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Southern blotting of bovine genomic DNA indicated the presence of at least 3 bovine tIFN genes. The full DNA sequence of one of these genes, thought to be expressed in trophoblast, has been determined, including 193 bp of 5' non-coding region. The inferred amino acid sequence of bovine tIFN is more similar to ovine tIFN (80%) than to bovine IFN-alpha II (70%). The 5' flanking sequence has some similarity with bovine IFN-alpha II, and may contain a viral response element. A recombinant bovine alpha I interferon (Ciba Geigy; brIFN), resembling tIFN, extended oestrous cycle length in sheep when administered by intrauterine infusion over the period, Days 12-15 after oestrus, when maternal recognition of pregnancy occurs. Intramuscular injection was only effective at the doses used if given over a longer period (i.e. Days 9-15). Our experiments indicate that both tIFN and brIFN inhibit luteolysis by preventing a rise in endometrial oxytocin receptor concentrations, and suggest that tIFN achieves this by extending the time for which progesterone suppresses oxytocin receptor development. Further studies are required to confirm this hypothesis and to elucidate the interaction of the effects of progesterone and tIFN in endometrial cells.

A mathematical model was constructed to simulate the bovine oestrous cycle by using nonlinear differential equations to describe the biological mechanisms which regulate the cycle. The model predicts circulating concentrations of gonadotrophin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, oestradiol, inhibin and progesterone. These hormones collectively provide control and feedback mechanisms between the hypothalamus, pituitary gland and ovaries, which regulate ovarian follicular dynamics, corpus luteum function and ovulation. When follicular growth parameters are altered, the model predicts that cows will exhibit either two or three follicular waves per cycle, as seen in practice. Changes in other parameters allow the model to simulate: effects of nutrition on follicle recruitment and size of the ovulatory follicle; effects of negative energy balance on postpartum anoestrus; and effects of pharmacological intervention on hormone profiles and timing of ovulation. It is concluded that this model provides a sound basis for exploring factors that influence the bovine oestrous cycle in order to test hypotheses about nutritional and hormonal influences which, with further validation, should help to design dietary or pharmacological strategies for improving reproductive performance in cattle.

The United Kingdom, as in most countries using intensive dairy management programmes, is facing serious challenges in terms of dairy cow fertility, as highlighted by a rapidly increasing calving interval (CI). A mechanistic, mathematical model is described that predicts the size of the future national dairy herd required to supply domestic requirements and its inherent sustainability in terms of production of replacement female numbers. The results from the model suggest that continuing use of current management strategies may result in the national dairy herd being unsustainable due to increasing CI and reduced fertility in as few as 10 years. Adoption of nutritional, endocrine and genetic techniques that increase fertility can effectively and rapidly reverse this trend and reduce the required size of the national herd, thereby reducing methane emissions from dairy production.

Arachidonic acid is a potential paracrine agent released by the uterine endometrial epithelium to induce PTGS2 [PG (prostaglandin)-endoperoxide synthase 2] in the stroma. In the present study, bovine endometrial stromal cells were used to determine whether PTGS2 is induced by arachidonic acid in stromal cells, and to investigate the potential role of PPARs (peroxisome-proliferator-activated receptors) in this effect. Arachidonic acid increased PTGS2 levels up to 7.5-fold within 6 h. The cells expressed PPARalpha and PPARdelta (also known as PPARbeta) (but not PPARgamma). PTGS2 protein level was increased by PPAR agonists, including polyunsaturated fatty acids, synthetic PPAR ligands, PGA1 and NSAIDs (non-steroidal anti-inflammatory drugs) with a time course resembling that of arachidonic acid. Use of agonists and antagonists indicated PPARalpha (but not PPARdelta or PPARgamma) was responsible for PTGS2 induction. PTGS2 induction by arachidonic acid did not require PG synthesis. PTGS2 levels were increased by the PKC (protein kinase C) activators 4beta-PMA and PGF(2alpha), and the effects of arachidonic acid, NSAIDs, synthetic PPAR ligands and 4beta-PMA were blocked by PKC inhibitors. This is consistent with PPAR phosphorylation by PKC. Induction of PTGS2 protein by 4beta-PMA in the absence of a PPAR ligand was decreased by the NF-kappaB (nuclear factor kappaB) inhibitors MG132 and parthenolide, suggesting that PKC acted through NF-kappaB in addition to PPAR phosphorylation. Use of NF-kappaB inhibitors allowed the action of arachidonic acid as a PPAR agonist to be dissociated from an effect through PKC. The results are consistent with the hypothesis that arachidonic acid acts via PPARalpha to increase PTGS2 levels in bovine endometrial stromal cells.

Summary Seven sequence variants (SVs) have been identified in exon 1 and in the promoter region upstream of the bovine gonadotrophin releasing hormone (GnRH) receptor gene, at nucleotides g.−331A>G, g.−108T>C, g.+206G>A, g.+260C>T, g.+341C>T, g.+383C>T and g.+410C>T relative to the translation start site. The SVs at nucleotides g.−108, g.260, g.341 and g.410 and those at g.206 and g.383 formed two groups with complete linkage disequilibrium within groups, but incomplete linkage disequilibrium between groups, and none of the SVs altered receptor amino acid sequence. The g.−108T>C allelic variants were associated with an approximately 0.4 day reduction in predicted transmitting ability for days to first service. None of the allelic variants affected the pattern of circulating LH following administration of GnRH. The g.260C>T alteration introduced a new transcription factor binding site in a region of DNA with relatively low nucleosome formation potential. The data suggest that selection for animals carrying the g.-108T>C group of alterations will improve fertility in the dairy cow.

