Age-related changes in circulating INSL3 concentrations and their associations with ovarian conditions in Japanese Black beef cattle.

Information on circulating levels of insulin-like peptide 3 (INSL3) in female domesticated animals is limited, as their concentrations are significantly lower than in males. The objectives of the present study were to 1) develop a sandwich time-resolved fluorescence immunoassay (TRFIA) with higher detectability to measure blood INSL3 concentrations in female cattle, 2) determine INSL3 concentrations in female cattle among age groups and reproductive conditions, and 3) explore associations between INSL3 levels and ultrasonographic ovarian measurements. Blood was collected repeatedly from Japanese Black beef female calves (n = 12; 0－8 mo), heifers (n = 10; 10－26 mo), and cows (n = 20; 27－200 mo). Blood was taken from the cows (n = 13) at follicular, post-ovulatory, and luteal phases, and from cows with follicular cysts (n = 12). Ultrasonography of ovaries was conducted in the calves (n = 12) and the cows without ovarian diseases (n = 9). The ovarian area, as well as the number and diameters of antral follicles ≥ 2 mm, were determined in each ovary. The proposed method detected a difference in plasma INSL3 between calves (0.01 ng/mL) and heifers (0.18 ng/mL). However, the conventional assay showed similar levels for calves and heifers (1.82 vs 2.07 ng/mL). Plasma INSL3 and testosterone concentrations increased from calves to heifers (P < 0.0001), but only INSL3 rose from heifers to cows (P < 0.0001). INSL3 and testosterone concentrations did not change across the estrus cycle in cows, and the levels of both hormones in follicular cystic cows did not differ from those in the follicular phase. Ovarian area, maximal and average follicular diameters, and total volume of all follicles per animal were higher in cows than calves (P < 0.001). Plasma INSL3 concentrations correlated positively with the total volumes of all follicles in calves (P < 0.05) and cows (P < 0.05), whereas testosterone concentrations did not correlate with ovarian follicular measurements. In conclusion, plasma INSL3 concentrations measured by the proposed sandwich TRFIA showed a clear increase from female calves to cows in beef cattle. These results suggest that circulating levels of INSL3, but not of testosterone, are associated with the total volume of all antral follicles in both ovaries per animal in female cattle.

Plasma estradiol-17ß, cortisol, and insulin concentrations and serum biochemical parameters surrounding puberty in Japanese Black beef bulls with normal and abnormal semen.

The associations of semen abnormalities with circulating hormones (estrogens, glucocorticoid, insulin) and common biochemical parameters are unclear in beef bulls. We compared plasma concentrations of estradiol-17ß, cortisol, and insulin and serum biochemical parameters surrounding puberty in Japanese Black beef bulls (n = 96) with normal post-thaw or abnormal semen (fresh and frozen). Blood samples were collected monthly from 4 to 24 months of age (n = 50) for the assays of plasma estradiol, cortisol, and insulin and every 3 months from 6 to 21 months of age (n = 92) for the serum biochemical analyses. Semen was collected weekly from 12 months until at least 18 months of age. Fresh semen was evaluated for semen volume, sperm progressive motility, concentrations, and morphological defects. The normal fresh semen was frozen by a standard method and examined for post-thaw sperm motility and fertility, which were evaluated for rates of transferable embryos. Bulls were classified as having either normal fresh semen or abnormal fresh semen (when at least one of the above test items was abnormal for 6 months). The normal fresh semen was categorized as having either normal post-thaw semen or low fertility post-thaw semen. The abnormal fresh semen was categorized as having sperm morphological defects, low motility, or morphological defects plus low motility. Plasma cortisol concentrations in the abnormal fresh semen group were higher than those of the normal fresh semen group (p < 0.0001). Plasma estradiol-17ß and insulin concentrations in the low-fertility post-thaw semen group were lower than those of the normal post-thaw semen group (p < 0.0001). Serum aspartate aminotransferase and magnesium concentrations were greater for the abnormal fresh semen group vs. the normal fresh semen group (p < 0.005). These results suggest that fresh semen abnormality in pubertal beef bulls might be associated with increased circulating aspartate aminotransferase, magnesium and cortisol. Low-fertility post-thaw semen could have been involved with the lower peripheral estradiol and insulin levels in beef bulls.

Plasma insulin-like peptide 3 concentrations are acutely regulated by luteinizing hormone in pubertal Japanese Black beef bulls.

