R. A. Cushman

Evaluation of antral follicle count and ovarian morphology in crossbred beef cows: investigation of influence of stage of the estrous cycle, age, and birth weight.

Depletion of the ovarian reserve is associated with reproductive senescence in mammalian females, and there is a positive relationship between the size of the ovarian reserve and the number of antral follicles on the surface of the ovary. Therefore, we conducted a series of experiments to investigate the influence of stage of the estrous cycle, age, and birth weight on antral follicle counts (AFC) in beef cows and heifers. Pairs of ovaries were collected from crossbred beef cows at slaughter (n = 72) or at necropsy (n = 333; 0 to 11 yr of age); all visible antral follicles were counted, the ovaries were weighed, and stage of the estrous cycle was estimated based on ovarian morphology. There was no influence of estimated stage of the estrous cycle on AFC (P = 0.36). There was a small but positive effect of birth weight on AFC [AFC = -1.7 + 0.31(birth weight); P = 0.007, r(2) = 0.05]. When antral follicle counts were regressed on age, there was a quadratic effect of age such that AFC increased until 5 yr of age and decreased thereafter [AFC = 12.9 + 9.0(yr) - 0.86(yr(2)); P < 0.001, r(2) = 0.22]. In a third experiment, crossbred beef heifers (n = 406; 353 to 463 d of age) at 3 locations were subjected to ovarian ultrasonography on unknown day of the estrous cycle. Heifers were classified as low AFC (<15 follicle, n = 84) or high AFC (>24 follicles, n = 178). Whereas estimated stage of the estrous cycle did not influence AFC (P = 0.62), heifers classified as low AFC had smaller ovaries (P = 0.001), decreased birth weight (P = 0.003), and a decreased heifer pregnancy rate (P = 0.05) compared with heifers in the high AFC group. From these results, we conclude that AFC in beef cows and heifers is influenced by birth weight and age but not by stage of the estrous cycle. In beef cows, the number of antral follicles increases to 5 yr of age and then begins to decline. This may indicate that a decrease in fertility due to decline of the ovarian reserve may begin earlier than previously thought in beef cows.

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.

Heifer calving date positively influences calf weaning weights through six parturitions

Longevity and lifetime productivity are important factors influencing profitability for the cow-calf producer. Heifers that conceive earlier in the breeding season will calve earlier in the calving season and have a longer interval to rebreeding. Calves born earlier in the calving season will also be older and heavier at weaning. Longevity data were collected on 2,195 heifers from producers in South Dakota Integrated Resource Management groups. Longevity and weaning weight data were collected on 16,549 individual heifers at the U.S. Meat Animal Research Center (USMARC). Data were limited to heifers that conceived during their first breeding season. Heifers were grouped into 21-d calving periods. Heifers were determined to have left the herd when they were diagnosed not pregnant at the end of the breeding season. Heifers that left the herd for reasons other than reproductive failure were censored from the data. Heifers that calved with their first calf during the first 21-d period of the calving season had increased (P < 0.01) longevity compared with heifers that calved in the second 21-d period, or later. Average longevity for South Dakota heifers that calved in the first or later period was 5.1 ± 0.1 and 3.9 ± 0.1 yr, respectively. Average longevity for USMARC heifers that calved in the first, second, or third period was 8.2 ± 0.3, 7.6 ± 0.5, and 7.2 ± 0.1 yr, respectively. Calving period as a heifer influenced (P < 0.01) unadjusted weaning BW of the first 6 calves. Estimated postpartum interval to conception as a 2-yr-old cow was greater for females that calved in the first period as heifers but did not differ between heifer calving periods in subsequent calving seasons. In summary, heifers that calved early in the calving season with their first calf had increased longevity and kilograms weaned, compared with heifers that calved later in the calving season.

BREEDING AND GENETICS SYMPOSIUM: Networks and pathways to guide genomic selection