Both the production of cytokines and the distribution of immune cells within the uterus change during early pregnancy. Evidence obtained mainly from mice indicates that these changes are important for implantation and in preventing a maternal immune response to the conceptus. The ruminant embryo also produces interferon tau at this time, the signal for the maternal recognition of pregnancy. The relationship between these events in cows was studied using uteri from three groups of animals on day 16 after observed oestrus: (i) cyclic controls, (ii) pregnant and (iii) inseminated but with no embryo present. Embryo size and the antiviral activity in uterine flushings (indicative of the interferon tau concentration) were measured. Sections of intact uterus were frozen for the localization and quantitation of CD4(+) (T lymphocytes), CD14(+) (macrophages) and CD21(+) (B lymphocytes) uterine cells by immunohistochemistry. The expression of interleukin (IL)-1alpha, IL-2, IL-6 and IL-10 mRNAs in uterine extracts was measured by RT-PCR. Neither embryo size, interferon tau concentration nor pregnancy status influenced the distribution of CD4(+), CD14(+) or CD21(+) cells in the day 16 uterus. Endometrial IL-1alpha mRNA was detected in most cows across the groups, whereas IL-2 mRNA was only present in the non-pregnant uterus. IL-6 and IL-10 mRNAs were not detectable in any uteri. In conclusion, IL-2 mRNA expression is detectable in the non-pregnant but not the pregnant uterus on day 16 and interferon t is unlikely to play a role in the redistribution of immune cells in the uterus during early bovine pregnancy.

The role of interferon in early pregnancy in red deer was investigated by (a) measuring production of interferon by the conceptus, (b) testing the anti-luteolytic effect of recombinant interferon-tau in non-pregnant hinds, and (c) treatment of hinds with interferon after asynchronous embryo transfer. Blastocysts were collected from 34 hinds by uterine flushing 14 (n = 2), 16 (n = 2), 18 (n = 8), 20 (n = 13) or 22 (n = 9) days after synchronization of oestrus with progesterone withdrawal. Interferon anti-viral activity was detectable in uterine flushings from day 16 to day 22, and increased with duration of gestation (P < 0.01) and developmental stage (P < 0.01). When interferon-tau was administered daily between day 14 and day 20 to non-pregnant hinds to mimic natural blastocyst production, luteolysis was delayed by a dose of 0.2 mg day(-1) (27.3 +/- 1.3 days after synchronization, n = 4 versus 21 +/- 0 days in control hinds, n = 3; P < 0.05). Interferon-tau was administered to hinds after asynchronous embryo transfer to determine whether it protects the conceptus against early pregnancy loss. Embryos (n = 24) collected on day 6 from naturally mated, superovulated donors (n = 15) were transferred into synchronized recipients on day 10 or day 11. Interferon-tau treatment (0.2 mg daily from day 14 to 20) increased calving rate from 0 to 64% in all recipients (0/11 versus 7/11, P < 0.005), and from 0 to 67% in day 10 recipients (0/8 versus 6/9, P < 0.01). The increased success rate of asynchronous embryo transfer after interferon-tau treatment in cervids may be of benefit where mismatched embryo-maternal signalling leads to failure in the establishment of pregnancy.

A type I interferon secreted by early sheep and cow conceptuses is responsible for the maternal recognition of pregnancy in these species. Interferon-tau (IFN tau) acts locally on the maternal endometrium to prevent luteolysis and prolong progesterone secretion. The production of IFN tau was investigated in early pregnancy in red deer, Cervus elaphus. The oestrous cycles of 14 hinds were synchronized using intra-vaginal controlled internal progesterone-releasing devices. Hinds were run with a fertile stag, then slaughtered on either day 20 (n = 10) or day 22 after withdrawal of progesterone (n = 11). Conceptuses were recovered after uterine excision and flushing with sterile saline. Conceptus RNA was reverse transcribed and amplified by PCR using primers designed from highly conserved regions of ovine and bovine IFN tau genes. The resulting PCR products were cloned and fully sequenced. Sequence comparisons indicate that the transcript characterized is closely related to the IFN tau and interferon-omega genes of bovids and giraffe, showing > 85% nucleotide sequence homology and > 74% predicted amino acid similarity with previously cloned genes. Northern blot analysis of total conceptus RNA using a homologous IFN tau probe confirmed the high expression of IFN tau which is encoded by a transcript of approximately 1 kilobase. Anti-viral activity was measured in uterine flushes from pregnant hinds using a cytopathic effect inhibition assay (4.3 x 10(3) +/- 0.78 x 10(3) iu ml-1; n = 14), but was not detectable in flushes from non-pregnant hinds (n = 7), confirming that preimplantation red deer conceptuses release interferons. This is the first demonstration of IFN tau expression in a cervid conceptus and provides evidence that IFN tau may be involved in the maternal recognition of pregnancy in red deer.

Summary Mutations within a number of genes have been associated with variations in fertility in various mammals. However, to date there have been no such associations reported for cattle. Herein, we describe three single nucleotide polymorphisms (SNPs) in the luteinizing hormone/choriogonadotropin receptor gene of cattle (Bos taurus). These polymorphisms include two missense mutations and one sense mutation, and all are located in areas of conserved synteny. When assessed in terms of haplotypes, these SNPs were significantly associated with variations in cattle fertility and production traits, most notably on calving interval, days to first service and production index (the UK economic index of milk yield measured in £GB).

