Chiho Kawashima

Peripheral administration of kisspeptin-10 increases plasma concentrations of GH as well as LH in prepubertal Holstein heifers

This study was conducted to estimate the effects of kisspeptin-10 on blood concentrations of LH and GH in prepubertal dairy heifers. Heifers received a single injection of 1 mg kisspeptin-10 (n=5) or saline (n=5) intravenously, and serial blood samples were collected at 15-min intervals to analyze the response curves of both LH and GH after injection. Peak-shaped responses were observed for concentrations of LH and GH, and the peaks were observed at 27+/-3 and 75+/-9 min, respectively, after injection, only in heifers injected with kisspeptin-10. These data suggest various possible important links among kisspeptin, the reproductive axis, and also the somatotropic axis in prepubertal Holstein heifers.

Insulin-like growth factor-I (IGF-I) system during follicle development in the bovine ovary: relationship among IGF-I, type 1 IGF receptor (IGFR-1) and pregnancy-associated plasma protein-A (PAPP-A).

Insulin-like growth factor-I (IGF-I) system that is exerted mainly through the type 1 IGF receptor (IGFR-1) and releasing of free IGF-I is regulated by the proteases of IGF-binding proteins (IGFBPs), an important factor in follicle development of bovine ovary. The aims of the present study were to examine the mRNA expressions of IGF-I, IGFR-1 and pregnancy-associated plasma protein-A (PAPP-A) in granulosa cells and theca tissues during bovine follicular development and the effects of follicle-stimulating hormone (FSH) and estradiol (E2) on the expression of these genes in cultured bovine granulosa cells. Follicles were classified into four groups such as small follicle (SF), estrogen inactive dominant follicle (EID), estrogen active dominant follicle (EAD) and preovulatory follicle (POF). The concentration of free IGF-I in follicular fluid of POF was significantly higher than those in EID, whereas the total IGF-I in follicular fluid did not change at all developmental stages. The expression of IGF-I mRNA was not detected in the granulosa cells at all at any developmental stages but the expression was detected in the theca tissues. The amount of IGFR-1 mRNA in granulosa cell showed the constant level at all developmental stages except EID. The expressions of IGFR-1 and PAPP-A in cultured bovine granulosa cells were stimulated with FSH but not with E2. The PAPP-A mRNA expression was stimulated by FSH in presence of 1 ng/ml E2. These results indicate that IGF-I in follicular fluid is mainly derived from the circulation and that FSH is an inducer for the expression of IGFR-1 and PAPP-A genes in granulosa cells. Therefore, we suggest that PAPP-A stimulated with FSH play a crucial role for IGF-I system in bovine follicular development.

Effect of β-carotene supply during close-up dry period on the onset of first postpartum luteal activity in dairy cows.

The aim of this study was to examine the effect of β-carotene supply during the close-up dry period on the onset of first postpartum luteal activity in dairy cows. Twelve cows were supplied with 2000 mg of β-carotene (20 g Rovimix(®) β-Carotene containing 10% β-carotene; DSM Nutrition Japan K.K., Tokyo, Japan) by oral administration daily from day 21 before expected calving date to parturition. Fourteen cows (control) did not receive β-carotene supplementation. Blood samples were obtained on days 21, 14 and 7 before expected calving date and on days 1, 7, 14, 21 postpartum. When the plasma progesterone concentration exceeded 1 ng/ml by day 21 postpartum, luteal activity was assumed to have been initiated. The result showed that serum β-carotene concentrations in the β-carotene cows were higher than in the control cows during the experimental period (p < 0.01). The number of cows with the onset of luteal activity by day 21 postpartum was 9/12 in the β-carotene cows and 4/14 in the control cows (p < 0.05). Retinol, certain metabolic parameters and metabolic hormones concentrations did not differ between β-carotene and control cows. In addition, serum retinol concentration in β-carotene cows without luteal activity was lower than in β-carotene cows with luteal activity (p < 0.05), and serum gamma-glutamyl transpeptidase concentration in β-carotene cows with luteal activity (p < 0.05) and control cows without luteal activity (p < 0.01) was higher than in control cows with luteal activity. In conclusion, β-carotene supply during the close-up dry period may support the onset of luteal activity during early lactation in dairy cows.

