Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births.

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.

Effect of postweaning diet on ovarian development and fertility in replacement beef 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.

Identification of an ionotropic glutamate receptor AMPA1/GRIA1 polymorphism in crossbred beef cows differing in fertility.

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.

Increased abundance of aromatase and follicle stimulating hormone receptor mRNA and decreased insulin-like growth factor-2 receptor mRNA in small ovarian follicles of cattle selected for twin births.

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.

MicroRNA expression profile in bovine cumulus-oocyte complexes: possible role of let-7 and miR-106a in the development of bovine oocytes.

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 16-27 bp 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.

Factors affecting pregnancy rate to estrous synchronization and fixed-time artificial insemination in beef cattle.

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.

Size of ovulatory follicles in cattle expressing multiple ovulations naturally and its influence on corpus luteum development and fertility.

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.

Confirmation of quantitative trait loci using a low-density single nucleotide polymorphism map for twinning and ovulation rate on bovine chromosome 5.

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.

Increased calf production in cattle selected for twin ovulations.

The effects of increasing fetal numbers and their distribution between the left and right uterine horns on calf survival, calf BW at birth and weaning, gestation length, dystocia, and calf sex ratio were evaluated for single (n = 1,587), twin (n = 2,440), and triplet calves (n = 147) born to primiparous and multiparous females in the Twinner population at the US Meat Animal Research Center between 1994 and 2004. Cattle were distributed equally between the spring and fall breeding seasons. Fetal number and distribution in utero were determined by real-time ultrasonography at 40 to 70 d postbreeding. For cows and heifers combined, number of calves per parturition increased from 1.34 in 1994 to 1.56 in 2004. Gestation length was 6.8 d shorter (P < 0.01) for twins compared with singles (277.5 +/- 0.2 vs. 284.3 +/- 0.2 d) and 12.7 d shorter for triplets (271.6 +/- 0.8 d). Survival and BW of individual calves at birth decreased (P < 0.01) but total calf BW per dam increased (P < 0.01) as fetal number increased from single to triplet births. Twins resulting from bilateral twin ovulations had increased (P < 0.01) survival and BW at birth, a longer (P < 0.01) gestation length, and less (P < 0.01) dystocia than twins resulting from unilateral twin ovulations. Calf survival and BW at birth were 97.2 +/- 0.3% and 48.0 +/- 0.1 kg for singles, 92.0 +/- 0.4% and 39.0 +/- 0.2 kg for bilateral twins, 83.2 +/- 0.4% and 36.7 +/- 0.2 kg for unilateral twins, 73.8 +/- 1.4% and 30.6 +/- 0.7 kg for bilateral triplets, and 51.9 +/- 3.2% and 31.7 +/- 1.6 kg for unilateral triplets. Birth weight of single calves increased by 0.51 kg/d for each additional day of gestation length vs. 0.38 kg/d for individual twins. Calf BW at birth increased (P < 0.01) with age of dam from 2 to 4 yr. Twin and triplet births had a greater (P < 0.01) incidence of dystocia than single births. The ratio of male:female calves (0.52:0.48) at birth was not affected by type of birth. Postnatal calf survival was similar for all 3 types of birth. Total progeny BW at weaning for single, twin, and triplet births was 217.7 +/- 2.5, 328.3 +/- 3.2, and 378.4 +/- 15.0 kg, respectively (P < 0.01). Although most bovine females have the uterine capacity to gestate twin calves, decreased survival and BW of unilateral twins and of all triplets indicate that their growth and development may have been compromised by uterine crowding.

Effects of ovulation rate and fetal number on fertility in twin-producing cattle.

