The freemartin condition occurs in cattle when a heifer is born twin to a male where the early exposure of testosterone impairs female tract and gonadal development resulting in infertility. Loss of gonadal development and function could have implications in future performance. Over two seasons, growth and carcass traits were compared between freemartins (FM; n = 48) and intact heifers that were born twin to another heifer (HF; n = 21) from a selected population of beef cows selected for twinning at the U.S. Meat Animal Research Center (MARC; Clay Center, NE). Heifers were weaned at an average of 173 days of age and were placed directly into a feedlot. Calves were fed a starter diet, which was gradually adjusted to a high concentrate finishing diet (3.19 Mcal ME/kg DM, 88.29% TDN, and 13.02% CP). Heifers were harvested at an average of 462 days of age at MARC. Data were analyzed using the mixed procedure of SAS with sex and dam age as fixed effects and age as a covariate. Means for weights and carcass traits, with the associated P-values, of FM and HF are presented in Table 031. Weaning, yearling, and harvest weight for FM (184, 409, and 470 kg respectively) and HF (184, 402, and 477 kg respectively) were not different (P > 0.05). Carcass weight (304 vs. 306; P > 0.05), ribeye area (69.8 vs. 72.3; P > 0.05), fat thickness (1.19 vs. 1.16; P > 0.05), and yield grade scores (3.22 vs. 3.07; P > 0.05) were not different between FM and HF, respectively; however, numerical marbling scores tended to be greater (P = 0.082) for freemartins. The data in our study showed that weights and carcass traits were similar for freemartin and intact twin-heifer calves managed equally. LS Means (± SE) of Weights and Carcass Traits of Freemartin and Intact Heifers LS Means (± SE) of Weights and Carcass Traits of Freemartin and Intact Heifers

Programs for developing replacement heifers are designed for heifers to calve at 2 yr of age and to extend their stayability in the herd and minimize feed cost. The experimental objective was to determine whether developing prepubertal heifers on less dietary energy and to a BW of 55% rather than 65% of mature BW at 14 mo of age would compromise ovarian development and reduce fertility. In a 3-yr study, 8-mo-old Angus (n = 60/yr) and composite MARC II (n = 60/yr) heifers were assigned equally by age, BW, and breed to receive either a low (LG) or high (HG) BW gain diet fed to achieve an ADG of either 0.45 or 0.8 kg/d from 8 to 15 mo of age, including the first 21 d of breeding, and then transferred to pasture. At 14 mo, heifers were housed with fertile bulls for 47 d. Estrus was monitored for 21 d. Within 12 h after detection of estrus, ovarian length and height, preovulatory follicle diam., and antral follicle count (AFC) were measured by transrectal ultrasonography. Corpus luteum (CL) volume and plasma progesterone concentration were measured 5 to 15 d after estrus. Data were analyzed by ANOVA with treatment, breed, and year and their 2-way interactions as independent variables. At breeding, HG heifers were heavier than LG heifers (419.9 vs. 361.8 ± 7.5 kg; P < 0.01); ADG for the treatment period was 0.79 vs. 0.47 ± 0.04 kg/d (P < 0.01), respectively. In 2010 and 2011, 97.2% of heifers were cyclic by 21 d of breeding. Size of the ovary, preovulatory follicle, CL, and AFC did not differ between HG and LG, but preovulatory follicle diam. and ovarian length were greater (P ≤ 0.05) for MARC II vs. Angus heifers. Progesterone concentrations were less for LG vs. HG heifers (P ≤ 0.02), whereas CL volume was not affected by treatment or breed but was correlated positively with preovulatory follicle size (P < 0.01). Total AFC ranged from 5 to 49 and was correlated positively with ovarian volume but was not associated with fertility. A greater proportion of HG vs. LG heifers conceived within the first 21 d of the breeding period (64.4% vs. 49.2% ± 3.8%, respectively; P < 0.01), but overall pregnancy rate was not affected by treatment (83.0% vs. 77.7% ± 3.1%, respectively; P > 0.10). Pregnancy rate was 10% less (P < 0.01) for Angus vs. MARC II heifers. Developing beef heifers at a lesser ADG to a lighter BW (55% vs. 64% of mature BW) at breeding did not influence postweaning ovarian development or AFC or compromise pregnancy rate during the 47-d breeding period.

