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.

Transcription profiling of ovarian follicles. Understanding the mechanisms by which a single follicle is selected for further ovulation is important to control fertility in mammals. However, development of new treatments is limited by our poor understanding of molecular mechanisms regulating follicular selection. Our hypothesis is that genes involved in the control of cell proliferation and apoptosis are differentially regulated during follicular selection. Our objective was to identify these new genes. Bovine follicles were collected and gene expression levels were measured using microarrays. First, follicles were allocated to three groups, according to the time spent from the initiation of follicular wave to surgery (24 H, 36 H, and 48-60 H). Fifty-seven genes are differentially expressed at a false discovery rate of 5%. These genes are involved in the control of lipid metabolism (P-value = 0.0005), cell proliferation (0.007), cell death (0.003), cell morphology (0.003), and immune response (0.003). Follicles were also grouped into four categories, according to the expected time of deviation (early deviation; 8 mm, mid-deviation; 8.5 mm, late deviation; 9 mm, dominant follicles; 10 mm). One hundred and twenty eight genes are differentially expressed between these four groups, including genes involved in cell proliferation (0.00002), cell death (0.0006), cell-to-cell signaling (0.003) cell, morphology (0.003), lipid metabolism (0.0004), and immune response (0.00007). The expression levels of 10 genes were confirmed using quantitative real time PCR. As expected, we identified new differentially regulated genes involved in the control of cell growth and apoptosis. We also discovered a potential role for immune cells, and in particular macrophages, in follicular selection.

Our objective was to determine the accuracy of identifying noncycling lactating dairy cows before the application of a timed artificial insemination (AI) protocol [with or without progesterone supplementation via a controlled internal drug-release (CIDR) insert and 2 different timings of AI] by using heatmount detectors and a single ovarian ultrasound examination. At 6 locations in the Midwest, 1,072 cows were enrolled in a Presynch protocol (2 injections of PGF2α 14 d apart), with the second injection administered 14 d before initiating the Ovsynch protocol (injection of GnRH 7 d before and 48h after PGF2α injection, with timed AI at 0 or 24h after the second GnRH injection). Heatmount detectors were applied to cows just before the first Presynch injection, assessed 14 d later at the second Presynch injection (replaced when activated or missing), and reassessed at initiation of the Ovsynch protocol. Ovaries were examined for the presence of a corpus luteum (CL) by ultrasound before the initiation of treatment. Treatments were assigned to cows based on the presence or absence of a CL detected by ultrasound: 1) no CL + no CIDR; 2) no CL + CIDR insert for 7 d; and 3) CL present. Further, alternate cows within the 3 treatments were assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0h) or 24h later. Pregnancy was diagnosed at 33 and 61 d after the second GnRH injection. By using low (<1 ng/mL) concentrations of progesterone in serum as the standard for noncycling status, heatmount detectors were activated on a large percentage of noncycling cows (>60%), whereas the single ultrasound examination incorrectly classified noncycling cows only 21% of the time. Conversely, cycling cows (progesterone ≥1 ng/mL) were correctly identified 70 to 78% of the time by heatmount detectors, but 85 to 92% were correctly identified by ultrasound. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced the incidence of ovulation but increased the proportions of pregnancies per AI at d 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Percentages of cows pregnant and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL and treated with progesterone. Compared with no response, when a follicle ovulated in response to the first GnRH injection, percentage of cows becoming pregnant after the timed AI increased from 33.3 to 41.6%. Timing of AI at 0 or 24h after the second GnRH injection did not alter pregnancies per AI, but cows having luteal activity before treatment had improved pregnancies per AI compared with noncycling cows. We conclude that identifying noncycling cows by ultrasound was more accurate than by heatmount detectors. Subsequent progesterone treatment of previously cycling cows not having a CL at the onset of Ovsynch increased the proportion of pregnant cows, equal to that of cows having a CL but not treated with progesterone.

