Adenylate kinase 9 is essential for sperm function and male fertility in mammals.

Despite passing routine laboratory tests for semen quality, bulls used in artificial insemination exhibit significant variation in fertility. Routine analysis of fertility data identified a dairy bull with extreme subfertility (10% pregnancy rate). To characterize the subfertility phenotype, a range of in vitro, in vivo, and molecular assays were carried out. Sperm from the subfertile bull exhibited reduced motility and severely reduced caffeine-induced hyperactivation compared to controls. Ability to penetrate the zona pellucida, cleavage rate, cleavage kinetics, and blastocyst yield after IVF or AI were significantly lower than in control bulls. Whole-genome sequencing from semen and RNA sequencing of testis tissue revealed a critical mutation in adenylate kinase 9 (AK9) that impaired splicing, leading to a premature termination codon and a severely truncated protein. Mice deficient in AK9 were generated to further investigate the function of the gene; knockout males were phenotypically indistinguishable from their wild-type littermates but produced immotile sperm that were incapable of normal fertilization. These sperm exhibited numerous abnormalities, including a low ATP concentration and reduced motility. RNA-seq analysis of their testis revealed differential gene expression of components of the axoneme and sperm flagellum as well as steroid metabolic processes. Sperm ultrastructural analysis showed a high percentage of sperm with abnormal flagella. Combined bovine and murine data indicate the essential metabolic role of AK9 in sperm motility and/or hyperactivation, which in turn affects sperm binding and penetration of the zona pellucida. Thus, AK9 has been found to be directly implicated in impaired male fertility in mammals.

Effect of enhanced plane of nutrition in early life on the transcriptome and proteome of the anterior pituitary gland in Angus heifer calves.

BACKGROUND: Enhanced nutrition during the early calfhood period has been shown to lead to earlier pubertal development in heifer calves. This is of interest as earlier pubertal onset can subsequently facilitate earlier calving which can economically benefit production systems. Reproductive development in heifers is regulated by the hypothalamic-pituitary-ovarian signalling pathway. In particular the anterior pituitary gland is central to reproductive development, through the dynamics of gonadotropic pulsatility. However, despite clear knowledge of the influence of enhanced dietary intake on subsequent reproductive development, the molecular control governing this response in the pituitary gland within the hypothalamic-pituitary-ovarian signalling axis in heifer calves is not fully understood. The objective of this study was to examine the effect of an enhanced plane of nutrition during early life on the anterior pituitary gland of heifer calves through both transcriptomic and proteomic analyses. Between 3 and 21 weeks of age, heifer calves were offered either a high (HI, n = 14) or moderate (MOD, n = 14) plane of nutrition, designed to elicit target growth rates of 1.2 and 0.5 kg/d for HI and MOD groups, respectively. All calves were euthanised at 21 weeks of age and anterior pituitary tissue harvested for subsequent use in global transcriptomic and proteomic analyses. RESULTS: Average daily gain was affected by diet (P < 0.001) and was 1.18 and 0.50 kg/day, for HI and MOD calves, respectively. RNAseq analysis resulted in the identification of 195 differentially expressed genes (Padj<0.05; fold change > 1.5), with 277 proteins identified as differentially abundant (Padj<0.05; fold change > 1.5) between contrasting dietary treatment groups. Biochemical pathway analysis of differentially affected genes and proteins revealed an enrichment for both growth hormone and GnRH signalling pathways (Padj.<0.05). Additionally, pathway analysis predicted an effect of enhanced dietary intake on endocrine function within the anterior pituitary gland as well as on reproductive system development and function (Padj.<0.05). CONCLUSIONS: Results from this study show that an enhanced dietary intake during early calfhood affected the molecular control of the anterior pituitary gland in heifer calves in early life.

Sperm DNA methylation patterns at discrete CpGs and genes involved in embryonic development are related to bull fertility.

