W. R. Lamberson

Dominant Bovine Ovarian Follicular Cysts Express Increased Levels of Messenger RNAs for Luteinizing Hormone Receptor and 3β-Hydroxysteroid Dehydrogenase Δ4,Δ5 Isomerase Compared to Normal Dominant Follicles

Abstract The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17α-hydroxylase, cytochrome P450 aromatase, 3β-hydroxysteroid dehydrogenase Δ4,Δ5 isomerase, LH, and FSH receptors and estrogen receptor-β in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17β, progesterone, and androstenedione concentrations were determined in follicular fluid using specific RIAs. Dominant cysts were larger than young cysts or dominant follicles, whereas nondominant cysts were intermediate. Estradiol-17β (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs was higher in granulosa cells of dominant cysts than in dominant follicles. Nondominant cysts had higher follicular concentrations of progesterone, lower estradiol-17β concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-β mRNAs than other groups. In summary, increased expression of LH receptor and 3β-hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17β concentrations were associated with dominant cysts compared to dominant follicles. Study of cysts at known developmental stages is useful in identifying alterations in follicular steroidogenesis.

Concentrations of nonesterified fatty acids and glucose in blood of periparturient dairy cows are indicative of pregnancy success at first insemination

Greater blood concentrations of nonesterified fatty acids (NEFA) and lesser blood concentrations of glucose are indicative of the normal process of nutrient partitioning that occurs in early postpartum dairy cows. The objective was to determine the relationship between blood NEFA and glucose concentrations and subsequent conception at first insemination in postpartum dairy cows. Holstein (n=148) and Guernsey (n=8) dairy cows were blood sampled at approximately d 10, 7, and 3 prepartum, on the day of calving and 3, 7, 14, and 21 d postpartum for measurement of NEFA and glucose concentrations. Serum and plasma were harvested and used for measurement of NEFA and glucose concentrations, respectively. Cows were given a presynchronization treatment (2 injections of PGF(2α) 14 d apart) with the second PGF(2α) injection occurring 14 d before the initiation of the timed AI (TAI) protocol. Blood for determination of progesterone concentrations was collected at each presynchronization injection and at the initiation of the TAI protocol that was used for first insemination (74±7 d postpartum). Cows were considered noncycling if serum progesterone concentrations at the 2 presynchronization PGF(2α) injections (d 37 and 51±7 postpartum) and at the initiation of the TAI protocol (d 65±7 postpartum) were ≤1 ng/mL, and there was no indication of ovulation or presence of a corpus luteum by ultrasound examination at the initiation of the TAI protocol. Pregnancy was determined at 33 d and again at 61 d after first insemination by using ultrasound. Across all days, serum NEFA and plasma glucose concentrations were not different between cows that ovulated before the initiation of the TAI program (cycling) compared with those that did not ovulate (noncycling). Serum NEFA concentrations, however, were less and plasma glucose concentrations were greater during the early postpartum period for cows that subsequently became pregnant at first insemination compared with those that failed to become pregnant. Logistic regressions were used to predict the probability of pregnancy based on NEFA and glucose concentrations from individual days. The prediction with the greatest likelihood ratio was for d 3 postpartum NEFA and glucose concentrations. Nutritional status during the early postpartum period (within 1 wk after calving), as indicated by blood NEFA and glucose concentrations, may affect subsequent fertility by a mechanism that is independent from interval to first ovulation.

Developmental Expression of 2489 Gene Clusters During Pig Embryogenesis: An Expressed Sequence Tag Project

