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Adoption of optimal management techniques for rearing dairy calves has significant effects on their health, welfare, and productivity. Despite much published literature on best practice, calf morbidity and mortality rates remain high. This survey aimed to establish current calf management practices in the United Kingdom, along with farmer perceptions surrounding different housing types. A survey containing 48 questions was distributed online to UK farmers via social media, online forums, and a convenience sample of veterinary practices and was completed by 216 participants. A descriptive analysis with frequency distributions was calculated, with chi-squared tests, linear regression and multinomial regression performed to assess associations between variables. There was a low level of regular veterinary involvement in day-to-day health decision making for calves (3/216, 1.4%), highlighting the need for appropriate staff training and standard operating procedures to ensure prudent antimicrobial usage. Restricted calf milk feeding remains highly prevalent in the United Kingdom, with most calves fed milk replacer (114/216, 52.8%), twice daily (189/216, 87.5%), initially given milk at 4 L/d (66/216, 30.6%) or 6 L/d (47/216, 21.8%). There was, however, a small number of farmers initially feeding only 2 to 3 L/d (28/216, 13.0%). Euthanasia of bull calves (5/216, 2.3%) and feeding antimicrobial waste milk to calves (8/216, 3.7%) both occurred on some farms. With regard to housing, use of individual calf pens has reduced from around 60% in 2010 to 38.4% in this study (83/216), with this reduction being partly driven by the policy of UK milk buyers. Farmer perceptions indicated that individual housing was thought to help to improve calf health and feed monitoring of calves, suggesting that successful use of group housing requires a higher level of stockmanship. The majority of farmers did not provide fresh bedding to calves on a daily basis (141/216, 65.3%), and relatively few disinfected both the calf housing (38.0%) and ground (47.7%) between calves, suggesting that hygiene practices may require additional attention in farm management protocols.

Growth rates in pre-weaned calves influence their health, age at first calving and lifetime productivity. Many farms restrict milk rations to encourage solid feed intake and facilitate early weaning, but this can compromise growth. This study determined the milk feeding policies and associated growth rates on 11 commercial dairy farms in South East England, each following their normal management regime. Between 26 and 54 heifers were recruited per farm, providing a final cohort of 492, of which 71% were pure Holstein. Information on calf rearing practices (feeding, weaning, housing) and health was collected via questionnaires and weekly observations. Estimates of actual milk fed (kg solids) between 1 and 63 days were calculated for individual calves. Morphometric data (weight, height, length) were taken at weeks 1, 5 and 9 and at a median age of 7.5 months and growth rates were calculated. Most calves were fed milk replacer via automated feeders (four farms), teat feeder (one) or buckets (four) whereas two farms provided drums of acidified waste milk. Farms fed between 4 and 6 l/day of milk at mixing rates of 10% to 15%, providing 400 to 900 g/day of milk solids. Both skeletal growth rates and average daily weight gain (ADG) increased in the second month of life compared with the first: height growth from 0.17±0.14 to 0.25±0.16 cm/day and ADG from 0.48±0.25 to 0.71±0.28 kg/day. Post-weaning heifers up to 7.5 months had height increases of 0.16±0.035 cm/day and ADG of 0.83±0.16 kg/day. From 1 to 63 days 70% of calves had growth rates

During luteal regression episodic pulses of oxytocin secretion become coupled to the release of prostaglandin F2 alpha (PGF2 alpha) following synthesis of endometrial oxytocin receptors, but in early pregnancy the inhibition of oxytocin receptor formation by the conceptus prevents the development of the pulsatile pattern of PGF2 alpha release needed to achieve luteolysis. Oxytocin receptors are present on the luminal epithelium in ovariectomized and anoestrous ewes, in pregnant animals throughout most of gestation (day 21 to term) and in explants of endometrial tissue cultured in vitro. These receptors can be downregulated for a brief period by progesterone (10-12 days in sheep, 12-14 days in cattle). This period of inhibition can be extended by infusion of interferon tau (IFN-tau) (which probably inhibits oxytocin receptor gene transcription) or of oxytocin into the systemic circulation (which may act further downstream, possibly at the level of translation). Oxytocin receptors also develop on the caruncular stroma and deep glands at oestrus, but these need positive upregulation and appear dependent on an oestrogenic environment. Only epithelial receptors are needed to achieve a maximal PGF2 alpha response to an oxytocin challenge, but the presence of oxytocin receptors does not necessarily confer responsiveness as other factors may influence intracellular coupling mechanisms and precursor availability. The duration of the luteal phase is regulated by the time of the initial post-ovulatory rise in progesterone and the duration of exposure to progesterone.(ABSTRACT TRUNCATED AT 250 WORDS)