Oestradiol Enhances Plasma Growth Hormone and Insulin‐like Growth Factor‐I Concentrations and Increased the Expression of their Receptors mRNAs in the Liver of Ovariectomized Cows

Many metabolic hormones, growth hormone (GH), insulin-like growth factor-I (IGF-I) and insulin affect ovarian functions. However, whether ovarian steroid hormones affect metabolic hormones in cattle remains unknown. This study aimed to determine the effect of sex steroids on the plasma profiles of GH, IGF-I and insulin and their receptors in the liver and adipose tissues of dairy cows. Ovariectomized cows (n = 14) were randomly divided into four groups: control group (n = 3) was treated with saline on Day 0; oestradiol (E2) group (n = 3), with saline and 1 mg oestradiol benzoate (EB) on Day 0 and 5, respectively; progesterone (P4) group (n = 4) with two CIDRs (Pfizer Inc., Tokyo, Japan) from Day 0; and E2 + P4 group (n = 4) with two CIDRs on Day 0 that were removed on Day 6 and were immediately injected with 1 mg EB. The animals were euthanized after the experiment, and liver and adipose tissues samples were quantitatively analysed using real-time PCR for the expression of mRNA for the GH (GHR), IGF-I (IGFR-I) and insulin (IR) receptor mRNAs. Oestradiol benzoate significantly increased the number of peaks (p < 0.05), pulse amplitude (p < 0.05) and area under the curve (AUC; p < 0.01) for plasma GH; moreover, it increased plasma IGF-I concentration (p < 0.05), but it had no effect on the plasma insulin profile. P4 significantly decreased the AUC (p < 0.01), compared with the control group, whereas it did not affect the number of peaks and the amplitude of GH pulses. P4 + E2 did not affect the GH pulse profile. E2 increased the mRNA expression of GHR, IGFR-I and IR in the liver (p < 0.05), whereas both P4 and E2 + P4 did not change their expressions. Our results provide evidence that the metabolic and reproductive endocrine axes may regulate each other to ensure optimal reproductive and metabolic function.

Supplementation with difructose anhydride III promotes passive calcium absorption in the small intestine immediately after calving in dairy cows.

The incidence of hypocalcemia increases in high-parity dairy cows because resorption of bone Ca is delayed in these animals, and they appear to have a reduced ability to absorb Ca from the intestine during the early postpartum period. Difructose anhydride (DFA) III has been shown to promote the absorption of intestinal Ca via a paracellular pathway. However, past studies have not reported this effect in peripartum dairy cows. Therefore, we investigated the effect of DFA III supplementation on Ca metabolism during the peripartum period to determine whether DFA III promotes intestinal Ca absorption via this route. Seventy-four multiparous Holstein cows were separated into DFA and control groups based on their parity and body weight. The feed of the DFA group was supplemented with 40g/d of DFA III from -14 to 6d relative to calving. The control group did not receive DFA III. At calving (0h relative to calving), serum Ca declined below 9mg/dL in both groups. However, serum Ca concentrations were greater in the DFA group than in the control group at 6, 12, 24, and 48h relative to calving, and the time required for serum Ca to recover to 9mg/dL during the postpartum period was shorter in the high-parity cows in the DFA group than in those in the control group. Parathyroid hormone concentrations increased immediately after calving in both groups and were greater in the control group than in the DFA group at 12 and 24h relative to calving. Serum 1,25-dihydroxyvitamin D concentrations increased at 0 and 12h relative to calving in both groups and were higher in the control group than in the DFA group at 72h relative to calving. Serum concentrations of the bone-resorption marker cross-linked N-telopeptide of type I collagen (NTX) were not different between the groups during peripartum period, and serum NTX in all cows was lower at 0, 6, 12, 24, 48, and 72h relative to calving than at -21, 4, and 5d relative to calving. Thus, DFA treatment induced faster recovery of serum Ca, although bone resorption was restrained. In conclusion, DFA III promotes intestinal passive Ca absorption via the paracellular pathway during the early postpartum period; this absorption is unaffected by aging.