Abstract Identification of mRNAs that are present at early stages of embryogenesis is critical for a better understanding of development. To this end, cDNA libraries were constructed from germinal vesicle-stage oocytes, in vivo-produced four-cell- and blastocyst-stage embryos, and from in vitro-produced four-cell- and blastocyst-stage embryos. Randomly picked clones (10 848) were sequenced from the 3′ end and those of sufficient quality (8066, 74%) were clustered into groups of sequence similarity (>95% identity), resulting in 2489 clusters. The sequence of the longest representative expressed sequence tag (EST) of each cluster was compared with GenBank and TIGR. Scores below 200 were considered unique, and 1114 (44.8%) did not have a match in either database. Sequencing from the 5′ end yielded 12 of 37 useful annotations, suggesting that one third of the 1114 might be identifiable, still leaving over 700 unique ESTs. Virtual Northerns compared between the stages identified numerous genes where expression appears to change from the germinal vesicle oocyte to the four-cell stage, from the four-cell to blastocyst stage, and between in vitro- and in vivo-derived four-cell- and blastocyst-stage embryos. This is the first large-scale sequencing project on early pig embryogenesis and has resulted in the discovery of a large number of genes as well as possible stage-specific expression. Because many of these ESTs appear to not be in the public databases, their addition will be useful for transcriptional profiling experiments conducted on early pig embryos.

Growth hormone receptor, insulin-like growth factor (IGF)-1, and IGF-binding protein-2 expression in the reproductive tissues of early postpartum dairy cows.

The growth hormone/insulin-like growth factor (IGF) system plays a critical endocrine role controlling nutrient metabolism in dairy cattle. In liver, growth hormone receptor (GHR) and IGF-1 are dynamically regulated by lactation and energy balance. Less is known about the regulation of GHR, IGF-1, and IGF-binding protein mRNA in reproductive tissues (uterus, ovarian follicle, and corpus luteum). The objective was to determine expression patterns for GHR, IGF-1, and IGF-binding protein (IGFBP)-2 mRNA in the liver, uterus, dominant follicle, and corpus luteum in Holstein cows (n = 21) sampled at 3 times during early lactation. The first postpartum ovulation was induced with an injection of GnRH within 15 d of calving. Nine days after ovulation [23 +/- 1 d postpartum; 20 d in milk (DIM)], the liver, uterus, dominant follicle, and corpus luteum were biopsied. Prostaglandin F(2alpha) and GnRH were injected 7 and 9 d after each biopsy to synchronize the second (41 +/- 1 d postpartum; 40 DIM) and third (60 +/- 1 d postpartum; 60 DIM) tissue collections. Total RNA was isolated and used for mRNA analysis by real-time quantitative reverse transcription PCR. Liver had more GHR, IGF-1, and IGFBP-2 mRNA than the reproductive tissues that were tested. Gene expression for GHR, IGF-1, and IGFPB-2 within tissues did not change across the sampling interval (20 to 60 DIM). The only detected change in gene expression across days was for cyclophilin in uterus (increased after 20 DIM). Parity had an effect on gene expression for GHR in corpus luteum. Neither level of milk production nor body condition score affected the amount of GHR, IGF-1, or IGFBP-2 mRNA in the respective tissues. The repeatability of gene expression within a tissue was 0.25 to 0.5 for most genes. In most instances, expression of a single gene within a tissue was correlated with other genes in the same tissue but was not correlated with the same gene in a different tissue. We did not find evidence for major changes in gene expression within reproductive tissues in postpartum cows. Differences between cows (independent of their BCS and milk production) accounted for a major portion of the variation that we observed.

The players may change but the game remains: Network analyses of ruminal microbiomes suggest taxonomic differences mask functional similarity

By mapping translated metagenomic reads to a microbial metabolic network, we show that ruminal ecosystems that are rather dissimilar in their taxonomy can be considerably more similar at the metabolic network level. Using a new network bi-partition approach for linking the microbial network to a bovine metabolic network, we observe that these ruminal metabolic networks exhibit properties consistent with distinct metabolic communities producing similar outputs from common inputs. For instance, the closer in network space that a microbial reaction is to a reaction found in the host, the lower will be the variability of its enzyme copy number across hosts. Similarly, these microbial enzymes that are nearby to host nodes are also higher in copy number than are more distant enzymes. Collectively, these results demonstrate a widely expected pattern that, to our knowledge, has not been explicitly demonstrated in microbial communities: namely that there can exist different community metabolic networks that have the same metabolic inputs and outputs but differ in their internal structure.

Effects of dietary aflatoxin on the hepatic expression of apoptosis genes in growing barrows.