Many traits affecting profitability and sustainability of meat, milk, and fiber production are polygenic, with no single gene having an overwhelming influence on observed variation. No knowledge of the specific genes controlling these traits has been needed to make substantial improvement through selection. Significant gains have been made through phenotypic selection enhanced by pedigree relationships and continually improving statistical methodology. Genomic selection, recently enabled by assays for dense SNP located throughout the genome, promises to increase selection accuracy and accelerate genetic improvement by emphasizing the SNP most strongly correlated to phenotype although the genes and sequence variants affecting phenotype remain largely unknown. These genomic predictions theoretically rely on linkage disequilibrium (LD) between genotyped SNP and unknown functional variants, but familial linkage may increase effectiveness when predicting individuals related to those in the training data. Genomic selection with functional SNP genotypes should be less reliant on LD patterns shared by training and target populations, possibly allowing robust prediction across unrelated populations. Although the specific variants causing polygenic variation may never be known with certainty, a number of tools and resources can be used to identify those most likely to affect phenotype. Associations of dense SNP genotypes with phenotype provide a 1-dimensional approach for identifying genes affecting specific traits; in contrast, associations with multiple traits allow defining networks of genes interacting to affect correlated traits. Such networks are especially compelling when corroborated by existing functional annotation and established molecular pathways. The SNP occurring within network genes, obtained from public databases or derived from genome and transcriptome sequences, may be classified according to expected effects on gene products. As illustrated by functionally informed genomic predictions being more accurate than naive whole-genome predictions of beef tenderness, coupling evidence from livestock genotypes, phenotypes, gene expression, and genomic variants with existing knowledge of gene functions and interactions may provide greater insight into the genes and genomic mechanisms affecting polygenic traits and facilitate functional genomic selection for economically important traits.

Physiology and Endocrinology Symposium: How single nucleotide polymorphism chips will advance our knowledge of factors controlling puberty and aid in selecting replacement beef females.

The promise of genomic selection is accurate prediction of the genetic potential of animals from their genotypes. Simple DNA tests might replace low-accuracy predictions for expensive or lowly heritable measures of puberty and fertility based on performance and pedigree. Knowing with some certainty which DNA variants (e.g., SNP) affect puberty and fertility is the best way to fulfill the promise. Several SNP from the BovineSNP50 assay have tentatively been associated with reproductive traits including age at puberty, antral follicle count, and pregnancy observed on different sets of heifers. However, sample sizes are too small and SNP density is too sparse to definitively determine genomic regions harboring causal variants affecting reproductive success. Additionally, associations between individual SNP and similar phenotypes are inconsistent across data sets, and genomic predictions do not appear to be globally applicable to cattle of different breeds. Discrepancies may be a result of different QTL segregating in the sampled populations, differences in linkage disequilibrium (LD) patterns such that the same SNP are not correlated with the same QTL, and spurious correlations with phenotype. Several approaches can be used independently or in combination to improve detection of genomic factors affecting heifer puberty and fertility. Larger samples and denser SNP will increase power to detect real associations with SNP having more consistent LD with underlying QTL. Meta-analysis combining results from different studies can also be used to effectively increase sample size. High-density genotyping with heifers pooled by pregnancy status or early and late puberty can be a cost-effective means to sample large numbers. Networks of genes, implicated by associations with multiple traits correlated with puberty and fertility, could provide insight into the complex nature of these traits, especially if corroborated by functional annotation, established gene interaction pathways, and transcript expression. Example analyses are provided to demonstrate how integrating information about gene function and regulation with statistical associations from whole-genome SNP genotyping assays might enhance knowledge of genomic mechanisms affecting puberty and fertility, enabling reliable DNA tests to guide heifer selection decisions.

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.

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.

Altered theca and cumulus oocyte complex gene expression, follicular arrest and reduced fertility in cows with dominant follicle follicular fluid androgen excess.

Aspiration of bovine follicles 12–36 hours after induced corpus luteum lysis serendipitously identified two populations of cows, one with High androstenedione (A4; >40 ng/ml; mean = 102) and another with Low A4 (<20 ng/ml; mean = 9) in follicular fluid. We hypothesized that the steroid excess in follicular fluid of dominant follicles in High A4 cows would result in reduced fertility through altered follicle development and oocyte maternal RNA abundance. To test this hypothesis, estrous cycles of cows were synchronized and ovariectomy was performed 36 hours later. HPLC MS/MS analysis of follicular fluid showed increased dehydroepiandrosterone (6-fold), A4 (158-fold) and testosterone (31-fold) in the dominant follicle of High A4 cows. However, estrone (3-fold) and estradiol (2-fold) concentrations were only slightly elevated, suggesting a possible inefficiency in androgen to estrogen conversion in High A4 cows. Theca cell mRNA expression of LHCGR, GATA6, CYP11A1, and CYP17A1 was greater in High A4 cows. Furthermore, abundance of ZAR1 was decreased 10-fold in cumulus oocyte complexes from High A4 cows, whereas NLRP5 abundance tended to be 19.8-fold greater (P = 0.07). There was a tendency for reduction in stage 4 follicles in ovarian cortex samples from High A4 cows suggesting that progression to antral stages were impaired. High A4 cows tended (P<0.07) to have a 17% reduction in calving rate compared with Low A4 cows suggesting reduced fertility in the High A4 population. These data suggest that the dominant follicle environment of High A4 cows including reduced estrogen conversion and androgen excess contributes to infertility in part through altered follicular and oocyte development.