Absent or irregular ovarian cycles in lactating dairy cows are caused by failure to ovulate the dominant follicle at the appropriate time. The follicle then either regresses or develops into a cyst. This process can be triggered by a variety of metabolic and disease factors that act at the hypothalamus and pituitary gland to inhibit pulsatile LH secretion and the LH surge, and at the ovary to reduce follicular growth and oestradiol production. Cows of poor energy status have low circulating concentrations of insulin-like growth factor I (IGF-I). Predisposing factors include calving difficulties, inappropriate diet, reduced intake of dry matter and a high rate of body condition score loss. Various stressors predispose the follicle to cyst development by inhibiting the LH surge and ovulation; these include common infections, such as mastitis. Even when ovulation does occur, poor follicular development may result in production of an inadequate corpus luteum. The timing of the increase in progesterone in the early luteal phase (days 4-5) appears to be a key determinant of fertility, probably because it alters the secretory activity of the reproductive tract, thus influencing embryonic growth and interferon-tau production. A period of negative energy balance after calving can reduce fertility even though metabolic parameters have apparently improved at the time of service.

Early pregnancy loss (EPL) between days 15-65 after breeding has been shown to occur in 7.9% of equine pregnancies with substantial economical, welfare and safety implications. Whilst maternal age has been recognised as an important risk factor in relation to the incidence of EPL, few other risk factors have been conclusively identified. Further, multivariable data analysis of risk factors for EPL is sparse. A prospective cohort investigation of thoroughbred broodmares in the United Kingdom was conducted over the 2013 and 2014 breeding seasons. Information relating to 28 factors including mare, stallion, pregnancy and therapeutic interventions was collected using questionnaires and entered into a custom-designed Microsoft Access database. Mixed effects logistic regression was used to determine risk factors for EPL, including 'mare' as a random effect to account for repeat pregnancies in the same mare. Stallion, stud and veterinarian were also evaluated as random effects. Variables with a p-value of0.25 in univariable analysis were taken forward for consideration in the multivariable model which was built using a forward stepwise approach. Data were collected on 2245 pregnancies in 1753 mares. Increasing mare age (OR = 1.11, 95% confidence interval (CI) = 1.04, 1.18, p = 0.001), having had one previous foal (OR = 3.52, 95% CI = 1.56, 7.95, p = 0.002) and presence of uterine cysts (OR = 1.76, 95% CI = 1.07, 2.91, p = 0.03) were all associated with increased odds of EPL following multivariable analysis. Increasing day 15/16 scan vesicle size (OR = 0.24, 95% CI = 0.16, 0.38, p 0.001) and the use of ovulatory induction agents (OR = 0.31, 95% CI = 0.17, 0.55, p 0.001) were negatively associated with EPL. Stallion, stud and veterinarian were not significantly associated with EPL. Analysis of a subpopulation of 344 multiple (twin and triplet) pregnancies found that the use of flunixin meglumine at the time of manual reduction of a multiple pregnancy resulted in reduced odds of EPL (OR = 0.34, 95% CI = 0.14, 0.84, p = 0.02). Results from this study can be used by stud farm personnel when assessing their broodmare population and by clinicians when deciding upon therapeutic strategies. Additional work can be focused around these risk factors to further our understanding of the pathophysiology of EPL.