An abnormal isoform of the prion protein (PrP) appears to be the agent responsible for transmissible spongiform encephalopathies (TSE). The normal isoform of PrP is host-encoded and expressed in the central nervous system. The recent bovine spongiform encephalopathy (BSE) epidemic in the UK and the incidence of prion-related diseases in other animals could indicate that ruminants are highly susceptible to infection via ingestion of prion-contaminated food. Sequence analysis of PrP gene open reading frames from red deer and camel was carried out to investigate sequence variability of these genes among ruminants.

We wished to confirm the presence of phosphorylation sites in the N‐terminal end of the estrogen receptor (ER) in several mammalian species. Sequencing PCR‐derived genomic fragments coding for the A/B region showed that the bovine receptor contained an extra proline between Gin116 and Leu117 in a ProGlnLeuSerPro motif which is otherwise highly conserved across vertebrate species. The extra proline was also present in red deer but not in the sheep, camel or pig. A motif of five hydrophobic amino acids at positions 64‐68 occurred in all species studied, with the exception of the rabbit, suggesting that the sequence of nine or ten hydrophobic residues at this point in the rat and mouse may be atypical of mammals.

In a forest population of roe deer Capreolus capreolus (Linnaeus, 1758) studied in eastern Poland during the rut, does spent a greater proportion of time feeding and selecting food than bucks, during the dawn and dusk periods of grazing on meadows at the forest boundary. Bucks spent a greater proportion of time in movement (walking and running) and in orientation. These differences occurred at 3 of 4 study sites, and were unaffected by occurrence of social interactions. Although these findings contradict previous data they are predictable on the basis of optimal resource utilization, female reproductive success being determined by access to food, male reproductive success by access to mates.

A sheep type I interferon receptor (oIFNAR1) cDNA was isolated from a λ-ZAP library using a reverse transcription (RT)-PCR product probe generated from oestrous endometrial RNA. The oIFNAR1 cDNA was 79, 66 and 95% homologous to human, murine and bovine IFNAR1 cDNAs respectively. The encoded receptor was a 560-amino acid transmembrane protein 80, 66 and 95% similar to human, murine and bovine IFNAR1 respectively. Northern blot analysis of endometrial mRNA revealed the presence of 6·5, 4·3 and 3·7 kb transcripts. Using semi-quantitative RT-PCR the oIFNAR1 mRNA was not found to be down-regulated after 72 h treatment with bovine recombinant IFN-αI in in vitro experiments with endometrial explants.

The entire coding region of an ovine endometrial oxytocin receptor (OTR) cDNA was generated by PCR, subcloned into the SV40 major late promoter expression vector pSVLJ and transiently expressed in Cos-7 cells. A specific OTR antagonist, 125I-labelled d(CH2)5 [Tyr(Me)2,Thr4,Tyr-NH29]-vasotocin (OTA), was used to describe the binding kinetics of the expressed receptor which had a Kd of 4·5 nm and Bmax of 2·4 nm/mg protein (6·8 × 105 receptor molecules/transfected cell). The functional properties of the expressed OTR were determined by measuring oxytocin-induced phosphoinositide (PI) hydrolysis. Oxytocin increased PI turnover in OTR transfected cells fourfold in excess of residual endogenous activity, and stimulated phospholipase C (PLC) activity in a dose- and time-dependent manner, confirming that the expressed OTR cDNA was functional. Arginine vasopressin also stimulated PI turnover in a dose-dependent manner; thresholds of responses to oxytocin and arginine vasopressin were 10−9 m and 10−7 m respectively. OTA did not increase PI turnover and competitively inhibited the oxytocin-induced response. Direct activation of the pathway by aluminium fluoride and guanosine (3′-Othio)-triphosphate (GTPγS) confirmed that the OTR was G-protein linked. Co-incubation of GTPγS with oxytocin shifted the PI-response threshold from 10−7 m to 10−9 m and significantly increased the level of response, suggesting that maximum PI turnover was agonist-dependent. The G-protein involved in mediating the signal transduction pathway was pertussis toxin-insensitive and, therefore, probably a member of the Gq subfamily. The PLC inhibitor, U73122, had no effect on oxytocin-induced PI turnover, consistent with the response in endometrial tissue. These data suggest that the signalling pathway mediated by expressed OTR is similar to that attributed to OTR occupancy in ovine endometrium. Journal of Endocrinology (1996) 149, 389–396

A bovine trophoblast interferon (IFN-τ) gene promoter sequence (− 450 to +26 bp relative to the transcription start site) led to expression of reporter gene (CAT) constructs transfected into L929 (murine fibroblast) or JAR (human choriocarcinoma) cells. Expression depended on the presence of an exogenous (SV40) enhancer. Poly(I)(C) activated endogenous IFN production in L929 and JAR cells but had no consistent effect on CAT expression. Similar results were obtained in L929 cells with inactivated Newcastle disease virus. There was no 'priming' effect of exogenous Type I IFN. Deletion mutants revealed sites exerting negative control on expression between −338 and −247 bp, and between −150 and −71 bp; these regions contained sequences resembling previously identified negative regulatory domains. In the absence of viral inducibility it is proposed that negative regulation contributes towards the stringent control of expression characteristic of IFN-τ genes.