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E2), progesterone (P4), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E2-active (EA) and E2-inactive (EI) follicles were selected according to their E2-to-P4 ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E2 and P4 concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.

A proposed functional polymorphism in the ionotropic glutamate receptor AMPA1 (GRIA1) has been reported to influence antral follicle numbers and fertility in cows. Repeat breeder cows that fail to produce a calf in multiple seasons have been reported to have reduced numbers of small (1 to 3 mm) antral follicles in their ovaries. Therefore, we tested the hypothesis that this GRIA1 polymorphism was affecting antral follicle numbers in repeat breeder cows. Repeat breeder cows (n = 64) and control cows (n = 72) that had always produced a calf were housed in a dry lot and observed twice daily for behavioral estrus. Blood samples were collected, and cows were genotyped for this GRIA1 polymorphism and for a polymorphism in the GnRH receptor (GnRHR) that was proposed to influence age at puberty. On d 3 to 8 after estrus cows were slaughtered, and reproductive organs were collected to determine antral follicle count, ovary size, and uterine horn diameter. Repeat breeder cows were older at first calving than control cows (P = 0.006). The length (P = 0.03) and height (P = 0.02) of the ovary contralateral to the corpus luteum (CL) were greater in control cows than repeat breeder cows. The endometrial diameter in the horn ipsilateral to the CL was greater in the control cows than the repeat breeder cows. Repeat breeder cows had fewer small (1 to 5 mm) antral follicles than control cows (P = 0.003); however, there was no association between GRIA1 genotype and antral follicle number. The GnRHR polymorphism was associated with age at first calving because cows that were homozygous for the C allele had a greater age at first calving than heterozygous cows or cows that were homozygous for the T allele (P = 0.01). In the granulosa cells from small (1 to 5 mm) antral follicles, mRNA abundances of 2 markers of oocyte quality, anti-Müllerian hormone and pentraxin 3, did not differ between fertility groups (P ≥ 0.12). We conclude that this GRIA1 polymorphism exists in beef cows but that it does not influence antral follicle numbers. The association between GnRHR genotype and age at first calving is likely not causal as this polymorphism is not functional. The utility of this polymorphism as a genetic marker for early conception in heifers will require further validation. Screening postpartum cows by ultrasonography to determine antral follicle numbers may aid in making culling decisions.

Cattle genetically selected for twin ovulations and births (Twinner) exhibit increased ovarian follicular development, increased ovulation rate, and greater blood and follicular fluid IGF-1 concentrations compared with contemporary cattle not selected for twins (Control). Experimental objectives were to 1) assess relationships among aromatase (CYP19A1), IGF-1 (IGF1), IGF-2 receptor (IGF2R), and FSH receptor (FSHR) mRNA expression in small (≤5 mm) antral follicles and 2) determine their association with increased numbers of developing follicles in ovaries of Twinner females. Ovaries were collected from mature, cyclic (d 3 to 6) Twinner (n = 11), and Control (n = 12) cows at slaughter and pieces of cortical tissue were fixed and embedded in paraffin. Expression of mRNA was evaluated by in situ hybridization using (35)S-UTP-labeled antisense and sense probes for CYP19A1, FSHR, IGF1, and IGF2R mRNA. Silver grain density was quantified within the granulosa and theca cells of individual follicles (2 to 7 follicles/cow) by Bioquant image analysis. Follicles of Twinners tended to be smaller in diameter than Controls (1.9 ± 0.1 vs. 2.3 ± 0.1 mm; P = 0.08), but thickness of granulosa layer did not differ (P > 0.1) by genotype. Relative abundance of CYP19A1 (P < 0.01) and FSHR (P < 0.05) mRNA was greater in granulosa cells of Twinners vs. Controls, respectively, whereas IGF2R mRNA expression was less in both granulosa (P < 0.01) and theca (P < 0.05) cells in follicles of Twinners vs. Controls, respectively. Abundance of CYP19A1 mRNA in granulosa cells was correlated negatively with IGF2R mRNA expression in both granulosa (r = -0.33; P < 0.01) and theca (r = -0.21; P = 0.05) cells. Expression of IGF1 mRNA was primarily in granulosa cells, including cumulus cells, and its expression did not differ between Twinners vs. Controls (P > 0.10). Detected increases in CYP19A1 and FSHR, but not IGF1, mRNA expression along with decreases in IGF2R mRNA expression in individual follicles of Twinners support the hypothesis that increased follicular development and steroidogenesis in Twinner females result from increased extra-ovarian IGF-1 production. Furthermore, a reduction in follicular IGF2R mRNA expression accompanied by a reduction in receptor numbers would increase availability of free IGF-2 and its stimulation of follicular development in Twinners.

