 to 15 mo old, were utilized in two experiments. In Experiment 1, the reproductive performance of synchronized heifers was compared with that of untreated controls. The heifers in both groups were inseminated following the detection of estrus. In Experiment 2, all heifers were synchronized and inseminated following the detection of estrus. Half of the animals in this experiment also received one or two fixed-time inseminations 72 and 96 h after the last synchronization treatment. Synchronization of estrous cycles was performed by two prostaglandin F2alpha (PG) injections given 12 d apart. In the control group of Experiment 1, observation of estrous behavior and insemination of heifers detected in estrus were carried out daily throughout the experiment. In the synchronized groups of Experiments 1 and in 2, the management of reproduction consisted of estrus detection followed by the insemination of heifers in estrus carried out only during 6 d of every 3 wk. Five days following the second PG injection, 86% of the heifers were detected in estrus, 71% of them at 49 to 96 h after treatment. In Experiment 1, age at first insemination, age at conception, and conception rate were, respectively, 425 d, 446 d and 54% in the control group vs 432 d (P<0.02), 449 d and 62% in the PG-treated group. In Experiment 2, the respective figures were 436 d, 462 d and 59% in the group inseminated following the detection of estrus vs 427 d (P<0.002), 464 d and 51% (P<0.05) in the group in which heifers were inseminated at estrus and also received one or two fixed-time inseminations.

Seasonal and parity effects on ghrelin levels throughout the estrous cycle in dairy cows.

In dairy cows, heat stress depresses appetite, leading to decreased food intake, a negative energy balance, and modifies ghrelin levels. Ghrelin is a gut-brain peptide with two major forms: acylated, with an O-n-octanoylated serine in position 3, and nonacylated. To date, the effect of heat stress and estrous cycle on ghrelin secretion in dairy cows has not been studied. We characterized ghrelin secretion during the estrous cycle in each, the winter and the summer seasons. We further examined the effects of parity on ghrelin secretion. Blood was collected from 10 primiparous or multiparous Israeli-Holstein dairy cows throughout the estrous cycle, in both, the hot and cold seasons. The levels of acylated and total ghrelin were measured in the blood samples. We found that both acylated and total ghrelin levels during heat stress were lower than their respective levels in the winter in both, primiparous and multiparous cows. No differences in acylated and total ghrelin levels were found between primiparous and multiparous cows in both seasons. We further found that in multiparous but not primiparous cows acylated ghrelin secretion oscillated during the estrous cycle in both seasons. Its levels peaked on the last days of the first follicular wave and on the days before and during ovulation. Interestingly, we found that elevated acylated ghrelin levels correlated with conception success and increased total ghrelin levels were associated with successful conception from first insemination. Our data is the first to demonstrate seasonal variation in ghrelin secretion. This study provides evidence for the yet unfamiliar link between heat stress, ghrelin and fertility. Increased circulating acylated ghrelin may contribute to improved fertility in dairy cows. It further raises the possibility of a link between ghrelin levels and successful inseminations. Further research is required to determine the effects of ghrelin on dairy cow performance.