BACKGROUND: Despite a multifactorial approach being taken for the evaluation of bull semen quality in many animal breeding centres worldwide, reliable prediction of bull fertility is still a challenge. Recently, attention has turned to molecular mechanisms, which could uncover potential biomarkers of fertility. One of these mechanisms is DNA methylation, which together with other epigenetic mechanisms is essential for the fertilising sperm to drive normal embryo development and establish a viable pregnancy. In this study, we hypothesised that bull sperm DNA methylation patterns are related to bull fertility. We therefore investigated DNA methylation patterns from bulls used in artificial insemination with contrasting fertility scores. RESULTS: The DNA methylation patterns were obtained by reduced representative bisulphite sequencing from 10 high-fertility bulls and 10 low-fertility bulls, having average fertility scores of - 6.6 and + 6.5%, respectively (mean of the population was zero). Hierarchical clustering analysis did not distinguish bulls based on fertility but did highlight individual differences. Despite this, using stringent criteria (DNA methylation difference ≥ 35% and a q-value < 0.001), we identified 661 differently methylated cytosines (DMCs). DMCs were preferentially located in intergenic regions, introns, gene downstream regions, repetitive elements, open sea, shores and shelves of CpG islands. We also identified 10 differently methylated regions, covered by 7 unique genes (SFRP1, STXBP4, BCR, PSMG4, ARSG, ATP11A, RXRA), which are involved in spermatogenesis and early embryonic development. CONCLUSION: This study demonstrated that at specific CpG sites, sperm DNA methylation status is related to bull fertility, and identified seven differently methylated genes in sperm of subfertile bulls that may lead to altered gene expression and potentially influence embryo development.

Comprehensive functional analysis reveals that acrosome integrity and viability are key variables distinguishing artificial insemination bulls of varying fertility.

In vitro methods of assessing bull semen quality in artificial insemination (AI) centers are unable to consistently detect individuals of lower fertility, and attempts to reliably predict bull fertility are still ongoing. This highlights the need to identify robust biomarkers that can be readily measured in a practical setting and used to improve current predictions of bull fertility. In this study, we comprehensively analyzed a range of functional, morphological, and intracellular attributes in cryopreserved spermatozoa from a selected cohort of Holstein Friesian AI bulls classified as having either high or low fertility (n = 10 of each fertility phenotype; difference of 11.4% in adjusted pregnancy rate between groups). Here, spermatozoa were assessed for motility and kinematic parameters, morphology, acrosome integrity, plasma membrane lipid packing, viability (or membrane integrity), superoxide production, and DNA integrity. In addition, spermatozoa were used for in vitro fertilization to evaluate their capacity for fertilization and successful embryo development. The information collected from these assessments was then used to phenotypically profile the 2 groups of bulls of divergent fertility status as well as to develop a model to predict bull fertility. According to the results, acrosome integrity and viability were the only sperm attributes that were significantly different between high- and low-fertility bulls. Interestingly, although spermatozoa from low-fertility bulls, on average, had reduced viability and acrosome integrity, this response varied considerably from bull to bull. Principal component analysis revealed a sperm phenotypic profile that represented a high proportion of ejaculates from low-fertility bulls. This was constructed based on the collective influence of several sperm attributes, including the presence of cytoplasmic droplets and superoxide production. Finally, using the combined results as a basis for modeling, we developed a linear model that was able to explain 47% of the variation in bull field fertility in addition to a logistic predictive model that had a 90% chance of distinguishing between fertility groups. Taken together, we conclude that viability and acrosome integrity could serve as fertility biomarkers in the field and, when used alongside other sperm attributes, may be useful in detecting low-fertility bulls. However, the variable nature of low-fertility bulls suggests that additional, in-depth characterization of spermatozoa at a molecular level is required to further understand the etiology of low fertility in dairy bulls.

The effect of breed and diet type on the global transcriptome of hepatic tissue in beef cattle divergent for feed efficiency.