Insulin-like peptide 3 (INSL3) is a major secretory product of testicular Leydig cells. The mechanism of acute regulation of INSL3 secretion is still unknown. The present study was undertaken in pubertal beef bulls to (1) determine the temporal relationship of pulsatile secretion among LH, INSL3, and testosterone and (2) monitor acute regulation of INSL3 secretion by LH using GnRH analogue and hCG. Blood samples were collected from Japanese Black beef bulls (N = 6) at 15-minute intervals for 8 hours. Moreover, blood samples were collected at -0.5, 0, 1, 2, 3, 4, 5, and 6 hours after GnRH treatment and -0.5, 0, 2, 4, and 8 hours on the day of treatment (Day 0), and Days 1, 2, 4, 8, and 12 after hCG treatment. Concentrations of LH, INSL3, and testosterone determined by EIAs indicated that secretion in the general circulation was pulsatile. The frequency of LH, INSL3, and testosterone pulses was 4.7 ± 0.9, 3.8 ± 0.2, and 1.0 ± 0.0, respectively, during the 8-hour period. Seventy percent of these INSL3 pulses peaked within 1 hour after a peak of an LH pulse had occurred. The mean increase (peak per basal concentration) of testosterone pulses was higher (P < 0.001) than that of INSL3 pulses. After GnRH treatment, LH concentrations increased (P < 0.01) dramatically 1 hour after treatment and remained high (P < 0.05) until the end of sampling, whereas an elevated (P < 0.05) INSL3 concentration occurred at 1, 2, 5, and 6 hours after treatment. Testosterone concentrations increased (P < 0.01) 1 hour after the treatment and remained high until the end of sampling. After hCG treatment, an increase of INSL3 concentration occurred at 2 and 4 hours, and Days 2, 4, and 8 after treatment (P < 0.05), whereas in case of testosterone, concentrations remained high (P < 0.01) until Day 8 after treatment. The increase (maximum per pretreatment concentration) of INSL3 concentrations after injecting GnRH or hCG was much lower (P < 0.001) than that of testosterone. In conclusion, secretion of INSL3 in blood of bulls occurred in a pulsatile manner. We inferred an acute regulation of INSL3 by LH in bulls because INSL3 concentrations increased immediately after endogenous and exogenous LH stimulation. The increase of INSL3 concentrations by LH was much lower than that of testosterone in bulls.

Changes in plasma concentrations of insulin-like peptide 3 and testosterone from birth to pubertal age in beef bulls.

The objectives were to: (1) develop an enzyme immunoassay (EIA) for insulin-like peptide 3 (INSL3) or relaxin-like factor (RLF) in bovine plasma; (2) investigate changes of plasma INSL3 concentrations from birth to pubertal age of beef bulls; and (3) compare changes in plasma concentrations of INSL3, testosterone, and LH. Plasma samples were collected from beef bull calves (n = 15) at birth (0 d) and at 28, 56, and 84 d after birth. Furthermore, in beef bulls around pubertal age (n = 26; age range 3 to 22 mo), plasma samples were collected at 1 to 4 mo intervals. Plasma INSL3 concentrations increased (P < 0.05) from 0 to 28, 28 to 56, and from 56 to 84 d of age. Plasma testosterone concentrations increased (P < 0.001) from 0 to 28 d, and from 28 to 56 d, but did not change from 56 to 84 d. For bulls around pubertal age, plasma INSL3 concentrations did not change from the prepubertal phase (3 to 6 mo) to the early pubertal phase (6 to 12 mo), but increased (P < 0.05) from the early to late pubertal phases (12 to 18 mo), and from the late pubertal to postpubertal phases (18 to 22 mo). Plasma testosterone concentrations increased from the prepubertal to early pubertal phases (P < 0.001), but did not change thereafter. Plasma LH concentrations did not change from 0 d to 84 d, but decreased (P < 0.001) from prepubertal to early pubertal phase, with no significant change thereafter. Plasma INSL3 concentrations increased during the first 3 mo of life and throughout the pubertal age in beef bulls. There were similar dynamic patterns for INSL3 and testosterone during the first 3 mo of life, but patterns subsequently diverged in bulls around pubertal ages.

Preventive effects of CIDR-based protocols on premature ovulation before timed-AI in Ovsynch in cycling beef cows.

Ovsynch is a program developed to synchronize ovulation for timed breeding. In this paper, the authors investigate whether controlled internal drug release (CIDR)-based protocols prevent premature ovulation before timed-artificial insemination (AI) when Ovsynch is started a few days before luteolysis in cycling beef cows. Nine beef cows at 16 days after oestrus were treated with (1) Ovsynch, i.e. gonadotropin releasing hormone (GnRH) analogue on day 0, prostaglandin (PG) F(2alpha) analogue on day 7 and GnRH analogue on day 9 with timed-AI on day 10, (n=3); (2) Ovsynch+CIDR (Ovsynch protocol plus a CIDR for 7 days from day 0, n=3), or (3) oestradiol benzoate (OB)+CIDR+GnRH (OB on day 0 in lieu of the first GnRH treatment, followed by the Ovsynch+CIDR protocol, n=3). In the Ovsynch group (1) plasma progesterone concentrations fell below 0.5 ng/mL earlier (day 5) than in both CIDR-treated groups (2) and (3), where this occurred on day 8. Plasma oestradiol-17beta concentrations peaked on day 8 in the Ovsynch group and on day 9 in both CIDR-treated groups. The dominant follicle ovulated on day 10 in the Ovsynch group and on day 11 in both CIDR-treated groups. Thus, both CIDR-based protocols prevented premature ovulation before timed-AI in Ovsynch when the protocol was started a few days before luteolysis. This reflects the fact that progesterone levels remained high until the beef cattle were treated with PGF(2alpha).