Our objective was to determine accuracy of identifying anovulatory lactating dairy cows before the application of a timed AI protocol [with or without progesterone supplementation via a controlled internal drug release (CIDR) insert and 2 different timings of AI] by using heatmount detectors and a single ovarian ultrasound examination. At 6 Midwest locations, 1,072 cows were enrolled in a Presynch protocol (2 injections of prostaglandin F2α(PGF2α) 14 days apart) with the second injection administered 14 days before initiating the Ovsynch protocol (injection of gonadotropin releasing hormone (GnRH) 7 days before and 48 hours after PGF2αinjection, with timed AI at 0 or 24 hours after the second GnRH injection). Heatmount detectors were applied to cows at the time of the first Presynch injection, assessed 14 days later at the second Presynch injection and again at initiation of the Ovsynch protocol, and ovaries were examined for presence of a visible corpus luteum (CL) by ultrasound before initiation of treatment. Treatments were assigned to cows based on presence or absence of a visible CL: 1) anovulatory (no CL + CIDR insert for 7 d); 2) anovulatory (no CL + no CIDR); and 3) cycling (CL present). Further, every other cow in the 3 treatments was assigned to be inseminated concurrent with the second GnRH injection of Ovsynch (0 hour) or 24 hours later. Pregnancy was diagnosed at 33 and 61 days after the second GnRH injection. Heatmount detectors and a single ultrasound examination both underestimated proportions of cows classified as anovulatory or having no prior luteal activity compared with those classifications determined by concentrations of progesterone in blood serum. Overall accuracy of heatmount detectors and ultrasound was 71 and 84%, respectively. Application of progesterone to cows without a CL at the time of the first injection of GnRH reduced incidence of ovulation but improved pregnancy rates at day 33 or 61 compared with nontreated cows without a CL at the onset of the Ovsynch protocol. Pregnancy rates and pregnancy survival did not differ for cows having a CL before treatment compared with those not having a CL but treated with progesterone. Pregnancy rates were 1.5-fold greater for cows ovulating in response to the first GnRH injection. Timing of AI at 0 or 24 hours after the second GnRH injection did not alter pregnancy rates, but cows having prior luteal activity before treatment had improved pregnancy rates compared with anovulatory cows. We conclude that identifying anovulatory cows by ultrasound was more accurate than by heatmount detectors. Subsequent treatment of potential anovulatory cows with progesterone failed to improve fertility but had benefit for cows with prior estrous cycles at the onset of the timed AI (TAI) protocol, regardless of luteal status before the final luteolytic injection of PGF2α.; Dairy Day, 2007, Kansas State University, Manhattan, KS, 2007; Dairy Research, 2007 is known as Dairy Day, 2007

A study was conducted to examine the effects of gonadotropins on ovarian follicular development and differentiation in GnRH agonist (GnRHa)-treated cattle. Holstein cows were allotted into two pre-treatment groups: controls (n = 5) and GnRHa-treated (n = 9). Ovaries were removed from control cows on day 5 following a synchronized estrus. Treatment with GnRHa resulted in follicular arrest at <5 mm. Following follicular arrest, GnRHa-treated cows received a constant infusion of FSH for 96 h (GnRHa/FSH), with a randomly selected subset receiving hourly pulses of LH in addition to FSH during the last 48 h of infusion (GnRHa/FSH + LH). At the end of infusion, ovaries were removed, follicles were counted and measured, and follicular fluid samples were collected from large follicles (>10 mm). Differences in expression of mRNA for LH receptor, FSH receptor, cytochrome P450 side-chain cleavage, 3beta-hydroxysteroid dehydrogenase, cytochrome P450 17alpha-hydroxylase (P450c17) and cytochrome P450 aromatase were determined in large follicles using in situ hybridization. The number of large follicles did not differ between GnRHa/FSH-treated and GnRHa/FSH + LH-treated cows (P = 0.64), but was greater than control animals (P < or = 0.004). Follicular fluid concentrations of estradiol-17beta and androstenedione were highest in GnRHa/FSH + LH-treated cows (P < or = 0.04), intermediate in control cows, and lowest in GnRHa/FSH-treated cows. Hybridization intensity of P450c17 was greater in GnRHa/FSH + LH-treated versus control or GnRHa/FSH-treated cows (P < or = 0.03). These results indicate that while FSH can support bovine follicular growth >10 mm, LH increases androgen production and expression of P450c17.