Long-term impact of puerperal metritis on the profiles of peripheral blood leukocytes in peripartum dairy cows.

To determine the effects of puerperal metritis on the immune response, changes in the differential peripheral blood leukocyte counts were analyzed during the peripartum period in cows with or without metritis. Multiparous Holstein cows were examined for uterine health disorders and classified into two groups: healthy (n = 11) or metritis (n = 5) cows. The lymphocyte and monocyte counts and the proportion of CD8(+) lymphocytes were higher in cows with metritis compared to healthy cows. Moreover, the effects of puerperal metritis on the lymphocyte counts and CD4(+)/CD8(+) ratio persisted weeks after the uterine inflammation had self-resolved. Taken together, the findings of the present study indicate the possible long-term alterations of systemic immune responses in cows with puerperal uterine inflammation.

Short communication: Difructose anhydride III promotes calcium absorption from the duodenum in cattle

Difructose anhydride (DFA) III promotes the intestinal absorption of calcium via a paracellular pathway in rats. In dairy cows, DFA III reaches the duodenum without being degraded by ruminal bacteria and hence could be used to control hypocalcemia. The aims of the present study were to investigate the percentage of DFA III that appears in the duodenum of cows and to determine the effect of DFA III on calcium absorption from duodenal fluid. The first experiment was performed in 3 ruminally and duodenally cannulated dry Holstein cows in a 3 × 3 Latin square design. Each experimental period lasted 7 d. On the first day, the cows were ruminally fed one of the following treatments: 0 (DFA0), 50 (DFA50), or 100 (DFA100) g/d of DFA III, using cobalt-EDTA as a liquid phase marker. Difructose anhydride III was detected in duodenal fluid 1 h after feeding, and its concentration peaked 4 h after feeding, in a dose-dependent manner. The percentages of DFA III that appeared in the duodenum after the DFA50 and DFA100 treatments were 69.1 ± 7.0% and 67.9 ± 5.6%, respectively. The second experiment used the everted duodenal sacs of cattle (n = 7 in each group). Sacs were incubated in artificial mucosal fluid containing 1 mM DFA III or no DFA III (control) for 60 min with 100% O2 in a water bath at 37 °C. After incubation, the calcium concentration of the artificial serosal fluid in the everted sacs was measured. Calcium absorption was higher in the DFA III-treated group than in the control group (803 ± 161 and 456 ± 74 nmol/cm of sac, respectively). The above results demonstrate that approximately 70% of administered DFA III reached the duodenum of cows intact. Moreover, similar to its effects on calcium absorption in rats, DFA III promoted calcium absorption via a paracellular pathway in the duodenum of cows.

Relationship between the degree of insulin resistance during late gestation and postpartum performance in dairy cows and factors that affect growth and metabolic status of their calves