Therapeutic practices in equine reproductive medicine have dramatically evolved over the last 20 years but current usage is not described. The aims of this study were to provide a description of medication use and clinical findings of reproductive examinations alongside measures of reproductive efficiency in thoroughbreds. A prospective cohort study was conducted in the 2013 and 2014 breeding seasons. Mare and stallion details, information on veterinary interventions and findings of reproductive ultrasound scans were collected using questionnaires and entered into a custom-designed Microsoft Access database. Descriptive summary statistics were derived directly from the database and using Microsoft Excel. Information was collected from 2246 pregnancies in 1754 mares from 29 stud farms. Ovulatory induction agents were used in 91.8% of cases, oestrus induction agents in 38.4% and covering therapies in 62.7%. Intrauterine antimicrobials were used in 49.6% of mares. Single pregnancies accounted for 83.9% of pregnancies, twins for 15.3% and triplets for 0.7%. The overall incidence of pregnancy loss between days 15–42 was 6.4% (95% CI 5.4%, 7.4%) and 1.6% (95% CI 1.1%, 2.1%) between days 43–65. A further 1.3% of pregnancies were lost by October and 4.5% by birth (including stillbirths). Eighty-three percent of all pregnancies resulted in a live foal. In conclusion, there has been a considerable increase in the use of reproductive therapeutics over the last 12 years. Nonetheless, incidence of pregnancy loss and live foal percentages remain essentially unchanged. Risk factor studies are required to determine if the substantial increase in therapeutic usage is conferring positive benefits.

The rearing period has a key influence on the later performance of cattle, affecting future fertility and longevity. Producers usually aim to breed replacement heifers by 15 months to calve at 24 months. An age at first calving (AFC) close to 2 years (23 to 25 months) is optimum for economic performance as it minimises the non-productive period and maintains a seasonal calving pattern. This is rarely achieved in either dairy or beef herds, with average AFC for dairy herds usually between 26 and 30 months. Maintaining a low AFC requires good heifer management with adequate growth to ensure an appropriate BW and frame size at calving. Puberty should occur at least 6 weeks before the target breeding age to enable animals to undergo oestrous cycles before mating. Cattle reach puberty at a fairly consistent, but breed-dependent, proportion of mature BW. Heifer fertility is a critical component of AFC. In US Holsteins the conception rate peaked at 57% at 15 to 16 months, declining in older heifers. Wide variations in growth rates on the same farm often lead to some animals having delayed first breeding and/or conception. Oestrous synchronisation regimes and sexed semen can both be used but unless heifers have been previously well-managed the success rates may be unacceptably low. Altering the nutritional input above or below those needed for maintenance at any stage from birth to first calving clearly alters the average daily gain (ADG) in weight. In general an ADG of around 0.75 kg/day seems optimal for dairy heifers, with lower rates delaying puberty and AFC. There is some scope to vary ADG at different ages providing animals reach an adequate size by calving. Major periods of nutritional deficiency and/or severe calfhood disease will, however, compromise development with long-term adverse consequences. Infectious disease can also cause pregnancy loss/abortion. First lactation milk yield may be slightly lower in younger calving cows but lifetime production is higher as such animals usually have good fertility and survive longer. There is now extensive evidence that as long as the AFC is >23 months then future performance is not adversely influenced. On the other hand, delayed first calving >30 months is associated with poor survival. Underfeeding of young heifers reduces their milk production potential and is a greater problem than overfeeding. Farmers are more likely to meet the optimum AFC target of 23 to 25 months if they monitor growth rates and adjust feed accordingly.

This study was conducted to elucidate mare cervical dilation mechanisms by testing two hypotheses: (i) the proportion of collagen staining in histological samples of mare cervices and (ii) the abundance of hormone receptors in the equine cervix differ with stage of the oestrous cycle and site within the cervix. Tissues and jugular vein blood samples were collected from 15 mares. Collagen content was assessed using Masson’s Trichome staining. Receptor abundance was assessed using RT-PCR, qRT-PCR and immunohistochemistry. In sub-epithelial stroma, there was less collagen during the follicular than luteal phase, in the caudal- (P = 0.029), mid- (P = 0.0000) and cranial (P = 0.001) cervical tissue. In the deep stroma, there was less collagen staining during the follicular stage in the mid- (P = 0.004) and cranial- (P = 0.041) cervical regions. There were PTGER2, PTGER3, PGR and ESR1 mRNA transcripts in the cervix. A greater proportion of cells were positive for ESR1 protein during the follicular phase in sub-epithelial (P = 0.019) and deep (P = 0.013) stroma. The abundance of ESR1 in the epithelium was negatively correlated with collagen staining in sub-epithelial (P = 0.007) and deep (P = 0.005) stroma. The results of the study provide new information about the cervical biology of mares by increasing the knowledge about collagen content and the relationship between collagen content and ESR1 protein abundance during the oestrous cycle which indicates the ESR1 receptor is a candidate for involvement in control of cervical dilation.