A study was carried out to measure the growth and germinal tissue responses of young bull calf whose testicles were exposed to different levels of high energy, pulsed beams (X rays). Treatments (absorbed doses) were 0, 1530, 1980, 3060, or 6300 rads. Body weights were measured monthly for 10 mo; testosterone concentrations were measured in mo 2 and 5. At the end of the study, scrotal circumferences were measured, and testes were removed and weighed. Sections of testes were taken, processed, and evaluated for effects on germinal epithelium. Treatments did not affect body weights or weight gains. Testosterone concentrations at mo 5 generally decreased with increased energy dose. Testicular weights were not different among treatments; generally, scrotal circumferences decreased and germinal tissue degeneration increased as the absorbed dose was increased.

The objective was to compare ovarian steroids and expression of mRNAs encoding cytochrome P450 side-chain cleavage, cytochrome P450 17 alpha -hydroxylase, cytochrome P450 aromatase, 3 beta -hydroxysteroid dehydrogenase Delta (4), Delta (5) isomerase, LH, and FSH receptors and estrogen receptor-beta in ovaries of cows with dominant and nondominant ovarian follicular cysts and in normal dominant follicles. Estradiol-17 beta, 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-17 beta (ng/ml) and total steroids (ng/follicle) were higher in dominant cysts than in dominant follicles. Expression of LH receptor and 3 beta -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-17 beta concentrations, and lower expression of steroidogenic enzyme, gonadotropin receptor, and estrogen receptor-beta mRNAs than other groups. In summary, increased expression of LH receptor and 3 beta -hydroxysteroid dehydrogenase mRNAs in granulosa and increased follicular estradiol-17 beta 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.

The objectives were to compare expression of mRNA for cytochrome P450 cholesterol side-chain cleavage (P450scc), cytochrome P450 17α-hydroxylase (P450c17), cytochrome P450 aromatase (P450arom), 3β-hydroxysteroid dehydrogenase Δ4, Δ5 isomerase (3β-HSD), FSH receptor (FSHr) and LH receptor (LHr) in bovine ovarian follicles of the first and second waves of the bovine oestrous cycle and to determine if LH infusion changes growth, steroidogenesis and gene expression in second wave follicles. Transrectal ultrasonography was used to examine follicular size changes during the oestrous cycle in non-lactating Holstein cows (n=31). Saline or purified bovine LH was infused intravenously into cows at emergence of follicular waves for 2 or 4 days using a computer-controlled syringe pump (n=5–6 per treatment). Treatments were: wave 1, saline (W1S); wave 2, saline (W2S) or LH (25μg/h; W2LH). During infusion, blood samples were collected at 12min intervals for 8h via i.v. catheters for measurement of serum LH concentrations. Ovaries were removed from cows on days 2 or 4 after emergence of follicular waves. Follicles were frozen and stored at −80°C. Follicular fluid (FF, 50μl) was collected for determination of progesterone (P4), oestradiol-17β (E2) and androstenedione (A4) concentrations. Frozen sections (14μm) were used for in situ hybridization to measure expression of mRNA (% pixel intensity) for P450scc, P450c17, P450arom, 3β-HSD, FSHr, and LHr. LH infusion resulted in a serum LH pattern (high frequency) similar to the early luteal phase. There were no significant differences in size of follicles among the three treatment groups. Follicular fluid concentrations of E2 and A4 in W2S were lower than those of W1S on day 2 of a follicular wave. LH infusion into cows during the midluteal phase increased follicular fluid E2 and A4 concentrations in second wave follicles on day 2 of a follicular wave (W2LH) compared to those of W2S. The increase in follicular fluid E2 on day 2 in wave 2 follicles after LH infusion occurred possibly through an increase in mRNA expression of P450c17 and 3β-HSD. In conclusion, follicular fluid concentrations of E2 and A4 were lower in W2S than in W1S and E2 and A4 concentrations were restored by infusion of LH in W2LH with an increase in mRNA expression of P450c17 and 3β-HSD.