The most common and toxic form of aflatoxin, aflatoxin B(1) (AFB(1)), is produced by molds growing on crops. Use of moldy corn can result in high concentrations of AFB(1) in swine diets, which could potentially lead to an increased incidence of aflatoxicosis, a disease associated with decreased health and performance through reduced feed intake, reduced BW gain, and impaired liver function. The objective of this study was to determine the effects of AFB(1) on the hepatic gene expression of growing barrows. Ninety Duroc × Yorkshire crossbred barrows (age = 35 ± 5 d; initial BW = 14.2 ± 3.0 kg) were allocated to 9 pens with 10 pigs per pen, and randomly assigned in a 3 × 3 factorial arrangements of treatments to receive diets containing 0 µg/kg of AFB(1), 250 µg/kg of AFB(1), or 500 µg/kg of AFB(1) for 7, 28, or 70 d. Because performance was most affected in animals administered AFB(1) for an extended period, liver samples from d 70 animals were used for RNA-sequencing analysis. Of 82,744 sequences probed, 179 had transcripts that were highly correlated (r ≥ |0.8|; P < 0.0001) with treatment. Of the 179 significant transcripts, 46 sequences were negatively and 133 sequences positively related to treatment. Forty-three unique functional groups were identified. Genes within the apoptosis regulation functional group were selected for 1) confirmation of d 70 gene expression differences using real-time reverse-transcription (RT)-PCR (n = 4 genes), and 2) investigation of d 7 expression to identify early responses to AFB(1) (n = 15 genes) using real-time RT-PCR. Expression of the 4 apoptosis genes selected for confirmation, cyclin-dependent kinase inhibitor 1A, zinc finger matrin type 3, kininogen 1, and pim-1 oncogene, was confirmed with real-time RT-PCR. Of the 15 genes tested in d 7 liver samples, 4 were differentially expressed: cyclin-dependent kinase inhibitor 1A; zinc finger matrin type 3; tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; and apoptosis enhancing nuclease. Results from this study demonstrate that administration of an AFB(1)-contaminated diet to growing barrows alters hepatic gene expression, and in particular apoptosis genes.

Black Cohosh Does Not Exert an Estrogenic Effect on the Breast

Abstract Womens Health Initiative findings indicate that hormone replacement therapy may increase breast cancer and cardiovascular disease risk. Black cohosh extract (BCE) is a popular alternative that reduced menopausal symptoms in several clinical trials. Preclinical studies have addressed the estrogenic properties of BCE, with conflicting results. The estrogenic influence of BCE on the breast has not been investigated. Black cohosh is standardized to triterpenes, but the activity and mechanism of action of these compounds are unknown. The study goals were to determine 1) triterpene content of 2 commercially available BCE preparations and 2) the effect of BCE on circulating and breast-specific estrogenic markers. Two black cohosh preparations were analyzed for triterpene content. Postmenopausal women took BCE for 12 wk followed by a 12-wk washout. One BCE preparation contained trace amounts and another contained 2.5% triterpenes. Women taking BCE with 2.5% triterpenes experienced relief of menopausal symptoms, with reversion toward baseline after washout. BCE had no effect on estrogenic markers in serum and no effect on pS2 or cellular morphology in nipple aspirate fluid. Triterpene content in commercially available black cohosh preparations varies. BCE standardized to 2.5% triterpenes relieved menopausal symptoms without systemic or breast-specific estrogenic effects.

Factors affecting follicular populations on Day 3 postweaning and interval to ovulation in a commercial sow herd.