Effects of ovulation rate and of fetal number and distribution within the uterus on pregnancy rate and fetal survival were evaluated in nulliparous (n = 1,331) and parous (n = 3,517) cattle selected for twinning. Cattle were divided into a spring (70 d) and fall (60 d) breeding season and bred by a combination of AI and natural service. Ovulation rate, pregnancy status, and fetal number and distribution were determined by transrectal, real-time ultrasonography of the uterus and both ovaries at the end of the breeding season. Pregnancy was reconfirmed by rectal palpation at 75 to 135 d of gestation. For heifers and cows combined, ovulation rate increased (P < 0.01) from 1.46 +/- 0.4 in 1994 to 1.89 +/- 0.4 in 2004; number of calves per parturition increased (P < 0.01) from 1.34 +/- 0.3 to 1.56 +/- 0.3, respectively, which included an increase in triplet and quadruplet ovulations and triplet births. Bilateral twin ovulations yielded proportionately more (P < 0.01) twin births than unilateral twin ovulations. Ovulation rate was greater (P < 0.01) in the fall than spring breeding season. Pregnancy rate at ultrasound diagnosis did not differ among females with 1, 2, or 3 ovulations (89.1 +/- 0.7, 91.2 +/- 0.7, or 91.5 +/- 2.8%, respectively), but rates at calving decreased (P < 0.01) with increasing ovulation rate (85.1 +/- 0.6, 82.7 +/- 0.6, or 64.2 +/- 2.7%, respectively). Pregnancy rate was less (P < 0.01) after twin or triplet births than single births. For dams birthing twins or triplets, pregnancy rate was less in the fall vs. spring, but rates were similar between seasons for dams with a single birth (type of birth x season, P < 0.05). Cows 60 d, regardless of type of birth. Maintenance of pregnancy to term differed (P < 0.01) among females diagnosed with 1, 2, or 3 fetuses (95.7 +/- 0.6, 87.8 +/- 0.8, and 54.9 +/- 2.3%, respectively). The reduced survival of twin and triplet fetuses in heifers had occurred (P < 0.01) by d 75 to 135 of gestation, and fetal losses were greater (P < 0.01) for unilateral than bilateral twins or triplets, whereas loss of twin or triplet fetuses in cows occurred later in gestation, and losses were not affected by uterine location. Thus, increased calf production from selecting for increased ovulation rate in beef cattle is tempered by increased fetal mortality, partially associated with the crowding of 2 or 3 fetuses within 1 uterine horn, especially in heifers.

Localization of Period 1 mRNA in the ruminant oocyte and investigations of its role in ovarian function.

The clock gene Period 1 (Per1) may be a prolificacy gene, because it localized to the mouse oocyte and Per1-null drosophila shed fewer eggs. Because Per1 mapped to a region of mouse chromosome 11 syntenic to bovine chromosome 19 where a quantitative trait loci (QTL) for ovulation rate existed, we hypothesized that Per1 influenced folliculogenesis and ovulation rate in ruminants. Ovarian cortex was collected at slaughter on days 5, 12, 15, 17, and 20 after estrus for real-time RT-PCR evaluation of Per1 mRNA expression in Dorset (n = 18), Romanov (n = 10), Romanov/Dorset (n = 21), and Composite (n = 22) ewes. Ovarian cortex was also collected from cows selected for increased ovulation rate (n=37) or unselected controls (n = 28) on days 4, 5, and 6 of the estrous cycle for in situ hybridization and real-time RT-PCR. To examine the role of Per1 in early follicular development, ovarian cortex from neonatal calves (n = 5) was cultured for 10 days and Per1 mRNA levels were measured on day 0 and on day 10 of culture. The primers generated a 483bp amplicon with 100% sequence homology to bovine RIGUI-like protein (Per1). In silico mapping of this sequence placed Per1 on bovine chromosome 19; however, it was 20cM from the QTL. Per1 mRNA expression was unaffected by prolificacy, day of the cycle, or pregnancy status in ewes or cows. The riboprobe hybridized to oocytes of bovine preantral and antral follicles. In bovine ovarian cortical cultures on day 0, the tissue contained mostly primordial follicles (5.6+/-0.6 follicles/section); however, after 10 days in culture, the number of primordial follicles per section decreased (0.5 follicles/section) and the number of primary follicles increased as follicles activated (day 0 = 0.5+/- 0.6 versus day 10 = 10.4 +/-0.6 primary follicles/section; P < 0.001). Per1 mRNA did not change over time in culture. We conclude that Per1 mRNA is expressed by ruminant oocytes in preantral and antral follicles; however, its physiological role in mammalian ovarian function remains to be elucidated.

Association of a single nucleotide polymorphism in SPP1 with growth traits and twinning in a cattle population selected for twinning rate.