This study aimed to investigate the effects of insulin resistance (IR) during the close-up dry period on the metabolic status and performance of dairy cows as well as to determine the effects on body weight (BW) and metabolic status of their calves. An insulin tolerance test (ITT) was conducted by administering 0.05 IU/kg BW of insulin to 34 multiparous Holstein cows at 3 weeks prepartum. Blood samples were collected at 0, 30, 45 and 60 min after insulin injection, and cows were divided into two groups based on the time required for glucose to reach the minimum levels [non-IR (NIR), 45 min (n=28); and IR, 60 min (n=6)]. Blood or milk sampling and body condition score (BCS) estimation were performed twice weekly during the experimental period. Blood samples from calves were collected immediately after birth. Cows with IR showed lower BCS (P<0.05) and serum urea nitrogen (P<0.05) and glucose concentration (P=0.05) before calving, and lower serum non-esterified fatty acid concentration (P<0.05) and milk yield (P<0.05) and earlier resumption of luteal activity (P<0.05) after calving; their calves showed lower BW (P<0.05) and plasma insulin-like growth factor-I concentration (P<0.001) and higher plasma insulin concentration (P<0.05). In conclusion, IR at 3 weeks prepartum in dairy cows is related to postpartum metabolic status and performance along with growth and metabolic status of their calves.

Effects of dietary contamination by zearalenone and its metabolites on serum Anti-Müllerian hormone: Impact on the reproductive performance of breeding cows

We investigated the effects of in vivo exposure to low zearalenone levels on the anti-Müllerian hormone endocrine levels and the reproductive performance of cattle. Urine and blood samples and reproductive records were collected from two Japanese Black breeding female cattle herds with dietary zearalenone contamination below the threshold levels (<1 ppm) at 30 days after calving. Urinary zearalenone, α-zearalenol and β-zearalenol concentrations were measured by chromatography-tandem mass spectrometry, and serum anti-Müllerian hormone concentrations were determined along with serum biochemical parameters. Urinary concentrations of α-zearalenol were significantly higher (p < 0.05) in cattle in Herd 1 than in cattle in Herd 2, reflecting the different amounts of zearalenone in the diet of the two herds. Although the number of 5-mm and 10-mm follicles of the herds and their fertility after artificial insemination were similar, the serum anti-Müllerian hormone concentrations in herds 1 and 2 were 438.9 ± 48.6 pg/ml and 618.9 ± 80.0 pg/ml, respectively, with a trend towards a significant difference (p = 0.053), which may indicate differences in the antral follicle populations between herds. Thus, zearalenone intake from dietary feed, even when below the threshold zearalenone contamination level permitted in Japan, may affect the ovarian antral follicle populations, but not the fertility, of post-partum cows.

The Effect of Single Nucleotide Polymorphisms in the Tumor Necrosis Factor-α Gene on Reproductive Performance and Immune Function in Dairy Cattle

The present study aimed to assess the effect of polymorphisms in the tumor necrosis factor α (TNF-α) promoter (A/A, A/G and G/G) and exons (T/T, T/C and C/C) on immune function and reproductive performance in dairy cows. The occurrence of the first postpartum ovulation within 3 weeks in the cows with the TNF-α promoter A/G and G/G genotypes was higher than in the A/A group. Among the different TNF-α exon genotypes, the occurrence of early first postpartum ovulation was higher in the T/C and C/C genotype groups than in the T/T group. Single nucleotide polymorphisms (SNPs) in the TNF-α gene did not affect the rate of artificial insemination (AI) or duration from parturition to next conception (days open). The apoptosis rate of polymorphonuclear leukocytes (PMNs) did not differ among the TNF-α promoter genotypes, but the PMN transmigration rate was significantly higher for the A/A and A/G genotypes than for the G/G genotype. Interleukin 8 (IL-8) mRNA expression in PMNs and peripheral blood mononuclear cells (PBMCs) before culture was significantly higher for the A/A genotype compared with the G/G genotype. There were no significant differences between the genotypes in the mRNA expression of TNF-α, IL-6, IL-1β, and toll-like receptor 4 (TLR4) in PMNs and PBMCs before and 4 h after culture. IL-8 and IL-1β production by PBMCs cultured for 4 h was significantly higher for the animals with the A/A genotype than for those with the G/G genotype. On the other hand, no significant difference was observed in IL-8 and IL-1β production by PMNs among different TNF-α genotypes. Taken together, these results suggest that SNP in the TNF-α gene affects immune function and reproductive performance in dairy cows.