In a previous study, the ERbeta cDNA protein-coding region was utilised to clone bovine ERbeta. The objectives in this study were to examine (1) ERbeta mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERbeta mRNA expression during the second follicular wave. In experiment 1, heifers (4-5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows (n = 31; 5-6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 microg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2-19mm in size, were dissected, frozen, and stored at -80 degrees C for in situ hybridisation with two bERbeta cRNA probes. Expression of bERbeta mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERbeta mRNA expression did not change with time points of the wave showing no association of bERbeta mRNA expression with follicular selection and dominance. However, bERbeta mRNA expression decreased with increase in size of all follicles. Expression of bERbeta mRNA was greater in very small follicles (2-4 mm) than in large (> or = 9 mm) follicles. In experiment 2, expression of bERbeta mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERbeta mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERbeta mRNA expression.

Endometrial periglandular fibrosis (EPF) has been proposed as a possible aetiology for equine embryonic and fetal loss. However, the pathophysiology of EPF is not well understood. Angiotensin-converting enzyme (ACE) is found in macrophages, endothelium (during angiogenesis) and myofibroblasts at sites of fibrosis in the heart, kidneys, liver and skin in several species. An increase in local tissue ACE-binding activity appears to be a critical event in the initiation and progression of fibrosis in these tissues. The aim of this study was to investigate the correlation between ACE activity in the equine endometrium and the degree of EPF, as determined by histological evaluation and morphometry based on a collagen-specific stain. ACE-binding activity values were significantly higher in the endometrial samples with moderate EPF (modified Kenney EPF category IIB) compared with endometria in all other categories. Ultrastructurally, the fibroblasts surrounding the glandular basal laminae in modified Kenney EPF category IIB and III endometria were undergoing myofibroblastic transformation-like changes. These observations indicate a possible link between ACE activity and the onset of EPF in mares.

Cows with ovarian follicular cysts were treated with progesterone to determine whether a reduction in LH concentrations and initiation of ovulatory follicular waves would occur. Cysts were diagnosed using transrectal ultrasonography when single follicular structures > 20 mm or multiple structures > 15 mm in diameter were present for 7 d in the presence of low progesterone concentrations. Three groups were studied: 1) cows with normal estrous cycles (CYC, n = 8); 2) cows with untreated cysts (CYST, n = 7); and 3) cows with cysts treated with two progesterone-releasing intravaginal devices (PRID, n = 8) for 9 d. Ovaries were examined with transrectal ultrasonography, and blood samples were collected daily for analysis of progesterone and FSH. Serial blood samples for determination of mean LH and LH pulse frequency were collected on d 0 (CYST and PRID cows only), 1, 5, 9, and 10. Progesterone concentrations were higher in PRID cows than in CYST cows throughout the PRID treatment period (P < .002). On d 0, LH pulse frequency was similar (P = .10) in PRID (6.6 +/- .6 pulses/8 h) and CYST cows (5.1 +/- .6 pulses/8 h), but mean LH tended to be higher (P = .054) on d 0 in PRID cows (2.5 +/- .2 ng/mL) than in CYST cows (1.9 +/- .2 ng/mL). Mean LH and LH pulse frequency decreased (P < .002) by d 1 in PRID cows (1.1 +/- .2 ng/mL, 1.8 +/- .6 pulses/8 h) compared with CYST cows (2.1 +/- .2 ng/mL, 5.6 +/- .6 pulses/8 h) and remained lower throughout most of the experimental period. The FSH concentrations were higher (P < .01) in PRID cows than;in CYC and CYST cows on d 3 and 4. The increase in FSH concentrations preceded emergence of the PRID-induced follicular wave. All PRID cows and four of seven CYST cows initiated new follicular waves during the period of PRID treatment. Follicular waves were initiated later (P < .05) in CYST cows (d 5.2 +/- 1.7) and PRID cows (d 5.5 +/- .6) than in CYC cows (d 1.8 +/- .3). Cysts were smaller (P < .01) at the end of the treatment period in PRID cows compared with CYST cows. No CYST cows ovulated, but all PRID cows ovulated newly developed follicles 3 or 4 d after PRID removal. Treatment with exogenous progesterone reduced LH in cows with cysts, and this was followed by development of normal ovulatory follicles.