The superovulation of red deer hinds with eCG is commonly associated with premature luteal regression. This study was an investigation of the endocrine mechanisms regulating luteal function after superovulation. Four groups of hinds (n = 7-8 per group) were treated with progesterone-impregnated intravaginal controlled internal drug-releasing (CIDR) devices for 12 days to synchronize estrus (CIDR device withdrawal = Day 0). Group 1 served as controls; group 2 received an i.m. injection of 0.25 mg estradiol benzoate (EDB) at 72, 84, 96, and 108 h after removal of the device; group 3 received an i.m. injection of 1200 IU eCG at -72 h; group 4 received both EDB and eCG treatments. Oxytocin-induced prostaglandin F2 alpha (PGF2 alpha) release was assessed on Day 4 by oxytocin challenge. Ovarian response was determined by laparoscopy on Day 14. Plasma steroid profiles were determined from thrice-weekly plasma samples collected from Day -13 to Day 35 (progesterone) and Days 0 to 14 (estradiol). EDB increased the incidence of premature luteal regression in monovulatory and eCG-treated animals (p < 0.05) and reduced the number of CL (p < 0.05) in eCG-treated animals. EDB and eCG each elevated plasma concentrations of estradiol and increased the incidence of significant oxytocin-induced PGF2 alpha release. These results support the hypothesis that eCG causes premature luteal regression by inducing prolonged estrogen secretion that sensitizes the endometrium to oxytocin, thereby eliciting PGF2 alpha release during the early luteal phase.

A sheep endometrial oxytocin receptor (OTR) cDNA (1·5 kb) was isolated from a λ-ZAP library using a reverse transcription-PCR product probe generated from oestrous endometrial mRNA. The sheep OTR cDNA shared an overall similarity of 82% with human OTR cDNA, 85% with pig OTR cDNA and 76% with rat OTR cDNA. The encoded receptor was a 391 amino acid polypeptide 94% similar to human OTR, 94% similar to pig OTR and 93% similar to rat OTR. The sheep OTR contained two additional amino acids compared with human OTR which were located in the highly GC-rich third intracytoplasmic loop. This region is thought to be associated with G protein coupling and signal transduction. Expression of the cDNA in Cos-7 cells and measurement of oxytocin-induced phosphoinositide turnover confirmed that it coded for a functional product. The affinity of the expressed receptor was comparable with that observed for the in vivo receptor.

A synthetic 45-mer oligonucleotide corresponding to part of the ovine endometrial oxytocin receptor cDNA was hybridized to sections of ovine uterus collected from 40 ewes at different stages during the oestrous cycle, the first 3 weeks of pregnancy and seasonal anoestrus. The quantity of oxytocin receptor mRNA was measured as the optical density (OD) value on autoradiographs using image analysis. Message first appeared in the luminal epithelium on days 14–15 of the cycle, increasing to a peak OD of 0·48 at oestrus and decreasing again between days 2 and 5. Oxytocin receptor mRNA in the superficial glands, deep glands and caruncular stroma increased between day 15 and oestrus to peak OD values of 0·17, 0·11 and 0·11 respectively, declining again by day 2 and reaching basal values (ODs.e.m.) on days 0–1, but the change was not significant. In pregnant ewes there was no detectable oxytocin receptor mRNA on days 14–15 in any region, but hybridization to the luminal epithelium was present in two of three ewes on day 21. In anoestrous ewes oxytocin receptor mRNA concentrations in all areas of the endometrium were approximately half those measured at oestrus. Optical density readings for oxytocin receptor mRNA in the various uterine compartments were compared with measurements of oxytocin receptors in the same regions as assessed by binding studies using the 125I-labelled oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]-vasotocin (125I-labelled OTA). In the endometrium, receptor mRNA and 125I-labelled OTA binding patterns changed in parallel, and both sets of measurements were significantly correlated (P125I-labelled OTA binding occurred at oestrus; this was not accompanied by a similar increase in oxytocin receptor mRNA hybridization. This study helps to confirm that the previously identified cDNA clone is derived from the ovine oxytocin receptor, as patterns of oxytocin receptor mRNA expression in the endometrium closely resembled those of oxytocin binding. Maximum expression and binding both occurred at oestrus, suggesting that regulation of the oxytocin receptor gene in the uterus occurs principally at the transcriptional, rather than at the translational, level. Failure to detect a significant increase in myometrial mRNA expression at oestrus may indicate that the endometrial and myometrial oxytocin receptors are of different isoforms.

The 5' end of exon C of the bovine estrogen receptor alpha gene (bov-ESR1) includes a unique G-rich insert, not found in other closely related mammalian genes, which lies close to both a double E-box transcription factor binding site and the site of a single nucleotide (G/A) polymorphism. Biophysical studies, using CD and UV absorbance measurements, show that this 22 base insert leads to the formation of a family of stable G-quadruplex structures which are unaffected by the G/A polymorphism. Multiplex PCR shows that the region including the G-quadruplex is transcribed into RNA, and studies with a synthetic RNA transcript sequence demonstrated formation of a highly stable parallel-folded quadruplex structure. Luciferase reporter constructs demonstrate that the G-rich sequence reduces rates of translation when present in the 5'-UTR of mRNA transcripts. Mutations (GGG to AAA) that destabilize the quadruplex lead to a 15-fold enhancement of translational efficiency, suggesting that a possible biological role of the insert in exon C of the bov-ESR1 is to regulate translation of this exon.

Tissue-specific expression of the human estrogen receptor alpha gene (ESR1) is achieved through multiple promoter sequences resulting in various mRNA transcripts encoding a common protein but differing in their 5'-untranslated region (5'-UTR). Many cancers are estrogen-sensitive with neoplastic growth stimulated through the estrogen receptor, a transcription factor that regulates developmental genes. We demonstrate that the human ESR1 gene is rich in potential quadruplex-forming sequences with 3 of 20 identified within exonic regions. In particular, we show using CD, UV, and NMR spectroscopy that a stable DNA G-quadruplex motif is formed within the exon C gene sequence. This motif, which PCR shows is transcribed in normal and neoplastic endometrium and in MCF-7 cells, forms a stable RNA quadruplex demonstrable by CD and UV analysis. Cloning the exon C G-quadruplex sequence upstream of a luciferase reporter gene caused a 6-fold reduction of enzymatic activity compared to a mutant sequence. We conclude that the exon C G-quadruplex motif is present in the 5'-UTR of the mRNA transcript, where it modulates the efficiency of translation.