Sows (n=146) in a commercial herd were studied to determine factors affecting follicular populations and interval to ovulation after weaning. Ovaries were examined daily by ultrasonography beginning on Day 3 postweaning and twice daily from Day 4.5 until ovulation. Ovarian images were recorded on videotape on Day 3 postweaning and follicles were counted. Subsequent ultrasounds were used to determine time of ovulation. Sows with short weaning to ovulation intervals (<or=6.5 days) had follicular populations on Day 3 postweaning that were more advanced (comprised of follicles with greater diameter) when compared to sows with long (>or=9 days) weaning to ovulation intervals (P<0.001). Follicular populations in sows with intermediate (7-8.5 days) intervals to ovulation were intermediate in diameter when compared to sows with short or long intervals to ovulation. Parity and body condition score (BCS) affected interval to ovulation; first parity and low body condition sows had longer intervals to ovulation (P<0.001 and 0.05, respectively). The longer intervals to ovulation in first parity and low body condition sows were associated with lesser follicular diameters on Day 3 after weaning. We conclude that follicular populations measured by ultrasonography on Day 3 after weaning were different for sows with different intervals to ovulation. Furthermore, production factors (i.e. parity and BCS) known to influence interval to ovulation were associated with differences in follicular growth within the first 3 days after weaning in sows.

Effect of time of ovulation and sperm concentration on fertilization rate in gilts

In normal production practices, sows and gilts are inseminated at least twice during estrus because the timing of ovulation is variable relative to the onset of estrus. The objective of this study was to determine if a normal fertilization rate could be achieved with a single insemination of low sperm number given at a precise interval relative to ovulation. Gilts (n=59) were randomly assigned to one of three treatment groups: low dose (LD; one insemination, 0.5 x 10(9) spermatozoa), high dose (HD; one insemination, 3 x 10(9) spermatozoa) or multiple dose (MD; two inseminations, 3 x 10(9) spermatozoa per insemination). Twice daily estrus detection (06:00 and 18:00 h) was performed using fenceline boar contact and backpressure testing. Transrectal ultrasonography was performed every 6 h beginning at the detection of the onset of standing estrus and continuing until ovulation. Gilts in the LD and HD groups were inseminated 22 h after detection of estrus; MD gilts received inseminations at 10 and 22 h after detection of estrus. Inseminations were administered by using an insemination catheter and semen was deposited into the cervix. The uterus was flushed on Day 5 after the onset of estrus and the number of corpora lutea, oocytes, and embryos were counted. Time of insemination relative to ovulation was designated as 40 to >24 h, 24 to >12 h, and 12 to 0 h before ovulation and >0 h after ovulation. The LD gilts had fewer embryos (P<0.04), more unfertilized oocytes (P<0.05) and a lower fertilization rate (P<0.07) compared to MD gilts. The effects of time of insemination relative to ovulation and the treatment by time interaction were not significant. We conclude that a cervical insemination with low spermatozoa concentration may not result in acceptable fertility even when precisely timed relative to ovulation.

Lifetime productivity in gilts previously selected for decreased age at puberty

A study was conducted to evaluate the effect of decreasing age of puberty on lifetime productivity in sows. Two lines of gilts from the Nebraska Gene Pool population were used in this study: a line that had been selected for decreased age at puberty (AP) and a line in which selection had been random (RS). The study was conducted in two parts. In part one, 75 gilts were mated at second oestrus and the productivity measured over five parities. A second experiment utilizing 68 gilts was conducted to provide further data for comparing litter size at parity 1, and also to compare ovulation rates in the two lines at second oestrus. Results showed that litter size was similar in both lines across parities. After five parities the percentage of sows farrowing relative to parity 1 was 58-8% for the AP line but only 39·4% for the RS line (P = 0·17). Litter birth weight, litter size and weight at 21 days, number weaned, and lactation food consumption were similar for both lines. Lactation weight loss was not significantly different between the two lines (60·9 (s.e. 5·9) v. 527 (s.e. 5·0) kg, for RS and AP gilts, respectively) but was consistent with the slightly longer weaning to remating intervals in the RS line (7·8 (s.e. 0·7) v. 6·6 (s.e. 0·7) days, P = 0·22). Ovulation rate at second oestrus did not differ between the two lines (14·1 (s.e. 0·9) v. 14·3 (s.e. 0·5), for RS and AP gilts, respectively). The regression of mean accumulative productivity on time was in favour of the AP line (P = 0·05). These results suggest that reproductive performance is not impaired in gilts which have been selected to reach puberty at earlier ages, and productivity at a specific age may be enhanced.