Kazuyuki Kaneko

Influence of lameness on follicular growth, ovulation, reproductive hormone concentrations and estrus behavior in dairy cows.

The objective of this study was to examine the effect of a chronic stressor, lameness, on reproductive parameters. Seventy cows 30–80 days post-partum were scored for lameness and follicular phases synchronized with GnRH followed seven days later by prostaglandin (PG). Fifteen Lame animals did not respond to GnRH ovarian stimulation. Milk progesterone for 5 days prior to PG was lower in the remaining Lame cows than Healthy herdmates. Fewer Lame cows ovulated (26/37 versus 17/18; P = 0.04) and the interval from PG to ovulation was shorter in Lame cows. In Subset 1 (20 animals), the LH pulse frequency was similar in ovulating animals (Lame and Healthy) but lower in Lame non-ovulators. An LH surge always preceded ovulation but lameness did not affect the interval from PG to LH surge onset or LH surge concentrations. Before the LH surge, estradiol was lower in non-ovulating cows compared to those that ovulated and estradiol concentrations were positively correlated with LH pulse frequency. In Subset 2 (45 cows), Lame ovulating cows had a less intense estrus than Healthy cows, although Lame cows began estrus and stood-to-be-mounted earlier than Healthy cows. In conclusion, we have identified several parameters to explain poor fertility in some chronically stressed animals. From 30 to 80 days post-partum, there was a graded effect that ranged from 29% Lame cows with absence of ovarian activity, whereas another 21% Lame cows failed to express estrus or ovulate a low estrogenic follicle; in 50% cows, many reproductive parameters were unaffected by lameness.

Effect of retained placenta on subsequent bacteriological and cytological intrauterine environment and reproduction in holstein dairy cows

To determine the effect of retained placenta on the characteristics of the intrauterine environment in dairy cows, bacteriological and cytological tests were performed on intrauterine perfusion fluid. The rate of cows with more than 70% neutrophils or fewer than 40% lymphocytes in inflammatory cells was 48.0% (12/25), while the rate and with more than 50 bacterial colonies/0.1 ml of perfusate was 96.0% (24/25) at 30 d after parturition. Actinomyces pyogenes was isolated from 56.0% (14/25). At 60 d after parturition, however, these values were significantly improved to 20.0% (5/25), 48.0% (12/25) and 12.0% (3/25), respectively. No significant differences in subsequent reproductive performance were observed between cows with and without retained placenta. The results suggest that injury to the intrauterine environment caused by retained placenta is largely healed by 60 d after parturition.

The roles of PGF2α and PGE2 in regression of the corpus luteum after intrauterine infusion of Arcanobacterium pyogenes in cows

To study the effect of bacteria in the uterus on the fate of the corpus luteum (CL), Arcanobacterium pyogenes was inoculated into the uteri of cows on Day 3 (Day 0=day of spontaneous ovulation). Plasma concentrations of 13,14-dihydro-15-keto-PGF(2alpha) (PGFM), 13,14-dihydro-15-keto-PGE(2) (PGEM) and progesterone (P(4)) were determined. In five cows, the developing CL regressed and first-wave dominant follicles, which normally become atretic, ovulated (Group OV) after bacterial inoculation. In another five cows (Group NOV) and five control cows, the developing CL did not regress and first-wave dominant follicles did not ovulate. In Group OV, PGFM concentrations increased by 126.2pg/mL (from 36.8+/-7.8pg/mL on Day 3 to 163+/-37.2pg/mL on Day 6), with an increase ratio of 5.8-fold. Conversely, in Group NOV, PGFM had a greater increase of 198.4pg/mL (from 128.2+/-27.8pg/mL on Day 3 to 326.6+/-115.1pg/mL on Day 5), but the increase ratio was only 2.3-fold. Although PGEM tended to increase in both groups, raw increases and increase ratios were small. Bacterial inoculation into the uterus stimulated the release of prostaglandins and affected the fate of the CL; in that regard, the CL was affected more by PGF(2alpha) than by PGE(2), and the increase ratio of PGF(2alpha) was more important than the raw increase.

Fertirelin and buserelin compared by LH release, milk progesterone and subsequent reproductive performance in dairy cows treated for follicular cysts

This field study compared the efficacy of a single injection of 200 microg fertirelin with that of 20 microg buserelin in shortening the recovery period of 68 laotating Holstein-Friesian cows with ovarian follicular cysts 65 d post partum or later. Differential diagnosis was based not only on palpation per rectum but also on skim milk progesterone profiles (<or= 1.0 ng/ml) spanning a 15-d period rather than the conventional 7-d period, and diagnostic accuracy was markedly enhanced by the 15-d progesterone profiles. At 2 to 2.5 h post treatment all the cows showed an LH increase four-fold or greater. Luteinization, indicated by progesterone levels (>or= 1.0 ng/ml) 1 wk after treatment, was evidenced in 75% of the fertirelin group and 72% of the buserelin group. A 74% conception rate was achieved in fertirelin-treated cows, with a mean interval of 71 d from treatment to conception. In the buserelin-treated cows, 65% conceived and the treatment-to-conception interval was 63 d. Differences were insignificant. At the doses used the two GnRH analogs were deemed equally effective in managing particularly stubborn follicular cysts.