BACKGROUND: Feed efficiency is an important economic and environmental trait in beef production, which can be measured in terms of residual feed intake (RFI). Cattle selected for low-RFI (feed efficient) have similar production levels but decreased feed intake, while also emitting less methane. RFI is difficult and expensive to measure and is not widely adopted in beef production systems. However, development of DNA-based biomarkers for RFI may facilitate its adoption in genomic-assisted breeding programmes. Cattle have been shown to re-rank in terms of RFI across diets and age, while also RFI varies by breed. Therefore, we used RNA-Seq technology to investigate the hepatic transcriptome of RFI-divergent Charolais (CH) and Holstein-Friesian (HF) steers across three dietary phases to identify genes and biological pathways associated with RFI regardless of diet or breed. RESULTS: Residual feed intake was measured during a high-concentrate phase, a zero-grazed grass phase and a final high-concentrate phase. In total, 322 and 33 differentially expressed genes (DEGs) were identified across all diets for CH and HF steers, respectively. Three genes, GADD45G, HP and MID1IP1, were differentially expressed in CH when both the high-concentrate zero-grazed grass diet were offered. Two canonical pathways were enriched across all diets for CH steers. These canonical pathways were related to immune function. CONCLUSIONS: The absence of common differentially expressed genes across all dietary phases and breeds in this study supports previous reports of the re-ranking of animals in terms of RFI when offered differing diets over their lifetime. However, we have identified biological processes such as the immune response and lipid metabolism as potentially associated with RFI divergence emphasising the previously reported roles of these biological processes with respect to RFI.

Effect of early calf-hood nutrition on the transcriptomic profile of subcutaneous adipose tissue in Holstein-Friesian bulls.

BACKGROUND: Adipose tissue is a major endocrine organ and is thought to play a central role in the metabolic control of reproductive function in cattle. Plane of nutrition during early life has been shown to influence the timing of puberty in both male and female cattle, though the exact biological mechanisms involved are currently unknown. The aim of this study was to investigate the effect of early calf-hood nutrition on the transcriptomic profile of subcutaneous adipose tissue in Holstein-Friesian bulls to identify possible downstream effects on reproductive physiology. RESULTS: Holstein-Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a high (n = 10) or low (n = 10) plane of nutrition. Calves were fed in order to achieve an overall growth rate of 1.08 and 0.57 kg/day for the high and low plane of nutrition treatments, respectively. At 126 days of age, the bulls were euthanized, subcutaneous adipose tissue samples were harvested and RNAseq analysis was performed. There were 674 genes differentially expressed in adipose tissue of calves on the low compared with the high plane of nutrition (P < 0.05; FDR < 0.05; fold change > 2.0). High plane of nutrition positively altered the expression of genes across an array of putative biological processes but the most dominant cellular processes affected were cellular energy production and branched chain amino acid degradation. A high plane of nutrition caused upregulation of genes such as leptin (LEP) and adiponectin (ADIPOQ), which are known to directly affect reproductive function. CONCLUSIONS: These results provide an insight into the effect of augmenting the plane of nutrition of Holstein-Friesian bull calves in the prepubertal period on the transcriptome of adipose tissue.

Experiments with More Than One Random Factor: Designs, Analytic Models, and Statistical Power.

Traditional methods of analyzing data from psychological experiments are based on the assumption that there is a single random factor (normally participants) to which generalization is sought. However, many studies involve at least two random factors (e.g., participants and the targets to which they respond, such as words, pictures, or individuals). The application of traditional analytic methods to the data from such studies can result in serious bias in testing experimental effects. In this review, we develop a comprehensive typology of designs involving two random factors, which may be either crossed or nested, and one fixed factor, condition. We present appropriate linear mixed models for all designs and develop effect size measures. We provide the tools for power estimation for all designs. We then discuss issues of design choice, highlighting power and feasibility considerations. Our goal is to encourage appropriate analytic methods that produce replicable results for studies involving new samples of both participants and targets.