Continued validation of genetic markers for economically important traits is crucial to establishing marker-assisted selection as a tool in the cattle industry. The objective of the current study was to evaluate the association of a SNP (T(9)/T(10)) in the osteopontin gene (SPP1) with growth rate in a large cattle population spanning multiple generations and representing alleles from 12 founding breeds. This population has been maintained at the US Meat Animal Research Center since 1981 and subjected to selection for twinning rate. Phenotypic records for this population included twinning rate and ovulation rate, providing an opportunity to examine the potential effects of SPP1 genotype on reproductive traits. A set of 2,701 animals was geno-typed for the T(9)/T(10) polymorphism at SPP1. The geno-typic data, including previously genotyped markers on chromosome 6 (BTA6), were used in conjunction with pedigree information to estimate genotypic probabilities for all 14,714 animals with phenotypic records. The genotypic probabilities for females were used to calculate independent variables for regressions of additive, dominance, and imprinting effects. Genotypic regressions were fit as fixed effects in a mixed model analysis, in which each trait was analyzed in a 2-trait model where single births were treated as a separate trait from twin births. The association of the SPP1 marker with birth weight (P < 0.006), weaning weight (P < 0.007), and yearling weight (P < 0.003) was consistent with the previously reported effects of SPP1 genotype on yearling weight. Our data supports the conclusion that the SNP successfully tracks functional alleles affecting growth in cattle. The previously undetected effect of the SNP on birth and weaning weight suggests this particular SPP1 marker may explain a portion of the phenotypic variance explained by QTL for birth and HCW on BTA6.

Large variation in steroid concentrations and insulin-like growth factor binding proteins exists among individual small antral follicles collected from within cows at random stages of the estrous cycle.

Variation in the biochemical status of individual small (< or = 5 mm diameter) antral follicles within the ovaries of a cow at any given time likely influences the capacity for undergoing recruitment, selection, and establishing dominance. The objectives of this study were to provide insight into the magnitude of variation in follicular fluid concentrations of steroids and activities of IGFBP that exists among individual small antral follicles within and between cows, and to determine the relationships between follicular fluid IGFBP and steroid concentrations in these follicles. A total of 108 small antral follicles were collected from 6 cows at random stages of the estrous cycle, with 10 to 26 follicles/cow. Concentrations of steroids (ng/mL of follicular fluid) in the overall population of follicles ranged from 0.1 (lowest detectable limit) to 51 for estradiol (E2), 4 to 1,149 for progesterone (P4), and 5 to 504 for androstenedione (A4). Concentrations of E2 and A4 were associated positively (r = 0.2; P < 0.02), but E2 (r = -0.4) and A4 (r = -0.4) were associated negatively, with P4. The proportion of variation in steroid concentrations accounted for by differences among animals (P < 0.05) was small for E2 (12%), moderate for P4 (43%), and greatest for A4 (74%). Least differences between minimum and maximum concentrations of steroids observed in follicles from within a cow were 21-, 5.5-, and 3.5-fold for E2, P4, and A4, respectively, whereas the greatest differences between minimum and maximum concentrations were 505-, 108-, and 26-fold for E2, P4, and A4, respectively. Ranges of IGFBP concentrations (arbitrary densitometer units) detected in fluid from a sub-sample of 43 follicles were 1.18 to 4.50 for IGFBP-3, 0.54 to 4.68 for IGFBP-2, 0.07 to 2.56 for IGFBP-4, and 0.01 to 6.71 for IGFBP-5. Concentrations of E2 were correlated negatively with each IGFBP (r = -0.4 to -0.8; P < 0.05) except IGFBP-3. In contrast, concentrations of A4 were correlated positively with IGFBP-3 (r = 0.4; P < 0.05) but were not correlated with other IGFBP. Concentrations of P4 were correlated positively (r > 0.4; P < 0.05) with IGFBP-4 and -5. The results indicate that steroid concentrations and IGFBP activities vary substantially among small antral follicles collected from within and among individual animals and that increasing production of E2, the hallmark of a developing follicle, was associated with reduced activity of all IGFBP except IGFBP-3, thereby implicating these IGFBP in the regulation of follicular recruitment.