Influence of Trueperella pyogenes in uterus on corpus luteum lifespan in cycling cows

To study ovarian responses to long-term intrauterine infusions of Trueperella pyogenes (T. pyogenes), 12 nonlacting Holstein cows were transcervically infused with 10 mL of a bacterial solution (8-19 × 10(8) colony-forming units/mL), and the uteri of another four cows (control) were similarly infused with sterile physiological saline. Infusions were done six times, every 3 days from Days 3 to 18 (Day 0 = day of spontaneous ovulation). Development of ovarian follicles and the CL were monitored with transrectal, real-time ultrasonography. In five of the experimentally infected cows (group A), the CL, which developed after Day 0, regressed without maturing, and the first dominant follicle (DF) ovulated (mean ± SEM interovulatory interval, 8.6 ± 0.5 days). In group A, plasma 13,14-dihydro-15-keto-PGF(2α) (PGFM) concentrations rose sharply on Day 6, but plasma progesterone concentrations did not increase substantially (as in the control) and were maintained at approximately 2.5 ng/mL after the first DF ovulated. In seven of the 12 infected cows (group B), the developing CL which formed after Day 0 matured and the second DF ovulated. However, the CL lifespan was shorter (P < 0.01) and the second DF ovulated earlier than in control (interovulatory interval, 16.0 ± 0.4 days and 22.3 ± 1.9 days; P < 0.01). Although there was no sharp increase in PGFM in group B, it tended to be high between Days 11 and 18. In conclusion, long-term, intrauterine infusions of T. pyogenes caused the CL to regress prematurely or to have a somewhat shorter lifespan because of release of endogenous PGF(2α).

Effects of high somatic cell counts in milk on reproductive hormones and oestrus behaviour in dairy cows with special reference to those with concurrent lameness.

The present investigation aims to establish the reason(s) why dairy cows with high somatic cell counts (SCCs; >100,000 cells/ml) are less fertile than cows with low SCCs alone. The objective of Study One was to determine whether differences in steroid hormone profiles could explain the low incidence of ovulation in cows with combined High SCC and lameness. Between 30 and 80 days post-partum, animals were scored for SCC and lameness and three groups were formed: Healthy (n=22), High SCC alone (n=12) or High SCC + Lame (n=9). The ovarian follicular phases of all cows were synchronised by administering gonadotrophin releasing hormone (GnRH) followed seven days later by prostaglandin F2alpha (PG). Milk samples were collected daily throughout the entire study period; twice daily during the follicular phase, blood samples were taken and the ovaries were monitored using ultrasonography. Progesterone concentrations were similar in all three groups during each of five specific time periods, i.e. throughout the five days before PG injection, the peri-ovulatory period, on Day 5 and on Day 7, and during the mid luteal phase 12-17 days after ovulation (P>0.13). Mean plasma oestradiol concentrations monitored every 12h during the 36h period before ovulation were similar in all groups (Healthy, 2.80±0.30pg/ml; High SCC alone, 3.82±0.48pg/ml; High SCC+Lame 2.94±0.51pg/ml; P=0.175). The objective of Study Two was to establish whether cows with High SCC (scored and synchronised as above) display different behaviours, especially the intensity and timing of oestrus. Intervals from PG to the onset of oestrus or to the first stand-to-be-mounted (STBM) were longer for the High SCC cows than the Low SCC animals (n=8 and 20; P=0.011 and 0.002, respectively). Also, cows with High SCC tended to have a less intense oestrus and a lower maximum oestrus score per 30-min period than Low SCC cows (P=0.063 and 0.066, respectively). In conclusion, High SCC±lameness did not affect progesterone or twice daily oestradiol profiles but the onset of oestrus was delayed and oestrus tended to be less intense in cows with High SCC. These factors could explain low fertility associated with High SCC.

An evaluation of the effect of age and the peri-parturient period on bone metabolism in dairy cows as measured by serum bone-specific alkaline phosphatase activity and urinary deoxypyridinoline concentration.