Context Pre-weaning growth in dairy heifers is highly dependent on the amount of milk fed. Both milk replacer (MR) and associated labour are costly, encouraging restricted milk rations and once-a-day feeding. Aims This study compared performance relating to the growth and health of calves receiving one of two commercial feeding regimens: High or Low. Methods All heifers born during the Spring (January–March) calving block on a commercial UK farm with mixed-breed genetics were recruited at birth, randomly assigned to the High (n = 104, receiving MR-A) or Low (n = 88, receiving MR-B) feed group and reared indoors on straw bedding, with free access to concentrate. Both groups initially received MR twice daily. The High group continued to receive MR twice daily throughout the experiment, whereas the Low group calves were reduced to a single MR feed daily during Weeks 4–8. Blood samples were taken in Weeks 1 and 6 to assess passive transfer and measure circulating insulin-like growth factor 1 (IGF1). The Wisconsin calf-scoring system was used to assess health of calves in Weeks 1, 2, 4, 6 and 8 and at 6 months and size was also measured at these times. Data were analysed by univariate and multivariate models. Key results Passive transfer was good in both groups (serum total protein (mean ± s.d.) 60.9 ± 9.1 mg/mL) with no differences in pre-weaning disease incidence; diarrhoea occurred in 64.5% and bovine respiratory disease in 26.3% of calves. High group calves were significantly heavier, taller and longer at all pre-weaning examinations except recruitment owing to more growth in the first month, and remained significantly larger at 6 months: weight 157 ± 8 vs 149 ± 7 kg, height 103 ± 5 vs 100 ± 5 cm, length 90 ± 4 vs 88 ± 5 cm. Plasma IGF1 concentrations at around Week 6 were doubled in the High group (101 ± 38.6 vs 55 ± 34.1 ng/mL). Bovine respiratory disease was associated with reduced weight gain. Heifers with diarrhoea were leaner at weaning. High feed group, weight at recruitment and good passive transfer were positively associated with weight at 6 months. Conclusions Higher feeding levels pre-weaning increased growth rates and IGF1, although the disease incidence was unaffected. Implications Previous studies have shown that more growth and higher IGF1 pre-weaning are associated with a lower age at first calving and an increased chance of reaching the end of first lactation. These in turn improve long-term performance.

Cyclic ewes were treated with control vehicle or progesterone receptor antagonist (onapristone; 100mg i.m. twice daily) during either early (day 3-5) or late (day 12-14) luteal phase and plasma samples collected for hormone analysis and to determine endogenous and oxytocin induced PGF2α release. On day 14 and 17, ewes were euthanised and reproductive tracts collected for ovarian morphology and endometrium for oxytoxin and steroid hormone receptor analysis. Early treatment increased LH, but not progesterone or oestradiol, while late treatment elevated all three hormones. Early treatment delayed the up-regulation of endometrial oxytocin receptors and responsiveness to oxytocin challenge, delaying luteolysis. Late treatment advanced development of oxytocin receptors and responsiveness to oxytocin though not timing of luteolysis. Patterns of hormone receptor mRNA were differentially disrupted by treatments. Results provide mechanistic insight into hormonal control of the oestrous cycle and identify the ability of the luteolytic mechanism to dissociate from functional luteolysis.

Early pregnancy loss occurs in 6–10% of equine pregnancies making it the main cause of reproductive wastage. Despite this, reasons for the losses are known in only 16% of cases. Lack of viable conceptus material has inhibited investigations of many potential genetic and pathological causes. We present a method for isolating and culturing placental cells from failed early equine pregnancies. Trophoblast cells from 18/30 (60%) failed equine pregnancies of gestational ages 14–65 days were successfully cultured in three different media, with the greatest growth achieved for cells cultured in AmnioChrome™ Plus. Genomic DNA of a suitable quality for molecular assays was also isolated from 29/30 of these cases. This method will enable future investigations determining pathologies causing EPL.