Increased vascular endothelial growth factor and pregnancy-associated glycoproteins, but not insulin-like growth factor-I, in maternal blood of cows gestating twin fetuses.

Differences in placental mass and vascularity exist between cows gestating single vs. multiple fetuses. Therefore, the association between fetal number and placental development or function was assessed by comparing concentrations of vascular endothelial growth factor (VEGF), pregnancy-associated glycoproteins (PAG), IGF-I, and progesterone in the maternal blood of cattle selected for twin births and gestating 1 (n = 23) vs. 2 (n = 17) fetuses. Samples of jugular venous blood were collected serially at a mean of 57, 121, 192, and 234 d (range within groups was 20 d) after AI. Plasma concentrations of VEGF, IGF-I, and progesterone were measured by double-antibody RIA, and of PAG by an indirect sandwich ELISA. Concentrations of VEGF and progesterone were greater (P < 0.05) in dams with twin vs. single fetuses. Maternal VEGF concentrations did not differ among collection times, but progesterone concentrations increased (P < 0.01) between d 192 and 234. Conversely, PAG concentrations were low at d 57 and 121 and did not differ between dams carrying singles or twins. However, the subsequent increase (P < 0.01) in PAG was greater in dams with twins, resulting in greater (P < 0.01) PAG concentrations for dams with twins at d 192 and 234 (type of birth x time; P < 0.01). Maternal IGF-I concentrations were unaffected by fetal number. Because corpora lutea persisted for the duration of the evaluation period, maternal progesterone concentrations were likely related to the number of corpora lutea rather than the number of fetuses. It is postulated that the greater PAG and VEGF concentrations in the blood of dams gestating twins are the result of a larger uteroplacental mass, including increased numbers of binucleate cells and increased angiogenesis and vasculogenesis associated with a twin pregnancy. Although PAG and VEGF were elevated in dams gestating twins, variability within and among birth groups limits the use of PAG or VEGF measurements for the diagnosis of twins.

Evaluation of ovulation rate and ovarian phenotype in puberal heifers from a cattle population selected for increased ovulation rate.

Long-term selection for increased ovulation rate (1984 to 2002) has resulted in a unique ovarian phenotype in the MARC Twinner cattle population. Ovulation rate and frequency of bilateral ovulations were examined by rectal palpation in 29,547 estrous cycles for 3,910 heifers (12 to 18 mo of age) in this population. Bilateral ovulations (one corpus lutuem [CL] on each ovary) were of interest because bilateral twin pregnancies result in decreased dystocia and increased calf survival. Ovulation rate increased linearly at a rate of 0.026 CL per year, and it currently averages 1.48 +/- 0.04 CL per estrous cycle. Concurrent with the increase in ovulation rate, the frequency of triplet ovulations increased from 0% to 2.3 +/- 0.8% (P < 0.001). Ovulation rate of both the right and left ovary increased equally at a rate of 0.013 CL per year, and mean ovulation rate of the right ovary remained greater than mean ovulation rate of the left ovary throughout the study (0.66 vs. 0.55 +/- 0.003 CL per estrous cycle; P < 0.001). Although correlations were low, ovulation rate of one ovary was negatively correlated (P < 0.001; r = -0.07) with ovulation rate of the same ovary in the previous estrous cycle, but positively correlated (P < 0.001; r = 0.13) with the contralateral ovary of the previous estrous cycle. The proportion of bilateral ovulations averaged 55.7 +/- 0.7%, a value greater than the predicted 49.5% (P < 0.001). In addition to dystocia and retained placenta, triplet pregnancies increase the incidence of pregnancies gestating fetuses of opposite sexes and subsequent incidence of freemartins; thus, selection pressure on ovulation rate may need to be adjusted in the MARC Twinner population. The proportion of bilateral ovulations in the population is greater than expected, and this may be an economically important trait, which will respond to selection and be beneficial for improving bovine reproductive efficiency. Understanding factors controlling the increased functional activity of the right ovary and bilateral ovulations may provide further insights into the mechanisms controlling follicle selection and methods to improve reproductive management of cattle.

Ovarian follicular development in cattle selected for twin ovulations and births.