Various biochemical markers help to evaluate the state of bone turnover in humans and could be used during the peri-parturient period in dairy cows when calcium (Ca) metabolism changes dramatically. To investigate this, the peri-partum characteristics of serum bone-specific alkaline phosphatase (BAP) and urinary deoxypyridinoline (DPD) were investigated. Both serum BAP activity and urinary DPD concentrations were increased and demonstrated wide variability in younger animals, and these findings were consistent with other bone turnover markers. Around the time of parturition, serum Ca concentration and serum BAP activity in multiparous cows were significantly lower than in primiparous cows, but urinary DPD concentration was unchanged. The use of BAP as a bone formation marker appears to be valuable for evaluating bone remodelling status in cows, but the specificity of the test needs to be confirmed. The DPD/BAP ratio around parturition demonstrated a clear difference in bone turnover status between the two parity groups with multiparous cows demonstrating increased signs of bone resorption compared with primiparous cows, corresponding to the Ca requirement for milk production. In future studies, the applicability of the ratio of bone resorption marker to bone formation marker should be evaluated for bone turnover assessment.

Influence of repeated dinoprost treatment on ovarian activity in cycling dairy cows

To study the ovarian response to the long-term effect of PGF2α, 16 cows were treated with 25 mg tromethamine dinoprost (Pronalgon F; Pfizer, Tokyo, Japan) for 21 days after natural ovulation. Five control cows were treated with sterile physiological saline. The follicle and corpus luteum (CL) development were monitored using a real-time ultrasound instrument. In addition, the plasma concentration of progesterone (P4) was determined. In nine of the 16 Pronalgon-treated cows, the first dominant follicle (1st DF), second dominant follicle (2nd DF), and third dominant follicle ovulated consecutively (group A). In five cows, the 1st and 2nd DFs ovulated consecutively (group B). The developing CL started to regress approximately 5 days after each ovulation without maturation in groups A and B. In the two remaining Pronalgon-treated cows, there was no further ovulation after natural ovulation (group C). In one cow in group C, the 1st DF became atretic and the 2nd DF became cystic with the diameter of the cystic follicle reaching 31.2 mm on Day 30. In another cow, the 1st DF became cystic with a diameter of 30.9 mm on Day 18. Although P4 began to increase after each ovulation in all of the Pronalgon-treated cows, it decreased immediately after each ovulation without a large increase, peaking at approximately 1 ng/mL. Furthermore, the number of days when P4 was >1 ng/mL from natural ovulation to Day 21 was 2.6 ± 0.7 days, which was significantly less than that in the control cows (16.0 ± 0.6 days). These results indicate that the long-term effect of PGF2α has an important role in ovulation of all dominant follicles and might induce cystic ovaries in cows.

Accurate ultrasonographic prediction of progesterone concentrations greater than 1 ng/ml in Holstein lactating dairy cows.

To develop an ultrasonographic assay for determining plasma progesterone concentration (P4 ) as < 1 ng/ml or ≥ 1 ng/ml, the corpus luteum (CL) area and P4 were measured in 1094 multiparous Holstein cows. The area-measuring function and frozen images were used to outline and measure CL imaged via ultrasonography, and CL area was estimated as a polygon of a continuation straight line. A significant correlation was found between CL area and P4 (p < 0.001), and this analysis resulted in the following correlation equation: y = -0.35 + 1.02x (r = 0.81). According to the correlation equation, a CL area of 1.3 cm(2) indicated a P4 of 1 ng/ml. Based on this relationship, each animal was categorized into one of six groups, groups differed based on CL area, and the area ranges were as follows: < 1.3 cm(2) (Group A), 1.3-2.2 cm(2) (Group B), 2.3-3.2 cm(2) (Group C), 3.3-4.2 cm(2) (Group D), 4.3-5.2 cm(2) (Group E) and > 5.2 cm(2) (Group F). For each group, the proportion of cows whose P4 was 1 ng/ml or more was 1.5% in Group A, 83.3% in Group B, 76.6% in Group C, 96.6% in Group D, 99.2% in Group E and 100% in Group F. There was a significant difference between Group A and the other five groups, and between Groups B or C and Groups D, E or F (p < 0.005). These results indicate that a functional CL does not exist when the CL area is less than 1.3 cm(2) and that it exists when the CL area is 3.3 cm(2) or more.

Relative concentrations of progesterone and estradiol-17β in blood serum, whole milk, skimmed milk and milk whey of Holstein cows