Culling for infertility remains the main reason for disposal of dairy cows, limiting productive lifespan. In extreme cases, ovulation is inhibited, preventing the possibility of conception. More often cows do conceive, but fail to remain pregnant owing to intrinsic problems in the embryo and/or to a poor-quality reproductive tract environment. Both aspects have a genetic component and are also influenced by management practices affecting nutrition and health. The relative importance of these factors varies among heifers, first-lactation and older cows. A common theme, however, is that an internal signalling system exists which reduces fertility when the cow is in an unsuitable metabolic state to sustain a pregnancy. This may be directly related to nutrient shortage caused by inadequate feed intake, or because available nutrients are being prioritized towards growth or milk production, away from reproduction. Evidence is presented for the involvement of the somatotrophic axis (GH, IGF1, insulin, IGFBP2) and leptin as key metabolic signalling molecules. Another emerging theme is the interaction between metabolism and disease that affects the fertility. Common examples include (i) calf diseases causing inadequate heifer growth and increased age at first calving; (ii) poor peripartum energy status reducing the capacity of the uterus to involute and mount an effective immune response, thereby increasing the likelihood of endometritis; and (iii) development of mastitis after conception, a contributory factor to both early and late embryo mortality. Finally, recent evidence suggests that times of metabolic stress cause mitochondrial damage that also contributes to a reduction in longevity.

The natural cervical relaxation which occurs at estrus in the ewe may be initiated by binding of hyaluronan (HA) to its receptor CD44. Indeed, we have previously shown that HA content and fragment size in the ovine cervix varies with the stage of the estrous cycle. Despite the importance of cervical relaxation in promoting sperm transport and facilitating the possible development of transcervical artificial insemination (AI), the mechanisms coordinating these changes in HA content remain to be defined. Hyaluronan synthases (HAS) 1, 2, and 3 regulate HA biosynthesis and herein, we describe the changing pattern of HAS isoform expression during the estrous cycle to determine whether this may underpin HA-mediated changes in relaxation of the ovine cervix. Accordingly, cervices were collected from 24 cyclic sheep (n = 8 / group) at the luteal, pre-luteinizing hormone (LH) and post-LH surge stages. Protein and mRNA expression for HAS 1, 2 and 3 was determined in five different tissue layers (epithelium, subepithelial stroma, and longitudinal, circular and transverse muscle) of the vaginal, mid and uterine regions of each cervix by immunohistochemistry and in situ hybridization, respectively. HA synthases were expressed in all the tissue layers and regions of the cervix, and the pattern of expression was similar for mRNA and protein. HAS1 protein and mRNA expression was significantly (P ≤ 0.05) higher at the pre-LH surge stage, while HAS 2 and 3 protein and mRNA expression was significantly (P ≤ 0.001) higher at the luteal stage. Overall, both HAS protein and mRNA expression was significantly (P ≤ 0.001) higher in the epithelial layer and the vaginal region. These findings are in accordance with our previous results and explain the differences observed in the HA content and differing HA fragment size at different stages of the estrous cycle.

Objective To use an in vitro model of the ovine placenta to determine effects of n-6 polyunsaturated fatty acid (PUFA) supplementation on prostaglandin (PG) production. PGs are key regulators of fetal maturation and parturition. Study design Fetal allantochorion tissue (FC) was collected in late pregnancy (day 135). FC cells were isolated and cultured with 0–100 μM of linoleic acid (LA), γ-linolenic acid (GLA) or arachidonic acid (AA) in serum free medium and challenged with control medium, lipopolysaccharide (LPS, 0.1 μg/ml), dexamethasone (DEX, 5 μM) or a combination of LPS (0.1 μg/ml) with DEX (5 μM). Spent medium was harvested at 2 h and 24 h post challenge for measuring PGs. Main outcome measures To assess the effects of treatment on placental 1- and 2-series PGE production. Results LA supplementation inhibited both PGE 1 and PGE 2 production. GLA predominantly stimulated PGE 1 generation, although it also increased PGE 2 production. AA supplementation predominantly increased PGE 2 production, but also stimulated PGE 1 . DEX treatment with or without LPS inhibited PG production. Supplementation with n-6 PUFAs attenuated or neutralised the stimulatory effect of LPS challenge on FC cells for both PGE 1 and PGE 2 production. Conclusion These data show that supplementation with n-6 PUFAs alters placental PG production, but their precise effects depend on their position in the biosynthetic pathway for PG synthesis. This study supports the possibility that GLA containing oils, widely promoted as dietary supplements, might reduce the risk of pre-term labour by inhibiting the responsiveness of PGE 2 production to LPS challenge in the placenta.