Comparisons of numbers of antral ovarian follicles and corpora lutea (CL), of blood hormone concentrations, and of follicular fluid steroid concentrations and IGFBP activity were conducted between cows selected (twinner) and unselected (control) for twin births to elucidate genetic differences in the regulation of ovarian follicular development. Ovarian follicular development was synchronized among cows by a single i.m. injection of PGF2alpha on d 18 of the estrous cycle; six cows per population were slaughtered at 0, 24, 48, and 72 h after PGF2alpha. Jugular vein blood was collected from each animal at PGF2alpha injection and at 24-h intervals until slaughter. Ovaries of twinner cows contained more small (< or = 5 mm in diameter, P < 0.05), medium (5.1 to 9.9 mm, P < 0.05), and large (> or = 10.0 mm, P < 0.01) follicles and more (P < 0.01) CL than ovaries of controls. Follicular fluid concentrations of estradiol, androstenedione, testosterone, and progesterone reflected the stage of follicular development and were similar for twinner and control follicles at the same stage. Earlier initiation of follicular development and/or selection of twin-dominant follicles in some twinner cows resulted in greater concentrations of estradiol in plasma at 0, 24, and 48 h and of estradiol, androstenedione, and testosterone in follicular fluid of large follicles at 0 h after PGF2alpha for twinner vs. control cows (follicular status x time x population, P < 0.01). Binding activities of IGFBP-5 and -4 were absent or reduced (P < 0.01) in follicular fluid of developing medium and large estro-gen-active (estradiol:progesterone ratio > 1) follicles but increased with atresia. Only preovulatory Graafian follicles lacked IGFBP-2 binding, suggesting a possible role for IGFBP-2 in selection of the dominant follicle. Concentrations of IGF-I were twofold greater (P < 0.01), but GH (P = 0.10) and cholesterol (P < 0.05) were less in blood of twinners. Three generations of selection of cattle for twin ovulations and births enhanced ovarian follicular development as manifested by increased numbers of follicles within a follicular wave and subsequent selection of twin dominant follicles. Because gonadotropin secretion and ovarian steroidogenesis were similar for control and twinner cattle, enhanced follicular development in twinners may result from decreased inhibition by the dominant follicle(s), increased ovarian sensitivity to gonadotropins, and/or increased intragonadal stimulation, possibly by increased IGF-I.

Characterization of gene expression in double-muscled and normal-muscled bovine embryos.

Myostatin, a member of the transforming growth factor-beta superfamily, is a negative regulator of skeletal muscle growth. Cattle with mutations that inactivate myostatin exhibit a remarkable increase in mass of skeletal muscle called double muscling that is accompanied by an equally remarkable decrease in carcass fat. Although a mouse knockout model has been created which results in mice with a 200% increase in skeletal muscle mass, molecular mechanisms whereby myostatin regulates skeletal muscle and fat mass are not fully understood. Using suppressive subtractive hybridization, genes that were differentially expressed in double-muscled vs. normal-muscled cattle embryos were identified. Genetic variation at other loci was minimized by using embryonic samples collected from related Piedmontese x Angus dams or Belgian Blue x Hereford dams bred to a single sire of the same breed composition. Embryos were collected at 31-33 days of gestation, which is 2-4 days after high-level expression of myostatin in the developing bovine embryo. The suppressive subtraction resulted in 30 clones that were potentially differentially expressed, 19 of which were confirmed by macroarray analysis. Several of these genes have biological functions that suggest that they are directly involved in myostatin's regulation of skeletal muscle development. Furthermore, several of these genes map to quantitative trait loci known to interact with variation in the myostatin gene.

Insulin-like growth factor binding proteins in granulosa and thecal cells from bovine ovarian follicles at different stages of development.