HORMONE diagnostic kits are frequently used for veterinary diagnoses, and the specimens for laboratory work in general are serum, whole milk and skimmed milk. However, studies on the differences between the concentrations of progesterone and estradiol-17β in these specimens are lacking. The aim of the present study was to examine the relationship between these concentrations in serum, whole milk, skimmed milk and milk whey. Twenty Holstein cows, comprising 10 pregnant cows with a mean age of 4·2 years (range 2·8 to 7·9 years) and two to six months’ gestation and 10 cows in oestrous, with a mean age of 5·1 years (range 3·8 to 6·2 years) that had been inseminated on the morning of the day of the study, were used. They were kept on standard dairy farms in the town of Hayakita, Hokkaido, Japan. All the animals were high milk producers, producing more than 8500 kg per lactation. Blood and milk were collected from all the cows at the evening milking on January 15, 2004. Blood was collected from tail veins into unanticoagulated vacuum tubes, centrifuged at 1900 g for 10 minutes at room temperature for the separation of serum, within two hours of collection, and stored at –20°C until analysis. Whole milk was collected into three 10 ml sterile plastic tubes from one quarter of each cow after discarding a few strips of foremilk, and taken to the laboratory in an ice bag for storage at –20°C until analysis. The whole milk was centrifuged at 1900 g for 10 minutes and a puncture was made in the wall slightly above the bottom of each plastic tube to release skimmed milk into another sterile plastic tube. Addition of 2 per cent HCl solution to the sample lowered the pH to 4·6, and the tube was centrifuged at 1900 g for 10 minutes to separate the milk whey. The concentrations of progesterone and estradiol-17β in the four samples were measured by radioimmunoassay (RIA), using a commercial kit (Diagnostic Products). The kit employed a solid-phase RIA designed for the direct, quantitative measurement of progesterone in serum and did not require extraction or predilution. All procedures followed the manufacturer’s instructions. In measuring the management sera containing known amounts of steroids, the recovery was 80 to 120 per cent. The cross-reactivities of the antiprogesterone antibody for progesterone, 5α-pregnan-3,20dione, 17α-hydroxyprogesterone, 5β-pregnan-3,20-dione, 20α-dihydroprogesterone, testosterone, 5β-pregnan-3α-ol20-one, androstenediol and estradiol-17β were 100, 9, 3·4, 3·2, 0·2, 0·1, 0·05, less than 0·05 and less than 0·05 per cent, respectively. The intra-assay and interassay coefficients of variation of these assays were less than 8·8 per cent and less than 9·7 per cent, respectively. The progesterone and estradiol-17β concentrations in the serum, whole milk, skimmed milk and milk whey of the 20 cows were determined by triplicate RIA, and 95 per cent confidence intervals (CI) were calculated for each type of specimen. The correlation coefficients that were calculated were tested by the F-test. The mean (95 per cent CI) progesterone concentrations in the serum, whole milk, skimmed milk and milk whey were 3·88 (1·97 to 5·78), 7·31 (3·87 to 10·76), 2·09 (1·14 to 3·04) and 0·19 (0·09 to 0·28) ng/ml, respectively; the concentration was highest in whole milk followed by serum, skimmed milk, and finally, milk whey. The mean (95 per cent CI) of estradiol17β concentrations in serum, whole milk and skimmed milk were 2·20 (1·24 to 3·16), 5·23 (2·23 to 8·24) and 7·77 (3·04 to 12·50) pg/ml, respectively. The estradiol-17β concentration was highest in skimmed milk, followed by whole milk and serum. Triplicate assays of estradiol-17β concentrations in the milk whey revealed the values to be below the minimum detectable activity, and results were expressed as 0 pg/ml. The correlation coefficients of progesterone and estradiol17β between the specimens are shown in Table 1. The difference in progesterone concentrations between whole milk and skimmed milk indicated that more progesterone was located in the milk fat of whole milk. Even though milk whey was selected in this study as one of the specimens, the progesterone concentrations in the serum, whole milk, skimmed milk and milk whey were highly correlated, and the correlation coefficients were in agreement with previous reports (Meistering and Dailey 1987, Kamboj and Prakash 1993). Therefore, any of the four types of specimens could be used to monitor the concentration of progesterone. The mean estradiol-17β concentration had a higher tendency to be higher in skimmed milk than in whole milk and serum. Since all the animals were high milk producers of more than 8500 kg per lactation, mammary excretion of estradiol17β (Janowski and others 2002) was possibly responsible for the rise in estradiol-17β concentration in the whole milk. The difference in estradiol-17β concentration between skimmed milk and whole milk suggests that more estradiol-17β is present in casein than in milk fat. Since the mean estradiol17β concentrations were only moderately correlated between serum, whole milk and skimmed milk, positive preference may not be given to these for the determination of estradiol17β. Milk whey was also selected as one of the specimens, but this was not regarded as an appropriate specimen for estradiol-17β determination, because the estradiol-17β concentration of milk whey was below the minimum detectable activity for all the cows. In conclusion, in Holstein cows, serum, whole milk, skimmed milk and milk whey are considered to be alternatives for reliable determination of the progesterone concentration. However, the use of serum, whole milk, and skimmed milk is not recommended as an alternative for estradiol-17β determination based on comparative study between estradiol-17β concentrations of the specimens. Milk whey is not useful for estradiol-17β determination.