The decline of dairy cattle fertility worldwide remains a major concern, with conception rates to first service commonly below 40%. The length and severity of negative energy balance postpartum are unfavorably correlated with fertility, suggesting that the length and severity of negative energy balance and fertility are linked via several hormones or metabolites. These compounds therefore have the potential to predict fertility at a genetic level. The addition of a predictor trait for fertility into present fertility indices would accelerate genetic gain, particularly if it was expressed before adulthood. The objective of this work was to estimate the genetic variation in several metabolites and hormones in calves, and to determine their genetic relationships with fertility and production through sire predicted transmitting abilities (PTA; sires of calves sampled). Circulating concentrations of free fatty acids (FFA), glucose, growth hormone (GH), insulin, and insulin-like growth factor 1 (IGF-1) in male and female UK Holstein-Friesian dairy calves (average age ± SD; 126 ± 12.7 d) were analyzed during 2 studies: data set 1 (n = 496 females; 1996-2001; 7 commercial dairy herds) and data set 2 (n = 326 females, n = 256 males; 2002-2006; multiple ovulation and embryo transfer breeding scheme). Univariate mixed models were fitted to the data using ASREML. Basal concentrations of FFA, glucose, GH, insulin and total IGF-1 were all moderately heritable in both sexes (heritability range ± SE; 0.09 ± 0.05 to 0.66 ± 0.14). The sire PTA for protein percentage had significant regression coefficients and approximate genetic correlations with FFA and insulin, and the sire PTA for calving interval had significant regression coefficients and approximate genetic correlations with GH. Additive genetic variance seems responsible for a moderate proportion of the phenotypic variation in important metabolites and regulatory hormones in male and female UK Holstein-Friesian dairy calves, therefore supporting further investigation into their use as juvenile predictors for fertility in the mature female The decline of dairy cattle fertility worldwide remains a major concern, with conception rates to first service commonly below 40%. The length and severity of negative energy balance postpartum are unfavorably correlated with fertility, suggesting that the length and severity of negative energy balance and fertility are linked via several hormones or metabolites. These compounds therefore have the potential to predict fertility at a genetic level. The addition of a predictor trait for fertility into present fertility indices would accelerate genetic gain, particularly if it was expressed before adulthood. The objective of this work was to estimate the genetic variation in several metabolites and hormones in calves, and to determine their genetic relationships with fertility and production through sire predicted transmitting abilities (PTA; sires of calves sampled). Circulating concentrations of free fatty acids (FFA), glucose, growth hormone (GH), insulin, and insulin-like growth factor 1 (IGF-1) in male and female UK Holstein-Friesian dairy calves (average age ± SD; 126 ± 12.7 d) were analyzed during 2 studies: data set 1 (n = 496 females; 1996-2001; 7 commercial dairy herds) and data set 2 (n = 326 females, n = 256 males; 2002-2006; multiple ovulation and embryo transfer breeding scheme). Univariate mixed models were fitted to the data using ASREML. Basal concentrations of FFA, glucose, GH, insulin and total IGF-1 were all moderately heritable in both sexes (heritability range ± SE; 0.09 ± 0.05 to 0.66 ± 0.14). The sire PTA for protein percentage had significant regression coefficients and approximate genetic correlations with FFA and insulin, and the sire PTA for calving interval had significant regression coefficients and approximate genetic correlations with GH. Additive genetic variance seems responsible for a moderate proportion of the phenotypic variation in important metabolites and regulatory hormones in male and female UK Holstein-Friesian dairy calves, therefore supporting further investigation into their use as juvenile predictors for fertility in the mature female

Polyunsaturated fatty acids (PUFAs) induce COX-2 in bovine endometrial stromal cells through activation of peroxisome-proliferator-activated receptor alpha (PPARalpha). We have investigated alternative (PPAR-independent) pathways to COX-2 induction using a reporter construct driven by a COX-2 gene promoter sequence lacking a PPAR response element. This construct was induced by PUFAs, but not by PPAR agonists. PPAR-independent reporter gene expression occurred 6h after PPAR-dependent induction of the endogenous COX-2 gene. In contrast to PPAR-dependent COX-2 induction, which is not affected by NF-kappaB inhibitors, the PPAR-independent pathway was blocked by the NF-kappaB inhibitor MG132 or following deletion of NF-kappaB sites in the COX-2 promoter. The PPAR-independent effect of PUFA was mimicked by the PKC activators 4beta-PMA and prostaglandin F(2alpha), but was not blocked by the PKC inhibitor RO318425. The results demonstrate a pathway to the induction of COX-2 by PUFAs requiring NF-kappaB but not PPAR or PKC.

We accept that we are responsible for the quality of life of animals in our care. We accept that the activities of man affect all the living things with which we share this planet. But we are slow to realize that as a result we have a duty of care for all living things. That duty extends to the breeding of animals for which we are responsible. When animals are bred by man for a purpose, the aim should be to meet certain goals: to improve the precision with which breeding outcomes can be predicted; to avoid the introduction and advance of characteristics deleterious to well-being; and to manage genetic resources and diversity between and within populations as set out in the Convention on Biological Diversity. These goals are summed up in the phrase precision animal breeding. They should apply whether animals are bred as sources of usable products or services for medical or scientific research, for aesthetic or cultural considerations, or as pets. Modern molecular and quantitative genetics and advances in reproductive physiology provide the tools with which these goals can be met.