This study investigated associations between single nucleotide polymorphisms (SNP) in the leptin, leptin receptor, and neuropeptide Y (NPY) genes with growth, milk production, and fertility traits. Holstein Friesian heifers from 19 UK dairy farms were recruited at birth, providing an initial population of 509. Animals were monitored until they either reached the end of their second lactation or were culled. Size (weight, height, length, girth) was measured at 1, 6, and 15 mo to assess growth traits. Heifer fertility was assessed by recording age at service, age at conception, age at first calving, and number of services. Cow fertility was assessed by recording days from calving to service and conception, services per conception, percentage of animals in calf at 100 d after calving and the calving interval in each lactation. Milk production was recorded as days in milk, total milk per lactation, 305-d yield, milk per day, and peak yield. Mixed model analyses revealed that leptin SNP were associated with early skeletal growth (height, A1457G; length, A59V), fertility (UASMS1, UASMS2, A1457G, A59V) and milk production (A59V). The leptin receptor SNP (T945M) was only weakly associated with milk per day and days to first service. The NPY SNP (NPY1) was associated with the prevalence of the animal being in calf 100 d after calving and 305-d milk yield in the first lactation. The association of leptin SNP with fertility traits in heifers, in addition to lactating cows, suggests that some effects on fertility are direct and not necessarily mediated via altered tissue mobilization. In accord with this, other work has shown that leptin can affect oocyte quality and early embryo development. These results support the use of leptin SNP to inform marker-assisted selection in dairy cows.

A short herd lifespan severely limits opportunities for on-farm selection of breeding cows in addition to causing financial losses on dairy farms and presenting welfare issues for individual animals. This prospective study monitored survival up to third calving and reasons for culling of a cohort of 468 Holstein-Friesian heifers on 18 dairy farms across southern England. Heifers born during 2003 and 2004 were monitored from 1 mo of age through to third calving. A longevity index was calculated as the proportion of days alive spent in milk production, a good measure of lifetime performance. On average, 11% of heifers recruited at 1 mo did not survive until first calving (0% longevity index). Of those that did calve, 19% were culled in lactation 1 (total average lifetime days in milk of 322 with a longevity index of 24%) and 24% were culled during lactation 2 (total average lifetime days in milk of 623 with a longevity index of 40%). The primary cause for culling was infertility. Only 55% of replacement heifers calved successfully for a third time, ranging from 80 to 32% across individual farms. These results show that on a selection of UK farms, a large number of heifers never become productive or are culled before they reach their full lactation potential. Increasing the productive lifetime of dairy cows would improve the efficiency of dairy production by lowering replacement costs and capturing a greater proportion of potential lactation milk yield from mature cows.

Dairy heifers, particularly when Cryptosporidium parvum and rotavirus were the dominant pathogens. The causative pathogens for BRD were harder to identify, with large differences between levels of farmer- and veterinarian-diagnosed disease and higher incidences in North America (around 16%) than Europe (around 3%). Analysis of the risk factors for disease and mortality highlighted that closed herds, good hygiene and availability of roughage were consistently associated with improved outcomes. Some common practices were challenged by the results of large, prospective studies. Calf diarrhoea was only weakly associated with good passive transfer of immunity via colostrum, suggesting other control strategies are required. Morbidity with small group housing demonstrated no disadvantages compared with contiguous single pens, so this system should be encouraged to improve welfare. Routine use of antibiotics in calf diarrhoea was associated with negative outcomes, so is actively discouraged.