The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus–oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16–27bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P<0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes.

The hedgehog signaling pathway is involved in the regulation of cell proliferation, differentiation, and turnover in a variety of mammalian embryonic and adult tissues including bovine ovarian granulosa and theca cells. Binding of hedgehog to the patch receptor derepresses smoothened resulting in transcription activation or repression. Cattle genetically selected for twin ovulations (Twinner) exhibit a greater number of antral follicles during the estrous cycle than cattle not selected for twins (Control), suggestive of enhanced folliculogenesis in Twinners. The objective was to further assess the relationship among gene expression for Indian hedgehog (IHH), its patch 1 receptor (PTCH1), and aromatase (CYP19A1) during development of small bovine follicles and their contribution to increased follicle numbers in Twinners. Ovaries were collected at 3, 6, or 9 days after estrus by ovariectomy from cyclic Twinner (n = 12) and Control (n = 12) cows. Pieces of cortical tissue were fixed in 4% formaldehyde, dehydrated in ethanol and then xylene, embedded in paraffin, and subsequently sectioned. Abundance of mRNA for IHH, PTCH1, and CYP19A1 was analyzed within individual small (< 5 mm) antral follicles by in situ hybridization using 35S-UTP-labelled bovine-specific antisense and sense probes. Slides were exposed to nuclear track emulsion for 4 wk followed by quantification of silver grain density in 4 areas within the intact granulosa and theca layer of each follicle (2 to 7 follicles/cow) using image analysis system. Antisense minus sense density measurements were averaged for the 4 replicates/follicle, and data were expressed as proportion of measured area occupied by specific pixels. Follicles were categorized as estrogen inactive (E-I) when area occupied by specific pixels for CYP19A1 mRNA was <10%. Expression data were analyzed by analysis of variance; independent variables were genetic line (Twinner vs. Control), estrogen status (E-A vs. E-I), follicle size, (< 1, 2-3, or 4-5 mm), and day of cycle (3, 6, or 9). Detection of IHH mRNA was localized to granulosa and cumulus cells; whereas, PTCH1 mRNA was detected primarily in theca cells of the same follicle. PTCH1 expression was also detected in granulosa cells of some E-I follicles. Abundance of mRNA for IHH was 37-fold greater in E-A compared with E-I follicles; whereas, PTCH1 mRNA was abundant in both E-A and E-I but twofold greater in E-A verses E-I follicles. In E-A follicles, CYP19A1 expression was correlated positively with IHH (r = 0.65) and PTCH1 (r = 0.37) expression. Expression of IHH or PTCH1 mRNA in E-A follicles did not differ between Twinner and Control cows or among days. Abundance of IHH, but not PTCH1, mRNA was twofold greater in E-A follicles >1 mm compared with < 1 mm (E status x size); however, a greater proportion of the 4-5 mm follicles were E-I compared with follicles <4 mm. Observed patterns for IHH and PTCH1 mRNA in granulosa and theca cells further indicate a potential paracrine role for the hedgehog signaling pathway in ovarian follicular development in cattle. Hedgehog signaling does not appear to contribute to increased folliculogenesis in adult Twinner ovaries but may have a role in germ cell proliferation during embryogenesis. USDA is an equal opportunity provider and employer. (poster)

