Effects of progesterone concentrations and follicular wave during growth of the ovulatory follicle on conceptus and endometrial transcriptome in dairy cows.

Objectives were to evaluate the effects of follicular wave and progesterone concentration on growth of the ovulatory follicle, conceptus elongation, uterine IFN-τ concentration, and transcriptome of conceptus and endometrium of pregnant cows on d 17 of gestation. Nonlactating nonpregnant Holstein cows were assigned randomly to one of 3 treatments: ovulation of a first-wave follicle (FW, n = 15); ovulation of a first-wave follicle and progesterone supplementation (FWP4, n = 12); and ovulation of a second-wave follicle (SW, n = 19). Ovulation of a first- or second-wave follicle was achieved by initiating the Ovsynch protocol (d -9 GnRH, d -2 and -1 PGF2α, d 0 GnRH and artificial insemination, d 0.7 artificial insemination) on d 0 or 6 of a presynchronized estrous cycle, respectively. Cows in FWP4 received 3 intravaginal inserts containing progesterone at 12, 24, and 48 h after the first GnRH injection that were removed on d -2. Cows were killed on d 17 for collection of the reproductive tract. Transcriptome was evaluated by microarray using the Affymetrix Bovine Array. Orthogonal contrasts were built to assess the effects of progesterone concentration during follicle growth (FW vs. FWP4 + SW) and follicular wave (FWP4 vs. SW). Progesterone concentrations (LSM ± SEM) from d -9 to -2 were greater for SW, followed by FWP4 and FW (5.38 ± 0.24, 4.26 ± 0.28, and 1.17 ± 0.27 ng/mL). Diameter of the ovulatory follicle (FW = 19.6 ± 0.6; FWP4 = 15.6 ± 0.6; SW = 15.2 ± 0.5 mm) and concentrations of estradiol from d -2 to 1 (FW = 4.05 ± 0.33; FWP4 = 2.73 ± 0.35; SW = 2.48 ± 0.30 pg/mL) were greater for FW compared with FWP4 and SW. Progesterone concentrations from d 3 to 16 were greater for FW compared with FWP4 and SW. A total of 28 singleton conceptuses were collected (FW, n = 8; FWP4, n = 8; SW, n = 12) and only intact conceptuses were included in the analyses of length (FW, n = 8; FWP4, n = 6; SW, n = 12). Although conceptuses were longer for FW compared with FWP4 and SW (FW = 16.6 ± 2.3; FWP4 = 9.8 ± 2.2; SW = 9.6 ± 2.0 cm), treatment did not affect the amount of IFN-τ in uterine flushing. Transcriptome of conceptuses and endometrium of pregnant cows was not extensively affected by follicular wave (8 and 1 differentially expressed transcripts) or concentration of progesterone during follicle growth (0 and 3 differentially expressed transcripts), showing that these factors did not affect conceptuses and endometrium transcriptome in pregnancies that are maintained to d 17.

Effect of induced subclinical hypocalcemia on physiological responses and neutrophil function in dairy cows.

The objectives were to study the effects of induced subclinical hypocalcemia [SCH, blood ionized Ca (iCa(2+)) <1.0mM, without recumbency] on physiological responses and function of immune cells in dairy cows. Ten nonpregnant, nonlactating Holstein cows were blocked by lactation and assigned randomly to a normocalcemic (NC; intravenous infusion of 0.9% NaCl i.v. plus 43 g of oral Ca, as Ca sulfate and Ca chloride, at -1 and 11h) or an induced SCH [SCHI, 5% ethylene glycol tetraacetic acid (EGTA), a selective iCa(2+) chelator, intravenous infusion] treatment for 24h, using a crossover design. The sequence of treatments was either NC-SCHI or SCHI-NC, with a 6-d washout period. Ionized Ca was evaluated before, hourly during the infusion period, and at 48 and 72 h, to monitor concentrations and adjust the rate of infusion, maintaining blood iCa(2+) <1.0mM in SCHI throughout the 24-h infusion period. Additional measurements included heart and respiratory rates, rectal temperature, dry matter intake, rumen contractions, whole-blood pH, concentrations of glucose and K in whole blood, concentrations of total Ca, Mg, nonesterified fatty acids, ß-hydroxybutyrate, and insulin in plasma, and urinary excretion of Ca. Total and differential leukocyte count in blood was also performed. The concentration of cytosolic iCa(2+) in neutrophils and lymphocytes was quantified and neutrophil function was assayed in vitro. Infusion of a 5% EGTA solution successfully induced SCH in all SCHI cows, resulting in decreased blood iCa(2+) concentrations throughout the 24-h treatment period (0.77 ± 0.01 vs. 1.26 ± 0.01 mM iCa(2+)). Induction of SCH reduced dry matter intake on the day of infusion (5.3 ± 0.8 vs. 9.1 ± 0.8 kg/d) and rumen contractions (1.9 ± 0.2 vs. 2.7 ± 0.2 contractions/2 min) for the last 12h of infusion. Cows in SCHI had decreased plasma insulin concentration (1.44 ± 0.23 vs. 2.32 ± 0.23 ng/mL) evident between 6 and 18 h after the beginning of the infusion, accompanied by increased concentrations of glucose (4.40 ± 0.04 vs. 4.17 ± 0.04 mM). Plasma nonesterified fatty acids concentration was greater for SCHI than NC cows (0.110 ± 0.019 vs. 0.061 ± 0.014 mM). Neutrophils of cows in SCHI had a faster decrease in cytosolic iCa(2+) after stimulation with ionomycin (9.9 ± 1.0 vs. 13.6 ± 1.4 Fluo-4:Fura Red post-end ratio) in vitro. Furthermore, induction of SCH reduced the percentage of neutrophils undergoing phagocytosis (22.1 ± 2.1 vs. 29.3 ± 2.1%) and reduced the oxidative burst response after incubation of pathogenic bacteria (16.1 ± 1.7 vs. 24.2 ± 1.7%). Subclinical hypocalcemia compromised appetite, altered metabolism, and impaired function of immune cells in dairy cows.