Effect of equine chorionic gonadotropin treatment during a progesterone-based timed artificial insemination program on reproductive performance in seasonal-calving lactating dairy cows.

The aim of this study was to investigate the effect of progesterone (P4)-based timed artificial insemination (TAI) programs on fertility in seasonal-calving, pasture-based dairy herds. A total of 1,421 lactating dairy cows on 4 spring-calving farms were stratified based on days in milk (DIM) and parity and randomly allocated to 1 of 3 treatments: (1) control: no hormonal treatment; cows inseminated at detected estrus; (2) P4-Ovsynch: cows received a 7-d P4-releasing intravaginal device (PRID Delta; CEVA Santé Animale, Libourne, France) with 100 µg of a gonadotropin-releasing hormone (GnRH) analog (Ovarelin; CEVA Santé Animale) at PRID insertion, a 25-mg injection of PGF2α (Enzaprost; CEVA Santé Animale) at PRID removal, GnRH at 56 h after device removal and TAI 16 h later; (3) P4-Ovsynch+eCG: the same as P4-Ovsynch, but cows received 500 IU of equine chorionic gonadotropin (eCG; Syncrostim; CEVA Santé Animale) at PRID removal. At 10 d before mating start date (MSD), all cows that were ≥35 DIM were examined by transrectal ultrasound to assess presence or absence of a corpus luteum; body condition score (BCS) was also recorded. Pregnancy diagnosis was performed by transrectal ultrasonography 30 to 35 d after insemination. Overall pregnancy/AI (P/AI) was not different between groups (50.9, 49.8, and 46.3% for control, P4-Ovsynch, and P4-Ovsynch+eCG, respectively) but the 21-d pregnancy rate was increased by the use of synchronization (35.0, 51.7, and 47.2%, respectively). Compared with the control group, synchronization significantly reduced the interval from MSD to conception (34.6, 23.0, and 26.5 d, respectively) and consequently reduced the average days open (98.0, 86.0, and 89.0 d). Across all treatment groups, DIM at the start of synchronization affected P/AI (42.3, 49.5, and 53.9% for <60, 60-80, and >80 DIM, respectively), but neither parity (46.5, 50.4, and 48.4% for parity 1, 2, and ≥3, respectively) nor BCS (44.0, 49.4, and 58.6% for ≤2.50, 2.75-3.25, and ≥3.50, respectively) affected the likelihood of P/AI. Two-way interactions between treatment and DIM, parity, or BCS were not detected. In conclusion, the use of TAI accelerated pregnancy establishment in cows in a pasture-based system by reducing days open, but eCG administration at PRID removal did not affect P/AI.

Effect of short term diet restriction on gene expression in the bovine hypothalamus using next generation RNA sequencing technology.

BACKGROUND: Negative energy balance (NEB) is an imbalance between energy intake and energy requirements for lactation and body maintenance affecting high-yielding dairy cows and is of considerable economic importance due to its negative impact on fertility and health in dairy herds. It is anticipated that the cow hypothalamus experiences extensive biochemical changes during the early post partum period in an effort to re-establish metabolic homeostasis. However, there is variation in the tolerance to NEB between individual cows. In order to understand the genomic regulation of ovulation in hypothalamic tissue during NEB, mRNA transcriptional patterns between tolerant and sensitive animals were examined. A short term dietary restriction heifer model was developed which induced abrupt onset of anoestrus in some animals (Restricted Anovulatory; RA) while others maintained oestrous cyclicity (Restricted Ovulatory; RO). A third control group (C) received a higher level of normal feeding. RESULTS: A total of 15,295 genes were expressed in hypothalamic tissue. Between RA and C groups 137 genes were differentially expressed, whereas between RO and C, 32 genes were differentially expressed. Differentially expressed genes were involved in the immune response and cellular motility in RA and RO groups, respectively, compared to C group. The largest difference between groups was observed in the comparison between RA and RO heifers, with 1094 genes shown to be significantly differentially expressed (SDE). Pathway analysis showed that these SDE genes were associated with 6 canonical pathways (P < 0.01), of which neuroactive ligand-receptor interaction was the most significant. Within the comparisons the main over-represented pathway functions were immune response including neuroprotection (CXCL10, Q1KLR3, IFIH1, IL1 and IL8; RA v C and RA v RO); energy homeostasis (AgRP and NPY; RA v RO); cell motility (CADH1, DSP and TSP4; RO v C) and prevention of GnRH release (NTSR1 IL1α, IL1ß, NPY and PACA; RA v RO). CONCLUSIONS: This information will assist in understanding the genomic factors regulating the influence of diet restriction on fertility and may assist in optimising nutritional and management systems for the improvement in reproductive performance.