Application of AI in extensive beef cattle production would be facilitated by protocols that effectively synchronize ovarian follicular development and ovulation to enable fixed-time AI (TAI). The objectives were to determine whether use of a controlled internal drug release (CIDR) device to administer progesterone in a GnRH-based estrous synchronization protocol would optimize blood progesterone concentrations, improve synchronization of follicular development and estrus, and increase pregnancy rates to TAI in beef cows. Beef cows (n = 1,240) in 6 locations within the US Meat Animal Research Center received 1 of 2 treatments: 1) an injection of GnRH [100 µg intramuscularly (i.m.)] followed by PGF(2α) (PGF; 25 mg i.m.) 7 d later (CO-Synch), or 2) CO-Synch plus a CIDR during the 7 d between GnRH and PGF injections (CO-Synch + CIDR). Cows received TAI and GnRH (100 µg i.m.) at 60 h after PGF. Progesterone was measured by RIA in blood samples collected 2 wk before and at initiation of treatment (d 0) and at PGF injection (d 7). Estrous behavior was monitored by Estrotect Heat Detectors. Pregnancy was diagnosed by ultrasonography 72 to 77 d after TAI. Plasma progesterone concentrations did not differ (P > 0.10) between synchronization protocols at first GnRH injection (d 0), but progesterone was greater (P < 0.01) at PGF injection (d 7) in cows receiving CO-Synch + CIDR vs. CO-Synch as a result of fewer CIDR-treated cows having progesterone ≤1 ng/mL at PGF (10.7 vs. 29.6%, respectively). A greater (P < 0.01) proportion of CO-Synch + CIDR vs. CO-Synch cows were detected in estrus within 60 h after PGF (66.7 vs. 57.8 ± 2.6%, respectively) and a greater (P < 0.01) proportion were pregnant to TAI (54.6 vs. 44.3 ± 2.6%, respectively). For both synchronization protocols, cows expressing estrus within 60 h before TAI had a greater pregnancy rate than cows without estrus. For cows with plasma progesterone ≤1 ng/mL at PGF injection, CO-Synch + CIDR increased pregnancy rate (65.2 ± 5.9 vs. 30.8 ± 3.4% with vs. without CIDR), whereas pregnancy rates did not differ (P > 0.10) between protocols (52.1 ± 2.1 vs. 50.0 ± 2.4%, respectively) when progesterone was >1 ng/mL (treatment × progesterone; P < 0.01). Inclusion of a CIDR in the synchronization protocol increased plasma progesterone concentration, proportion of cows detected in estrus, and pregnancy rate; however, the increase in pregnancy rate from inclusion of the CIDR was primarily in cows with decreasing or low endogenous progesterone secretion during treatment.

In rodents, disruption of the circadian clock genes results in increased incidence of anovulation, irregular estrous cycles, decreased luteal function, and accelerated reproductive aging. In cattle, reproductive ageing is associated with decreased numbers of follicles in the ovary, decreased luteal function and increased risk of anovulation. The clock gene, Period 1, is expressed in the rodent corpus luteum and may control steroidogenesis. Therefore, we tested the hypotheses that Period 1 mRNA is expressed in the bovine corpus luteum and that luteal Period 1 mRNA levels decrease as part of the reproductive ageing process of cows. Ovaries were collected from young (≤ 6 yrs; n = 6) and old (> 6 yrs; n = 9) crossbred beef cows by colpotomy on days 3, 6, or 9 after estrus. Immediately prior to surgery, a blood sample was collected by caudal venipuncture and processed for determination of serum progesterone concentration by radioimmunoassay. After removal, ovaries were transported to the laboratory where surface follicles were counted and ovaries were weighed. Corpora lutea were dissected, weighed, and frozen in liquid nitrogen. Total cellular RNA was extracted from luteal tissue and diluted to 50 ng/microliter. Relative levels of messenger RNA for Period 1, 3β-hydroxysteroid dehydrogenase (3β-HSD), and GAPDH were determined using previously validated primers in a one-step real-time RT-PCR reaction with 100 ng of tcRNA. Ovarian morphometric data were analyzed using the GLM procedure of SAS with age (young vs. old) as a fixed effect and day of the estrous cycle as a covariate. Relative levels of Period 1 and 3β-HSD mRNA were analyzed using the GLM procedure of SAS with age (young vs. old) as a fixed effect and day of the estrous cycle and relative levels of GAPDH as covariates. There was no difference in antral follicle number between young and old cows (42.0 ± 10.2 vs. 48.5 ± 8.2 follicles, respectively; P = 0.64); however, luteal weight was decreased in old cows (3.5 ± 0.2 vs. 2.8 ± 0.2 g; P = 0.01) and serum progesterone concentrations tended to be decreased in old cows (4.2 ± 0.3 vs. 3.4 ± 0.4 ng/ml; P = 0.1) when compared to young cows. Serum progesterone concentrations were positively associated with luteal weight and day of the estrous cycle (P < 0.0001). Luteal mRNA levels for 3β-HSD did not differ between age groups or days of the estrous cycle. However, luteal Period 1 mRNA levels tended to be greater in young cows than in old cows (823.9 ± 179.8 vs. 302.6 ± 223.4 relative units; P = 0.1) and increased with day of the estrous cycle (P = 0.04). From these data, we conclude that reproductive ageing results in decreased luteal function in cows as observed by a trend for decreased serum progesterone concentrations that agrees with previously published data in women and cows. Decreases in serum progesterone concentrations were not due to decreased levels of 3β-HSD mRNA. Novel findings of this study were that 1) Period 1 mRNA is expressed in the bovine corpus luteum, 2) luteal Period 1 mRNA levels increase during luteal development, and 3) luteal Period 1 mRNA levels tend to decrease with cow age. The exact mechanisms by which circadian clock genes influence steroidogenesis in mammalian corpora lutea remain to be elucidated. (poster)