This study describes a digital technique for uterine morphometry and its application to endometrial structure during the bovine oestrous cycle. Neither the number nor the size of uterine gland ducts changed during the cycle but a reduction in total endometrial area from days 0 to 8 after oestrus led to an increase in the proportion of the endometrium occupied by gland ducts (gland duct density). This effect on day 8 was maintained to day 16. When endometrial morphology was related to circulating progesterone concentrations on days 5 and 8 of the luteal phase, no relationships were found on day 5, but on day 8, a high progesterone concentration was associated with an increased number of gland ducts. Furthermore, in animals slaughtered on day 8, a high progesterone concentration on day 5 was associated with decreased gland duct size, though a simultaneous decrease in endometrial area led to an increase in gland duct density. The results suggest that contrary to expectation, endometrial glands do not grow and regress during the oestrous cycle, although cyclic changes in endometrial area controlled by progesterone lead to changes in gland duct density.

Second messenger signalling through cyclic AMP (cAMP) plays an important role in the response of the endometrium to prostaglandin (PG) E2during early pregnancy. Arachidonic acid, which is a by-product of the luteolytic cascade in ruminants, is a potential paracrine signal from the epithelium to the stroma. We investigated the effects of arachidonic acid on the response of the stroma to PGE2. cAMP was measured in bovine endometrial stromal cells treated with agents known to activate or inhibit adenylyl cyclase, protein kinase C (PKC) or phosphodiesterase (PDE). PGE2increased the intracellular cAMP concentration within 10 min, and this effect was attenuated by arachidonic acid and the PKC activator, 4β-phorbol myristate acetate (PMA). The inhibitory effect of arachidonic acid on PGE2-induced cAMP accumulation was prevented by the PKC inhibitor, RO318425, and was absent in cells in which PKC had been downregulated by exposure to PMA for 24 h. The effect of arachidonic acid was also prevented by the PDE inhibitor, 3-isobutyl-1-methylxanthine. Arachidonic acid was shown by immunoblotting to prevent induction of cyclooxygenase-2 by PGE2, forskolin or dibutyryl cAMP. The results indicate that arachidonic acid activates PDE through a mechanism involving PKC, counteracting a rise in intracellular cAMP in response to PGE2. The data suggest that arachidonic acid antagonizes PGE2signalling through cAMP in the bovine endometrium, possibly acting to ensure a rapid return to oestrus in the case of failure of the maternal recognition of pregnancy.

The incidence of low day 5 milk progesterone in dairy cows has been investigated and the efficacy of treating the problem assessed. The incidence of inadequate milk progesterone (empirically defined as

The objective of this study was to estimate genetic parameters for measures of luteal activity during the first 60 d postpartum. Analyses were made with different sampling intervals to investigate the possibility of combining progesterone measurement with routinely performed milk recording. Progesterone level in milk as an indicator of female fertility when selecting sires in a progeny-testing scheme was also examined. Data were collected from 1996 to 1999, and comprised 1,212 lactations from 1,080 British Holstein-Friesian cows at 8 commercial dairy farms in the United Kingdom. Milk samples for progesterone analysis were collected thrice weekly. Mixed linear animal models were used to analyze the data. Heritability for the percentage of samples with luteal activity during the first 60 d postpartum (PLA) was 0.30 and decreased with more infrequent sampling to 0.25, 0.20, and 0.14 for weekly, twice-monthly, and monthly sampling, respectively. Measures of PLA had a high negative genetic correlation with prolonged anovulation (-0.53 for monthly sampling, 0.65 if at least twice-monthly sampling). Genetic correlations with interval from calving to commencement of luteal activity were close to -1 for all PLA measurements and the selection index calculations showed that monthly progesterone sampling could be used with high accuracy (0.80 with 50 daughters per bull) to predict breeding values for commencement of luteal activity. Progesterone analysis at the time of regular milk recording could thereby be used to select for an early interval from calving to commencement of luteal activity and, at the same time, a decreased frequency of prolonged anovulation during the postpartum period.

In several transformed cell lines, the growth factors IGF-I and epidermal growth factor (EGF) activate second messenger systems that cause the phosphorylation of the estrogen receptor (ER). One kinase catalysing receptor phosphorylation is mitogen activated protein (MAP) kinase, and the result of phosphorylation is an increase in receptor transactivation function. EGF and IGF-I, secreted locally and systemically, are involved in uterine–conceptus interactions in early pregnancy, and therefore it is of interest to determine whether these growth factors affect ER function in the uterus. An estrogen response element, chloramphenicol acetyl transferase reporter gene construct (CATERE) was transfected into bovine endometrial epithelial and stromal cells in vitro , and CAT measured during transient expression. Growth factors were added at various times following transfection, and MAP kinase phosphorylation was monitored by western blotting of p42 and p44. The MEK inhibitor U 0126 was used to determine whether the effect of IGF-I on CATERE expression was mediated through MAP kinase, and the anti-estrogen ICI 182780 was used to identify effects involving the ER. In stromal cells, reporter gene activity was increased in a dose dependent manner by IGF-I or hEGF in the presence or absence of estradiol-17β. In the absence of estradiol the effect of IGF-I was not inhibited by ICI 182780. The effect of IGF-I occurred within an hour, before any detectable increase in cell proliferation, and the activation of CAT expression in response to IGF-I or EGF was blocked by U 0126. In contrast to their effects in stromal cells, neither IGF-I nor EGF affected CAT expression in bovine endometrial epithelial cells. Measurement of phosphorylated MAP kinases p42/p44 by western blotting showed that EGF but not IGF-I activated MAP kinase phosphorylation in both epithelial and stromal cells. In stromal cells, the fact that U 0126 blocked the CAT responses to IGF-I and EGF indicates the involvement of a MAP kinase. But since IGF-I did not activate p42/p44, a different MAP kinase, not detected by the antibody used here, is implicated. As the response was not blocked by ICI 182780, we conclude this effect is independent of ER activation. Therefore in bovine uterine cells in culture effects on MAP kinases p42/p44 can be dissociated from those on ERE-dependent gene expression, and reporter gene expression may be independent of ER activation.