Endometrial gene expression in high- and low-fertility heifers in the late luteal phase of the estrous cycle and a comparison with midluteal gene expression.

Embryonic mortality is a major constraint to improving reproductive efficiency and profitability in livestock enterprises. We previously reported differential expression of genes with identified roles in cellular growth and proliferation, lipid metabolism, endometrial remodeling, inflammation, angiogenesis, and metabolic exchange in endometrial tissue on day 7 of the estrous cycle (D7), between heifers ranked as either high (HF) or low (LF) for fertility. The aim of the current study was to further elucidate the underlying molecular mechanisms contributing to early embryo loss by examining differential endometrial gene expression in HF or LF heifers at a later stage of the estrous cycle;day 14(D14). A second objective was to compare these expression profiles with those from midluteal HF and LF endometrium. Using the same animal model as employed in the previous study, we slaughtered HF and LF animals on D14, harvested endometrial tissue, and carried out global gene expression analysis using the Affymetrix Bovine GeneChip. Microarray analysis detected 430 differentially expressed genes (DEG) between HF and LF animals. Ingenuity Pathway Analysis revealed enrichment for a host of biological pathways including lipid metabolism, molecular transport, immune response, cell morphology and development, and cell growth and proliferation. Important DEG includedALB, BMPR2, CCL28, COL4A3/4, FADS1, ITGA6, LDLR, PLCB3, PPARG, PTGS2, and SLC27A4 Furthermore, DEG expressed on both D7 and D14 included:PCCB,SLC25A24,DAP, and COL4A4 This study highlights some of the pathways and mechanisms underpinning late luteal bovine endometrial physiology and endometrial-related conception rate variance.

Effect of dietary restriction and subsequent re-alimentation on the transcriptional profile of hepatic tissue in cattle.

BACKGROUND: Compensatory growth (CG) is an accelerated growth phenomenon observed in animals upon re-alimentation following a period of dietary restriction. It is typically utilised in livestock systems to reduce feed costs during periods of reduced feed availability. The biochemical mechanisms controlling this phenomenon, however, are yet to be elucidated. This study aimed to uncover the molecular mechanisms regulating the hepatic expression of CG in cattle, utilising RNAseq. RNAseq was performed on hepatic tissue of bulls following 125 days of dietary restriction (RES) and again following 55 days of subsequent re-alimentation during which the animals exhibited significant CG. The data were compared with those of control animals offered the same diet on an ad libitum basis throughout (ADLIB). Elucidation of the molecular control of CG may yield critical information on genes and pathways which could be targeted as putative molecular biomarkers for the selection of animals with improved CG potential. RESULTS: Following a period of differential feeding, body-weight and liver weight were 161 and 4 kg higher, respectively, for ADLIB compared with RES animals. At this time RNAseq analysis of liver tissue revealed 1352 significantly differentially expressed genes (DEG) between the two treatments. DEGs indicated down-regulation of processes including nutrient transport, cell division and proliferation in RES. In addition, protein synthesis genes were up-regulated in RES following a period of restricted feeding. The subsequent 55 days of ad libitum feeding for both groups resulted in the body-weight difference reduced to 84 kg, with no difference in liver weight between treatment groups. At the end of 55 days of unrestricted feeding, 49 genes were differentially expressed between animals undergoing CG and their continuously fed counterparts. In particular, hepatic expression of cell proliferation and growth genes were greater in animals undergoing CG. CONCLUSIONS: Greater expression of cell cycle and cell proliferation genes during CG was associated with a 100 % recovery of liver weight during re-alimentation. Additionally, an apparent up-regulation in capacity for cellular protein synthesis during restricted feeding may contribute to and sustain CG during re-alimentation. DEGs identified are potential candidate genes for the identification of biomarkers for CG, which may be incorporated into future breeding programmes.