Long-term genetic selection of cattle for fraternal twins has increased the frequency of twin and triplet ovulations. In contrast, the ratio of fetal numbers to ovulation sites in pregnant females with twin (0.83) or triplet (0.73) ovulations is <1.0 and the number of calves per parturition is 1.6 and 2.0, respectively. Failure of individual twin or triplet ovulations to yield a conceptus in fertile females indicates a significant contribution of ovulation or oocyte anomalies to increased fertilization failure or early embryonic mortality. The present objective was to identify physiological traits affecting conception in cyclic cattle expressing multiple ovulations naturally, including the effect of ovulation rate on follicle or corpus luteum (CL) size, and their relationship to conception. Diameter of the individual ovulatory follicles was measured by transrectal ultrasonography at AI and ranged from 8 to 30 mm, with a trend for diameter of the individual follicles, and associated CL, to decrease with increasing ovulation rate. Independent of ovulation rate, ovulatory follicles were smaller (P < 0.05) for nulliparous heifers (1.5 yr) compared with parous cows (> or =2.5 yr). Pregnancy and fetal status were diagnosed by transrectal ultrasonography between 42 and 72 d after AI. Fertility was reduced (P < 0.01) for small twin or triplet ovulatory follicles (8 to 8.9 mm vs. 10 to 17.9 mm diam.), whereas fertility in monovular females was reduced (P < 0.01) for large ovulatory follicles (> or =22 vs. 14 to 17.9 mm). Plasma progesterone concentrations increased with ovulation rate and were correlated positively with total CL or ovulatory follicle volume per female, indicating that CL size and function were influenced by the size of the follicle of origin. Progesterone was greater (P < 0.05) in the blood of nulliparous heifers compared with parous cows. The increased proportion of small ovulatory follicles associated with twin and triplet ovulations indicates that some ovulatory follicles were either selected to ovulate at a lesser stage of maturity or rescued while undergoing atresia, thus compromising oocyte competency or ovulation. Of greatest importance for reduced fertility was the greater incidence of pregnancy losses occurring in the middle of gestation in females gestating 2 or more fetuses as an apparent effect of uterine crowding, especially when 2 or more fetuses were contained within 1 uterine horn.

Traditional genetic selection in cattle for traits with low heritability, such as reproduction, has had very little success. With the addition of DNA technologies to the genetic selection toolbox for live- stock, the opportunity may exist to improve reproduc- tive efficiency more rapidly in cattle. The US Meat An- imal Research Center Production Efficiency Population has 9,186 twinning and 29,571 ovulation rate records for multiple generations of animals, but a significant number of these animals do not have tissue samples available for DNA genotyping. The objectives of this study were to confirm QTL for twinning and ovulation rate previously found on BTA5 and to evaluate the ability of GenoProb to predict genotypic information in a pedigree containing 16,035 animals when using geno- types for 24 SNP from 3 data sets containing 48, 724, or 2,900 animals. Marker data for 21 microsatellites on BTA5 with 297 to 3,395 animals per marker were used in conjunction with each data set of genotyped ani- mals. Genotypic probabilities for females were used to calculate independent variables for regressions of addi- tive, dominance, and imprinting effects. Genotypic re- gressions were fitted as fixed effects in a 2-trait mixed model analysis by using multiple-trait derivative-free REML. Each SNP was analyzed individually, followed by backward selection fitting all individually significant SNP simultaneously and then removing the least sig- nificant SNP until only significant SNP were left. Five significant SNP associations were detected for twinning rate and 3 were detected for ovulation rate. Two of these SNP, 1 for each trait, were significant for imprint- ing. Additional modeling of paternal and maternal al- lelic effects confirmed the initial results of imprinting done by contrasting heterozygotes. These results are supported by comparative mapping of mouse and hu- man imprinted genes to this region of bovine chromo- some 5.