The objective of this study was to estimate the heritability of a number of traditional and endocrine fertility traits in addition to d-56 predicted milk yield (MY56), and the genetic and phenotypic correlations between these traits. Various fixed effects such as season, year, herd, lactation number, diet, percentage Holstein (PCH) of the cow, and occurrence of uterine infection (UI), dystocia (DYS), and retained placenta (RP) were also investigated. Data collected for 1212 lactations of 1080 postpartum (PP) Holstein-Friesian dairy cows in eight commercial farms between 1996 and 1999 included thrice weekly milk progesterone samples, calving and insemination dates, various reproductive health records, monthly/bimonthly production records, three-generation pedigrees, and PCH information. Genetic models were fitted to the data to obtain heritabilitites and correlations using ASREML. Estimates of heritability for interval to commencement of luteal activity PP (InCLA), length of the first luteal phase PP (lnLutI) and occurrence of persistent CL type I (PCLI) were 0.16, 0.17, and 0.13, respectively. Heritabilities for pregnancy to first service (PFS), interval to first service (IFS), and MY56 were 0.14, 0.13, and 0.50, respectively. Genetic regressions of InCLA and lnLut1 on PTA of the sire for milk, fat, and protein yields., and PIN95 were investigated. Regressions of InCLA were positive and significant on fat yield, while regressions of InLut1 on both protein yield and PIN95 were negative and significant. Genetic correlations of endocrine fertility traits (InCLA, lnLutI, and PCLI) with MY56 were high (0.36, P

The maternal recognition of pregnancy in ruminants requires the production of interferons by the preimplantation blastocyst. These proteins, the trophoblast interferons (IFN-tau), are the products of a number of similar genes, the expression of which is controlled by characteristic promoter regions. They are expressed for a short period in high concentrations, and have antiluteolytic, antiviral, antiproliferative and immunomodulatory effects, through receptors on the endometrial epithelium. The antiluteolytic effects of IFN-tau result from inhibition of endometrial expression of the oxytocin receptor, through which circulating oxytocin stimulates episodic prostaglandin F2a production. Some of the properties of IFN-tau differ from those of other type I interferons, and they may have novel therapeutic effects. Because of their central role in early gestation, these proteins have excited the interest of reproductive physiologists. However, their other properties, and the fact that their expression is controlled so precisely, have made them of interest to a wide range of biologists.

The calving rate of the modern dairy cow is declining at approximately 1%per annum. First service conception rates are now below 40%, and the average cow requires more than two inseminations to get her in calf. We review here genetic, endocrine and nutritional strategies for reducing subfertility, and we emphasize the role of milk progesterone data in the analysis of the condition. Milk progesterone data underpin three approaches to treating subfertility; firstly, they allow the identification of specific reproductive abnormalities which can be treated pharmacologically. Secondly, they show that at least one endocrinopathy is heritable, thereby providing the basis for a selective breeding strategy. Thirdly, they provide a means for monitoring ovarian (and indirectly uterine and conceptus) function during early pregnancy, which are central to the successful establishment of pregnancy, and are amenable to dietary modification. These approaches to reversing the falling fertility of dairy cows are characterized by different timescales.

Exposure to type I interferons (IFN) increased estrogen receptor (ER) ligand binding and induced protein kinase C (PKC) translocation within 30 min but had no effect on net incorporation of [32P] into ER in Madin Darby bovine kidney (MDBK) cells. Ligand binding was also increased within 30 min by phorbol ester and the protein phosphatase inhibitor okadaic acid. Mitogen-activated protein (MAP) kinase phosphorylation was initially inhibited between 2 and 30 min and subsequently activated between 30 and 60 min after treatment with IFN. The activatory response was blocked by the PKC inhibitor Ro 31-8220. Following transient transfection with an ERE-CAT reporter construct, IFN increased CAT expression after 6 h but decreased ER ligand binding, transcriptional activity and phosphorylation after 48 h, probably as a result of decreased ER concentrations. The results rule out rapid activation of ER ligand binding through phosphorylation at Ser118 by MAP kinase because (1) the increase in ligand binding preceded activation of MAP kinase, and (2) IFN had no short-term effect on [32P]incorporation or ER transcriptional activity. The rapid effect of IFN on ER ligand binding is postulated to reflect phosphorylation of the receptor at Tyr537 by p56lck, a member of the Src family of PKC-activated tyrosine kinases.

Combination of suppression polymerase chain reaction (PCR) and megaprimer method of site-directed mutagenesis allows specific reamplification of extended megaprimer. This modification is proposed for templates that are difficult to amplify when standard megaprimer procedures do not generate sufficient yield of mutated product.