The Combined Effects of Measurement Error and Omitting Confounders in the Single-Mediator Model.

Mediation analysis requires a number of strong assumptions be met in order to make valid causal inferences. Failing to account for violations of these assumptions, such as not modeling measurement error or omitting a common cause of the effects in the model, can bias the parameter estimates of the mediated effect. When the independent variable is perfectly reliable, for example when participants are randomly assigned to levels of treatment, measurement error in the mediator tends to underestimate the mediated effect, while the omission of a confounding variable of the mediator-to-outcome relation tends to overestimate the mediated effect. Violations of these two assumptions often co-occur, however, in which case the mediated effect could be overestimated, underestimated, or even, in very rare circumstances, unbiased. To explore the combined effect of measurement error and omitted confounders in the same model, the effect of each violation on the single-mediator model is first examined individually. Then the combined effect of having measurement error and omitted confounders in the same model is discussed. Throughout, an empirical example is provided to illustrate the effect of violating these assumptions on the mediated effect.

Insulin secretion and signaling in response to dietary restriction and subsequent re-alimentation in cattle.

The objectives of this study were to examine systemic insulin response to a glucose tolerance test (GTT) and transcript abundance of genes of the insulin signaling pathway in skeletal muscle, during both dietary restriction and re-alimentation-induced compensatory growth. Holstein Friesian bulls were blocked to one of two groups: 1) restricted feed allowance for 125 days (period 1) (RES, n = 15) followed by ad libitum feeding for 55 days (period 2) or 2) ad libitum access to feed throughout (periods 1 and 2) (ADLIB, n = 15). On days 90 and 36 of periods 1 and 2, respectively, a GTT was performed. M. longissimus dorsi biopsies were harvested from all bulls on days 120 and 15 of periods 1 and 2, respectively, and RNA-Seq analysis was performed. RES displayed a lower growth rate during period 1 (RES: 0.6 kg/day, ADLIB: 1.9 kg/day; P < 0.001), subsequently gaining more during re-alimentation (RES: 2.5 kg/day, ADLIB: 1.4 kg/day; P < 0.001). Systemic insulin response to glucose administration was lower in RES in period 1 (P < 0.001) with no difference observed during period 2. The insulin signaling pathway in M. longissimus dorsi was enriched (P < 0.05) in response to dietary restriction but not during re-alimentation (P > 0.05). Genes differentially expressed in the insulin signaling pathway suggested a greater sensitivity to insulin in skeletal muscle, with pleiotropic effects of insulin signaling interrupted during dietary restriction. Collectively, these results indicate increased sensitivity to glucose clearance and skeletal muscle insulin signaling during dietary restriction; however, no overall role for insulin was apparent in expressing compensatory growth.

Effect of feed restriction and subsequent re-alimentation on hormones and genes of the somatotropic axis in cattle.