During late oogenesis, the mammalian oocyte synthesizes and stores mRNA necessary to guide the early stages of embryo development before the activation of embryonic transcription. The oocyte also contains many post-transcriptional regulatory mechanisms that coordinate mRNA stability and translation before specific activation. MicroRNAs (miRNAs) are short noncoding RNAs (17–25 nucleotides) that repress translation of target genes through sequence complementation and have recently been identified in murine oocytes. The objective of the current study was to identify and characterize the expression of miRNAs in bovine cumulus–oocyte complexes (COC) during late oogenesis as a potential mechanism for post-transcriptional regulation of mRNA in developing bovine oocytes. Ovaries from beef cattle (mixed populations) were obtained at a local abattoir. The COC were aspirated from 2- to 10-mm follicles and were pooled from each of 5 replicate collections for RNA extraction (n = 2241 total COC). Small RNA in the 16- to 27-bp range was isolated and used to construct cDNA libraries for sequencing, producing 2529 successful sequences that were clustered based on matching 14 consecutive bases to the most common member of the cluster. The consensus sequences of the clusters were screened for mitochondrial RNA, rRNA, tRNA, and snoRNA contaminants, leading to removal of 774 (31%) sequences from consideration. The remaining 1755 putative miRNA sequences were compared with known miRNA in miRBase, revealing 62 bovine COC miRNA clusters matching previously known sequences and 4 with no match. The cluster with the largest number of sequences identified in bovine COC matched the sequence of the let-7 miRNA family (657 sequences or 37% of putative miRNA). Within the let-7 family, let-7b (459 sequences or 26%) was the most abundant followed by let-7i (135 sequences or 8%). The four clusters that did not match sequences in miRBase represent putative novel miRNA. One of these four clusters had relatively high expression in bovine COCs (308 sequences or 18%), whereas the other 3 clusters had relatively low expression (total of 55 combined sequences or 3%). Expression of several putative miRNAs (let-7b, let-7i, miR-106a, and the abundant novel miRNA) in bovine COC were confirmed using TaqMan miRNA assays. These results demonstrate the presence of miRNA within bovine COC during late oogenesis, which suggests that these post-transcriptional regulatory elements may play a role in coordinating mRNA stability and translation in bovine oocytes.

Traditional genetic selection in cattle for traits with low heritability, such as reproduction, has had very little success. With the addition of DNA technologies to the genetic selection toolbox for livestock, the opportunity may exist to improve reproductive efficiency more rapidly in cattle. The US Meat Animal Research Center Production Efficiency Population has 9,186 twinning and 29,571 ovulation rate records for multiple generations of animals, but a significant number of these animals do not have tissue samples available for DNA genotyping. The objectives of this study were to confirm QTL for twinning and ovulation rate previously found on BTA5 and to evaluate the ability of GenoProb to predict genotypic information in a pedigree containing 16,035 animals when using genotypes for 24 SNP from 3 data sets containing 48, 724, or 2,900 animals. Marker data for 21 microsatellites on BTA5 with 297 to 3,395 animals per marker were used in conjunction with each data set of genotyped animals. Genotypic probabilities for females were used to calculate independent variables for regressions of additive, dominance, and imprinting effects. Genotypic regressions were fitted as fixed effects in a 2-trait mixed model analysis by using multiple-trait derivative-free REML. Each SNP was analyzed individually, followed by backward selection fitting all individually significant SNP simultaneously and then removing the least significant SNP until only significant SNP were left. Five significant SNP associations were detected for twinning rate and 3 were detected for ovulation rate. Two of these SNP, 1 for each trait, were significant for imprinting. Additional modeling of paternal and maternal allelic effects confirmed the initial results of imprinting done by contrasting heterozygotes. These results are supported by comparative mapping of mouse and human imprinted genes to this region of bovine chromosome 5.