Blood and uterine concentrations of GH and insulin-like growth factor (IGF)-I are correlated with improved fertility in cattle. We tested incremental doses of a 14-d sustained release recombinant bovine GH (rbGH) to increase blood GH and IGF-I (Experiments 1 and 2). Conception rate after administration of an optimized rbGH dose was also tested (Experiment 3). In Experiment 1, lactating Holstein cows (n = 18) were randomly assigned to receive 0 (n = 5), 100 (n = 5), 200 (n = 5), or 500 (n = 3) mg sc rbGH. Increasing the doses of rbGH was associated with increased serum concentrations of GH and IGF-I. The 100- and 200-mg doses caused an IGF-I release that was below and above, respectively, the perceived optimum response. Therefore, Experiment 2 was designed to test a rbGH dose (167 mg), which was intermediate to the doses tested in Experiment 1. Lactating and nonlactating postpartum beef cows were treated with 0 (n = 9) or 167 (n = 9) mg rbGH at insemination. Plasma concentrations of GH and IGF-I were greater in rbGH-treated cows than in controls. Lactating cows had initial IGF-I concentrations that were lower than nonlactating cows. The 167-mg dose of rbGH increased plasma IGF-I concentrations in lactating cows to the levels of those of nonlactating cows. In Experiment 3, cows and heifers were administered either 0 or 167 mg rbGH at insemination. The conception rate for rbGH- treated and control cows was 54.4 and 49.5% (n = 617), and 46.0 and 46.3% for heifers (n = 1123), respectively. Herd (P < 0.01) and parity (P < 0.01) affected conception rate, but conception rates for rbGH and control cattle were similar. In summary, low doses of rbGH increased blood GH and restored blood IGF-I concentrations in lactating cows to those of nonlactating cows, but the conception rate in cows and heifers was not affected by administration of 14-d sustained-release rbGH at insemination.

OBJECTIVES To develop an objective, quantifiable assay for endometrial periglandular fibrosis (EPF) and correlate assay results with histologic and ultrastructural changes in equine endometrial biopsy specimens. SAMPLE POPULATION Endometrial biopsy specimens from 70 mares from 3 to 27 years old in estrus. PROCEDURE In a double-blinded study design, endometrial biopsy specimens were graded histologically (modified Kenney classification) for EPF and inflammation. Endometrial periglandular collagen volume fraction (%EPCVF) was determined by light microscopic image analysis of picrosirius red-stained sections. Specimens from selected mares were examined by transmission electron microscopy. RESULTS %EPCVF values varied significantly among the 4 modified Kenney EPF categories (I, IIA, IIB, and III) and increased with increasing age of mares. Morphologically, EPF consisted of concentric layers of transformed fibroblasts with myofibroblastic features and deposition of fibrillar collagen around unaltered glandular basal laminae. CONCLUSIONS AND CLINICAL RELEVANCE %EPCVF correlates well with morphologic changes in endometrial biopsy specimens. Determination of %EPCVF could be useful in evaluation and clinical management of subfertile mares and in investigations of the pathogenesis of EPF.

A decrease in insulin-like growth factor (IGF) binding protein (BP) amount occurs within the follicular fluid of dominant ovarian follicles. At the same time, concentrations of follicular fluid IGF-I do not change. The mRNA for IGF-I, IGF-II, IGFBP-2, and IGFBP-3 in dominant and subordinate follicles were measured to determine if changes in IGF or IGFBP gene expression are associated with follicular dominance. Heifers were ovariectomized during a follicular wave, either during early-dominance (emerging dominant follicle, 9 mm diameter) or mid-dominance (established dominant follicle, 14–16 mm diameter). Follicles were classified as either dominant (DF), subordinate (SF), or not-recruited (NRF; small antral follicles). mRNA was localized by in situ hybridization and measured by image analyses. The IGF-I mRNA (granulosa cells) was greatest in DF and increased in DF, SF, and NRF from early- to mid-dominance. Likewise, IGF-II mRNA (theca cells) was greatest in DF compared with SF or NRF. The IGFBP-2 mRNA (granulosa cells), however, was nearly undetectable in DF, whereas adjacent SF expressed abundant IGFBP-2 mRNA. The NRF were not uniform in their IGFBP-2 expression because only 5 of 13 NRF had IGFBP-2 mRNA. The IGFBP-3 mRNA (granulosa cells) was found only in two NRF, suggesting that local synthesis is not a predominant source of follicular fluid IGFBP-3. These data show that changes in gene expression for IGFBP-2 are opposite to those for IGF-I or IGF-II. Increased IGF-I and IGF-II mRNA and decreased IGFBP-2 mRNA within the DF may be one mechanism leading to follicular dominance. The opposite pattern of IGFBP-2 gene expression in SF and some NRF may lead to follicular atresia.