The objective of this study was to characterize the effect of feed restriction and compensatory growth during re-alimentation on the functionality of the somatotropic axis. We blocked 60 bulls into one of two groups: 1) restricted feed allowance for 125 days (period 1) (RES, n = 30) followed by ad libitum feeding for 55 days (period 2) or 2) ad libitum access to feed throughout (ADLIB, n = 30). A growth hormone releasing hormone (GHRH) challenge was performed during each period. At the end of each period, 15 animals from each treatment were slaughtered and hepatic tissue collected. Hepatic expression of 13 genes of the somatotropic axis was measured by qRT-PCR. RES displayed a lower growth rate during period 1 (0.6 vs. 1.9 kg/day; P < 0.001), subsequently gaining more than ADLIB animals during period 2 (2.5 vs. 1.4 kg/day; P < 0.001). Growth hormone response to GHRH was not different between treatments at either time-point (P > 0.05); however, resultant plasma IGF-1 was lower in period 1 and greater in period 2 in RES animals (P < 0.05). Expression of IGFBP2 was higher (P < 0.01) and IGF1 (P < 0.001) and GHRIA (P < 0.05) lower in RES compared with ADLIB during period 1, with no difference evident in period 2 (P > 0.05). Collectively, the results of this study are consistent with uncoupling of the somatotropic axis following feed restriction. However, there is no evidence from this study that the somatotropic axis per se is a significant contributor to compensatory growth.

Global gene expression in endometrium of high and low fertility heifers during the mid-luteal phase of the estrous cycle.

BACKGROUND: In both beef and dairy cattle, the majority of early embryo loss occurs within the first 14 days following insemination. During this time-period, embryos are completely dependent on their maternal uterine environment for development, growth and ultimately survival, therefore an optimum uterine environment is critical to their survival. The objective of this study was to investigate whether differences in endometrial gene expression during the mid-luteal phase of the estrous cycle exist between crossbred beef heifers ranked as either high (HF) or low fertility (LF) (following four rounds of artificial insemination (AI)) using the Affymetrix® 23 K Bovine Gene Chip. RESULTS: Conception rates for each of the four rounds of AI were within a normal range: 70-73.3%. Microarray analysis of endometrial tissue collected on day 7 of the estrous cycle detected 419 differentially expressed genes (DEG) between HF (n = 6) and LF (n = 6) animals. The main gene pathways affected were, cellular growth and proliferation, angiogenesis, lipid metabolism, cellular and tissue morphology and development, inflammation and metabolic exchange. DEG included, FST, SLC45A2, MMP19, FADS1 and GALNT6. CONCLUSIONS: This study highlights, some of the molecular mechanisms potentially controlling uterine endometrial function during the mid-luteal phase of the estrous cycle, which may contribute to uterine endometrial mediated impaired fertility in cattle. Differentially expressed genes are potential candidate genes for the identification of genetic variation influencing cow fertility, which may be incorporated into future breeding programmes.

Rumen methanogenic genotypes differ in abundance according to host residual feed intake phenotype and diet type.

Methane is an undesirable end product of rumen fermentative activity because of associated environmental impacts and reduced host feed efficiency. Our study characterized the rumen microbial methanogenic community in beef cattle divergently selected for phenotypic residual feed intake (RFI) while offered a high-forage (HF) diet followed by a low-forage (LF) diet. Rumen fluid was collected from 14 high-RFI (HRFI) and 14 low-RFI (LRFI) animals at the end of both dietary periods. 16S rRNA gene clone libraries were used, and methanogen-specific tag-encoded pyrosequencing was carried out on the samples. We found that Methanobrevibacter spp. are the dominant methanogens in the rumen, with Methanobrevibacter smithii being the most abundant species. Differences in the abundance of Methanobrevibacter smithii and Methanosphaera stadtmanae genotypes were detected in the rumen of animals offered the LF compared to the HF diet while the abundance of Methanobrevibacter smithii genotypes was different between HRFI and LRFI animals irrespective of diet. Our results demonstrate that while a core group of methanogen operational taxonomic units (OTUs) exist across diet and phenotype, significant differences were observed in the distribution of genotypes within those OTUs. These changes in genotype abundance may contribute to the observed differences in methane emissions between efficient and inefficient animals.

Power anomalies in testing mediation.