Because IGFBP inhibit IGF-stimulated cellular proliferation and differentiation, it is hypothesized that variations among IGFBP in individual follicles might contribute to the regulation of recruitment, selection, dominance, and turnover of ovarian follicles. Sources of IGFBP in fluid of bovine follicles are not well established; thus, objectives of this study were to determine levels of IGFBP binding activities and messenger RNA (mRNA) in granulosa and theca interna cells at different stages of follicular development (small [< 6 mm], medium [6 to < 8 mm], and large [> or = 8 mm]) and to characterize associations of these levels measured in the cells with levels of IGFBP and steroids in follicular fluid. Thecal and granulosa cells from large healthy follicles contained two- to twentyfold less (P < 0.05) IGFBP-2, -3, and -5 than cells from small, medium, and large atretic follicles. Thecal cells from small, medium, and large atretic follicles contained more (P < 0.05) IGFBP-3 and -4 than granulosa cells from these follicles, whereas granulosa cells from these follicles contained more IGFBP-2 activity than thecal cells. Differences in IGF binding activity were paralleled by differences in levels of mRNA for the respective IGFBP. Developmental differences in IGFBP activity in follicular fluid were positively associated with activity in granulosa and/or thecal cells, with the exception of IGFBP-4, which was low in fluid from large healthy follicles but markedly increased (mRNA and binding activity) in granulosa cells from these follicles. It is concluded that developmental changes in follicular fluid IGFBP-2 and -5 binding activities seem to be controlled in part by alterations in synthesis of these IGFBP by granulosa and thecal cells, whereas diminished IGFBP-4 in fluid from large healthy follicles occurs concomitantly with increased levels of IGFBP-4 mRNA and activity in granulosa cells, implicating posttranslational regulation by specific proteases.

Evaluation of numbers of microscopic and macroscopic follicles in cattle selected for twinning.

We hypothesized that the number of microscopic follicles present in the ovaries of cattle selected for twin births (Twinner) would be greater than in the ovaries of contemporary Controls. Ovaries were collected from seven Control and seven Twinner cows at slaughter. The number of Small (1 to 3.9 mm), Medium (4 to 7.9), and Large (> 8 mm) surface follicles was counted and one ovary was fixed for histological evaluation. Fifty to sixty consecutive 6-microm slices were taken from a piece of cortical tissue, approximately 1 cm x 1 cm in area, located between the surface follicles. Microscopic follicles were classified as primordial (oocyte surrounded by a single layer of squamous pregranulosa cells), primary (oocyte surrounded by a single layer of one or more cuboidal granulosa cells), secondary (oocyte surrounded by two or more layers of granulosa cells), or tertiary (oocyte surrounded by multiple layers of granulosa cells with initiation of antrum formation to < or = 1 mm in diameter). The total number of follicles was counted in 200 fields (2 mm x 2 mm) per ovary. A field containing no follicles was classified as empty. There were significantly more secondary follicles in Twinner compared with Control ovaries (12.9 vs 6.3; P < .05). Twinners also tended to have more small surface follicles (35.4 vs 49.0; P < 0.1). We conclude that ovaries of Control and Twinner cows do not differ in the number of primordial follicles or in the number of follicles activated into the growing pool; however, Twinner cows are able to maintain more growing follicles at the secondary and subsequent stages of development.

Initial results of genomic scans for ovulation rate in a cattle population selected for increased twinning rate.

Genomic scans were conducted with 273 markers on 181 sires from a cattle population selected for increased twinning rate to identify chromosomal regions containing genes that influence ovulation rate. Criteria used for selecting markers were number of alleles, ease of scoring, and relative position within linkage group. Markers were multiplexed or multiple-loaded on the gels to reduce the costs and labor required to obtain genotypic data. This approach reduced the number of gels by 45% when compared with running each marker independently. Male animals selected for the genomic scan sired the majority of the population. A modified interval analysis was used in a granddaughter design to compare effects of each allele within sire for 10 different sire families. The midparent deviation of the son's estimated breeding value for ovulation rate was used as the phenotype. Forty-one potential peaks were identified with a nominal significance level < or = 0.05. The 10 peaks with the highest significance levels (P < 0.02) were selected for further analysis. Markers were genotyped across daughters of the sire where nominal significance was found for each of the 10 peaks. One peak (BTA5, relative position 40 cM) was found to be nominally significant in the daughters. The nominal significance levels were P = 0.01 for the sons (n = 32) and P = 0.02 for the daughters (n = 94) of sire 784403. A combined genomewide significance value (P = 0.07) was calculated that accounted for the 10 analyses with sons and the 10 analyses with daughters. These results strongly suggest that this region contains a gene(s) that is involved in the follicular recruitment and development process.