Expression of mRNA encoding steroidogenic acute regulatory protein (StAR) in bovine follicles during recruitment and selection was examined. Dairy heifers (4-5/time period) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave (Time 0) after estrus. Follicles were collected and stored at -80 degrees C until sectioning. Expression of StAR mRNA was localized by in situ hybridization and quantified by image analysis. Expression of StAR mRNA was first detected in theca interna of antral follicles as small as 0.5 mm in diameter and increased with increasing follicular size (>/= 4 mm; r = 0.75; p < 0.001). StAR mRNA was undetectable in granulosa of healthy follicles at any size or stage of follicular wave examined. However, granulosa or luteinized granulosa of some advanced or late atretic follicles expressed StAR mRNA. During recruitment, StAR mRNA expression in theca cells was similar among recruited follicles (4-8 mm). During selection of dominant follicles (36-48 h), StAR mRNA was expressed in theca of more than one follicle (7-9 mm); therefore, expression of StAR mRNA may not be associated with dominant follicle selection. StAR mRNA in theca was higher (p < 0.05) at 48 h after initiation of the first follicular wave than at 12, 24, and 36 h, and it remained elevated thereafter through 96 h. Dominant follicles expressed more (p < 0.01) StAR mRNA in theca than did subordinate healthy follicles. Healthy follicles expressed higher (p < 0.05) StAR mRNA in theca than atretic follicles. In summary, levels of StAR mRNA increased in theca with stage of follicular wave and size of follicles. Follicular atresia was associated with reduced expression of StAR mRNA in theca cells. The results indicate that expression of StAR mRNA in theca may not be the primary limiting factor for follicular recruitment and selection.

The objective of this study was to investigate changes in expression of mRNAs encoding FSH receptor (FSHr), LH receptor (LHr), cytochrome P450 side-chain cleavage (P450(scc)), cytochrome P450 17 alpha-hydroxylase (P450(c17)), and cytochrome P450 aromatase (P450(arom)) during recruitment and selection of bovine ovarian follicles. Dairy heifers (4-5 per group) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, or 96 h after initiation of the first follicular wave following estrus as determined by ultrasonography (Time 0 = initiation of follicular wave; mean +/- SEM = 42.0 +/- 2.6 h after estrus). Expression of mRNAs encoding FSHr, LHr, P450(scc), P450(c17), and P450(arom) was detected by in situ hybridization and quantified by image analysis. Antral follicles were classified as healthy or atretic, Healthy follicles expressed higher (p < 0.01) amounts of mRNAs for gonadotropin receptors and steroidogenic enzymes than did atretic follicles, and expression of LHr, FSHr, P450(scc), P450(c17), and P450(arom) increased (p < 0.01) with follicular size and stage of the follicular wave. Expression of mRNAs for P450(scc), P450(arom), and LHr was time- and size-dependent during recruitment and selection. During recruitment, expression of mRNAs for P450(scc) and P450(arom) was first detected in granulosa cells of 16 of 21 of the follicles 4-6 mm in diameter at 12 h. At 24 and 36 h, almost all follicles 6-9 mm in diameter, but not those 4-5 mm in diameter, expressed both P450(scc) and P450(arom) mRNA in the granulosa cells. At 48 h and thereafter, P450(scc) and P450(arom) mRNA were expressed predominantly in one healthy large follicle per cow with a few exceptions. Expression of LHr mRNA was first detected in granulosa cells at 36 h and was always found in granulosa cells of one follicle greater than or equal to 8 mm per cow with exception of one cow at 36 h (no expression) and another two cows, one each at 36 and at 84 h (expression in 2 follicles). In addition, LHr mRNA expression in the granulosa cell layer was limited to follicles that also expressed mRNAs for P450(scc) and P450(arom) in the granulosa cells. In summary, follicular recruitment in cattle was associated with expression of P450(scc) and P450(arom) mRNA within granulosa cells, and the process of follicular selection was associated with initiation of LHr mRNA expression in granulosa cells.