Two rather surprising anomalies relating to statistical power occur in testing mediation. First, in a model with no direct effect for which the total effect and indirect effect are identical, the power for the test of the total effect can be dramatically smaller than the power for the test of the indirect effect. Second, when there is a direct effect of a causal variable on the outcome controlling for the mediator, the power of the test of the indirect effect is often considerably greater than the power of the test of the direct effect, even when the two are of the same magnitude. We try to explain the reasons for these anomalies and how they affect practice.

Effect of dietary n-3 polyunsaturated fatty acids on transcription factor regulation in the bovine endometrium.

Dietary n-3 polyunsaturated fatty acid (n-3 PUFA) supplementation is postulated to have positive effects on fertility. The impact of dietary n-3 PUFA supplementation on physiological and biochemical processes involved in reproduction is likely to be associated with significant alterations in gene expression in key reproductive tissues which is in turn regulated by transcription factors. Beef heifers were supplemented with a rumen protected source of either a saturated fatty acid or high n-3 PUFA diet per animal per day for 45 days and uterine endometrial tissue was harvested post slaughter. A microarray analysis was conducted and bioinformatic tools were employed to evaluate the effect of n-3 PUFA supplementation on gene expression in the bovine endometrium. Clustering of microarray gene expression data was performed to identify co-expressed genes. Functional annotation of each cluster of genes was carried out using Ingenuity Pathway Analysis. Furthermore, oPOSSUM was employed to identify transcription factors involved in gene expression changes due to supplementary PUFA. Gene functions which showed a significant response to n-3 PUFA supplementation included tissue development, immune function and reproductive function. Numerous transcription factors such as FOXD1, FOXD3, NFKB1, ESR1, PGR, FOXA2, NKX3-1 and PPARα were identified as potential regulators of gene expression in the endometrium of cattle supplemented with n-3 PUFA. This study demonstrates the complex nature of the alterations in the transcriptional regulation process in the uterine endometrium of cattle following dietary supplementation which may positively influence the uterine environment.

Multi-level modelling of physical activity in nuclear families.

BACKGROUND: Few studies focus on the different dyadic relations among family members to study physical activity (PA) levels. AIM: The aim was to investigate predictors and sources of variance of PA levels in nuclear families using multi-level modelling. SUBJECTS AND METHODS: The sample consisted of 2661 Portuguese four-member nuclear families (10 644 subjects). PA was measured using a questionnaire and socioeconomic status (SES) was assessed by parental occupation. Height and weight were measured in children, while parents self-reported their values. RESULTS: The results showed that intra-generational similarities were higher than inter-generational, with correlation values of 0.26 and 0.10, respectively. SES was unrelated to any family members' PA level. Being male (ß = 0.26, t = 21.77), being older (ß = -0.36, t = -4.73) and greater BMI for mothers (ß = 0.02, t = 2.55) had effects on individuals' PA. CONCLUSION: These results suggest a strong dyadic resemblance in PA, showed different effects of gender, age and BMI on individuals' PA and demonstrated that multi-level modelling is a useful strategy to study PA in families.

Examining the Contribution of Physical Cues for Same- and Cross-Race Face Individuation.

Face individuation involves sensitivity to physical characteristics that provide information about identity. We examined whether Black and White American faces differ in terms of individuating information, and whether Black and White perceivers differentially weight information when judging same-race and cross-race faces. Study 1 analyzed 20 structural metrics (e.g., eye width, nose length) of 158 Black and White faces to determine which differentiate faces within each group. High-utility metrics (e.g., nose length, eye height, chin length) differentiated faces of both groups, low-utility metrics (e.g., face width, eye width, face length) offered less individuating information. Study 2 (N = 4,510) explored Black and White participants' sensitivity to variation on structural metrics using similarity ratings. High-utility metrics affected perceived dissimilarity more than low-utility metrics. This relationship was non-significantly stronger for same-race faces rather than cross-race faces. Perceivers also relied more on features that were racially stereotypic of the faces they were rating.