Effect of CIDR-based protocols for timed-AI on the conception rate and ovarian functions of Japanese Black beef cows in the early postpartum period.

Our objectives were to compare: (1) conception rates (in early postpartum Japanese Black beef cows) to timed-artificial insemination (timed-AI) among Ovsynch and Ovsynch plus CIDR protocols, and a protocol that used estradiol benzoate (EB) in lieu of the first GnRH of the Ovsynch plus CIDR; and (2) the effects of these protocols on blood concentrations of ovarian steroids. Cows in the control group (Ovsynch; n=35) underwent a standard Ovsynch protocol (GnRH analogue on Day 0, PGF(2 alpha) analogue on Day 7 and GnRH analogue on Day 9), with timed-AI on Day 10, approximately 20 h after the second GnRH treatment. Cows in the Ovsynch+CIDR group (n=31) received a standard Ovsynch protocol plus a CIDR for 7 days (starting on Day 0). Cows in the third treatment group (EB+CIDR+GnRH; n=41) received 2mg of EB on Day 0 in lieu of the first GnRH treatment, followed by the same treatment as in the Ovsynch+CIDR protocol. The conception rate tended to be greater in the Ovsynch+CIDR group (67.7%, P<0.15) and was greater in the EB+CIDR+GnRH (73.2%, P<0.05) and CIDR-combined (both CIDR-treated groups were combined) groups (70.8%, P<0.05) than in the Ovsynch group (48.6%). Plasma progesterone concentrations were higher on Day 7 (P<0.01) and lower on Days 14, 17 and 21 (P<0.001) in the CIDR-combined group than in the Ovsynch group. Plasma estradiol-17beta concentrations were higher on Day 7 in the Ovsynch group of non-pregnant cows than in the CIDR-combined group of non-pregnant cows and in an all-combined group (all treatment groups combined) of pregnant cows (P<0.01). Furthermore, estradiol-17beta concentrations were lower on Day 9 in the Ovsynch and CIDR-combined groups of non-pregnant cows than in the all-combined group of pregnant cows (P<0.05). In conclusion, both protocols using CIDR improved conception rates following timed-AI in early postpartum suckled Japanese Black beef cows relative to the Ovsynch protocol. Treatment with a CIDR may prevent early maturation of follicles observed in non-pregnant cows treated with the Ovsynch protocol, by maintaining elevated blood progesterone concentrations until PGF(2 alpha) treatment.

Improved conception in timed-artificial insemination using a progesterone-releasing intravaginal device and Ovsynch protocol in postpartum suckled Japanese Black beef cows.

The primary objective was to determine the effect of supplemental progesterone, administered via an intravaginal device (CIDR), on conception rates to timed-artificial insemination (timed-AI) in postpartum suckled Japanese Black beef cows treated with the Ovsynch protocol. A secondary objective was to compare the effects of treatments on plasma concentrations of progesterone and estradiol. Cows in the control group (Ovsynch, n=38) received a standard Ovsynch protocol (100 microg GnRH analogue on Day 0, 500 microg PGF2alpha analogue on Day 7, and 100 microg GnRH analogue on Day 9), with AI on Day 10, approximately 20 h after the second GnRH treatment. Cows in the treatment group (Ovsynch+CIDR; n=40) received a standard Ovsynch protocol plus a CIDR for 7 days (starting on Day 0). Plasma progesterone concentrations were determined on Days 0, 1, 7, 9, 10, and 17 and plasma estradiol-17beta concentrations were determined on Days 7, 9, 10, and 17. The odds ratio for likelihood of conception was 3.29 times greater (P=0.02) in the Ovsynch+CIDR group compared to Ovsynch group. The conception rate was greater (P=0.03) in the Ovsynch+CIDR group than in the Ovsynch group (72.5% versus 47.7%). Insertion of a CIDR device significantly increased plasma progesterone concentrations only on Days 1 and 7 (P<0.001 and P=0.05, respectively), but had no significant effect on plasma estradiol-17beta concentrations. Including a CIDR with the Ovsynch protocol significantly improved conception rates in postpartum suckled Japanese Black beef cows.