Luteolysis in sheep is associated with uterine secretion of pulses of prostaglandin F2 alpha (PGF2 alpha) due to the action of luteal oxytocin on endometrial oxytocin receptors. For pregnancy to become established inhibition of oxytocin receptors is important as an antiluteolytic mechanism. The maternal recognition of pregnancy in cattle and sheep involves production, by the trophoblast, of a type 1 interferon (IFN-tau) that suppresses uterine development of oxytocin receptors and the generation of luteolytic episodes of PGF2 alpha. The action of IFN-tau in surgically prepared unilaterally pregnant ewes was investigated. Finn-Dorset ewes were anaesthetized on day 6 or 7 of the oestrous cycle and one uterine horn was surgically isolated at the uterine bifurcation from the body of the uterus. Ewes were mated at the subsequent oestrus either by a fertile or by a vasectomized ram and killed on day 13 or 16 after mating. On day 16, in the non-pregnant ewes, there was no measurable uterine IFN-tau but there were high concentrations of oxytocin receptors in both horns. In the pregnant ewes on day 16 after mating, the oxytocin receptor concentration was 45 +/- 11 fmol mg-1 protein in the pregnant horn and 585 +/- 131 fmol mg-1 in the non-pregnant horn. Antiviral activity was 5.8 x 10(7) +/- 5.2 x 10(7) U ml-1 in the pregnant horn and 2.9 x 10(3) +/- 1.2 x 10(3) U ml-1 in the non-pregnant horn. Thus, 16 days after mating, the pregnant horn exhibited high antiviral activity but oxytocin receptors were suppressed, while in the same endocrine environment (characteristic of pregnancy) there were low IFN-tau and high oxytocin receptor concentrations in the isolated horn equivalent to those expected at the onset of luteolysis. In situ hybridization to ovine mRNA encoding the oxytocin receptor and autoradiographic studies using the 125I-labelled oxytocin antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-vasotocin both showed that the large amount of oxytocin receptor message and binding sites in the endometrium of the isolated horn were localized in the luminal epithelium. Immunocytochemical studies showed that there was a suppression of oestradiol receptors in the pregnant horn but high concentrations equivalent to those at oestrus were present in the isolated horn. The content of progesterone receptors was low in the stromal tissue only in both horns, a pattern of localization similar to that seen in the late luteal phase and in early pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

The oxytocin receptor, a seven transmembrane domain, G protein-linked receptor molecule, plays a central role in determining the endocrine function of the ruminant uterine endometrium. During non- pregnant cycles the control of this molecule by circulating steroid hormones leads to regression of the corpora lutea. The kinetics of the mechanisms involved determine the time at which luteolysis occurs, and therefore the length of the oestrous cycle. In pregnancy, secretions of the trophoblast block endometrial oxytocin receptor gene expression and lead to luteal maintenance. An understanding of the molecular mechanisms involved in the steroidal control of oxytocin receptor gene expression will provide an explanation for the relative constancy of oestrous cycle lengths in non-pregnant animals. Unravelling the way in which trophoblast products block expression of the oxytocin receptor gene will lead to a better understanding of the reasons for the high rate of embryonic loss in domestic ruminants.

The oxytocin receptor (OTR), a member of the seven-transmembrane domain guanine-nucleotide-binding protein (G protein) coupled receptor family plays a central role in lactation, ovarian cyclicity and reproductive behaviour. Recent cloning and sequencing unexpectedly revealed that the third intracytoplasmic region (3ICR) of the sheep receptor has 3 and 2 additional amino acids (aa) relative to the rat and human receptors, respectively. We have now confirmed, by sequencing polymerase chain reaction (PCR)-derived genomic fragments coding for the OTR 3ICR from a variety of ruminant and non-ruminant species, that additional aa are a general phenomenon in ruminants.

Analysis of ovine conceptus RNA by slot blotting, Northern analysis and nested polymerase chain reaction failed to detect oxytocin–neurophysin prohormone mRNA. Probes used hybridized with both the 3' end of the prohormone mRNA and the oxytocin-coding sequence. Northern analysis of bovine and porcine conceptus RNA was also negative, and polymerase chain reaction demonstrated oxytocin–neurophysin mRNA in ovine corpus luteum, but not in human corpus luteum or decidua, or in ovine endometrium. Infusion of oxytocin into the uterine lumen in cyclic ewes between days 9 and 19 or 20 after oestrus failed to prolong the luteal phase of the cycle and had no effect on endometrial oxytocin receptor concentrations or uterine prostaglandin F secretion. Oxytocin administered systematically prevented luteolysis and reduced uterine prostaglandin F secretion. Taken together, these data suggest that blastocyst-derived oxytocin is unlikely to contribute to corpus luteum maintenance in early pregnancy. They are inconsistent with a previous report that the ovine blastocyst synthesizes and secretes oxytocin. Journal of Endocrinology (1991) 130, 443–449

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.

A cloned cDNA has been isolated by probing a sheep blastocyst cDNA library using a synthetic oligonucleotide representing the N-terminal amino acid sequence of the antiluteolytic protein, ovine trophoblast protein-1. Sequence analysis of the cDNA confirms the 70% homology between the antiluteolysin and the interferon-α family of proteins; however, the sequence reported here differs at several points from previously reported amino acid and cDNA sequences for the antiluteolysin. In-vitro translation of day-16 poly(A)+ RNA indicated that antiluteolysin mRNA is a major constituent of total mRNA at this stage of blastocyst development, and Northern blotting confirmed that antiluteolysin mRNA production occurred between days 13 and 22 after oestrus. This is consistent with the stage at which embryonic extracts are antiluteolytic on administration in vivo. These and other data confirm that the ovine trophoblast antiluteolysin is an interferon, and suggest that at least five isoforms of this protein may exist.