Artificial insemination (AI) using frozen semen is a key method to enable rapid genetic improvement but its use in the sheep industry is currently limited by poor fertility. Laparoscropic AI is most effective but has not gained popularity due to cost and welfare considerations. Transcervical intrauterine AI (TCAI) may offer a practical alternative but the complex anatomy of the ovine cervix limits adequate penetration of the inseminating pipette. Hyaluronan (HA) is a glycoaminoglycan whose content in the cervix increases at oestrus and which may contribute to the degree of natural relaxation observed at this time. This study investigated the effect of intracervical application of HA on the depth of cervical penetration in sheep. Oestrus was synchronised on three occasions in 48 Welsh mountain ewes with progesterone sponges and PMSG. Each animal initially served as its own untreated control. Ewes were subsequently treated intracervically with 25 mg of: (2i) low molecular weight (MW) HA; (2ii) high MW HA or (2iii) both low and high MW HA (n = 16/group) at 52 h after sponge removal or with low MW HA at: (3i) 50 h; (3ii) 52 h or (3iii) both 50 h and 52 h. Depth of cervical penetration measured at 54 h was increased from 1.22 cm to 3.66 cm by treatment with low MW HA (P ≤ 0.001), with no differences between the number of treatments (1 or 2) or the time at which the HA was administered (50 or 52 h). High MW HA alone or together with low MW HA had no effect. In conclusion, intracervical application of low MW HA 52 h after sponge removal increases cervical penetration up to 3.4cm to allow TCAI in sheep.

McCarthy SD, Waters SM, Kenny DA, Diskin MG, Fitzpatrick R, Patton J, Wathes DC, Morris DG. Negative energy balance and hepatic gene expression patterns in high-yielding dairy cows during the early postpartum period: a global approach. Physiol Genomics 42A: 188-199, 2010. First published August 17, 2010; doi:10.1152/physiolgenomics.00118.2010.-In high-yielding dairy cows the liver undergoes extensive physiological and biochemical changes during the early postpartum period in an effort to re-establish metabolic homeostasis and to counteract the adverse effects of negative energy balance (NEB). These adaptations are likely to be mediated by significant alterations in hepatic gene expression. To gain new insights into these events an energy balance model was created using differential feeding and milking regimes to produce two groups of cows with either a mild (MNEB) or severe NEB (SNEB) status. Cows were slaughtered and liver tissues collected on days 6-7 of the first follicular wave postpartum. Using an Affymetrix 23k oligonucleotide bovine array to determine global gene expression in hepatic tissue of these cows, we found a total of 416 genes (189 up- and 227 downregulated) to be altered by SNEB. Network analysis using Ingenuity Pathway Analysis revealed that SNEB was associated with widespread changes in gene expression classified into 36 gene networks including those associated with lipid metabolism, connective tissue development and function, cell signaling, cell cycle, and metabolic diseases, the three most significant of which are discussed in detail. SNEB cows displayed reduced expression of transcription activators and signal transducers that regulate the expression of genes and gene networks associated with cell signaling and tissue repair. These alterations are linked with increased expression of abnormal cell cycle and cellular proliferation associated pathways. This study provides new information and insights on the effect of SNEB on gene expression in high-yielding Holstein Friesian dairy cows in the early postpartum period.

The complex anatomy of the ovine cervix limits the success of trans-cervical artificial insemination in sheep. However, there is a degree of natural relaxation of cervix at oestrus that is accompanied by an increase in the water content. As hyaluronan (HA) has a high affinity for water molecules, in this study, we tested the hypothesis that the HA content of the cervix, the proportion of different size fragments of HA and expression of its receptor CD44 vary with the stage of the oestrous cycle. Oestrous was synchronized in 25 Welsh mountain ewes, and their cervices were collected either during luteal phase (n=8) or pre-LH (n=8) or post-LH (n=9) surge stage of the oestrous cycle. The pre-LH surge group had the highest HA content (2.96 ng/mg of cervical tissue), which was significantly (P≤0.05) higher than that observed for the post-LH surge (2.04 ng/mg) group. The luteal phase group had a mean HA content intermediate between the pre- and post-LH surge groups, and was significantly different from either. The frequency of cervical samples containing both sizes of HA fragments (small and large) was significantly higher (P≤0.05) in the pre-LH surge group compared with the luteal and the post-LH surge groups, whereas that in post-LH surge group was significantly (P≤0.05) higher than that in the luteal group. The number of cervical samples that contained only small HA fragments was significantly (P≤0.05) higher in the luteal group compared with both the pre- and post-LH surge groups, whereas the number of samples containing only large HA fragments was significantly (P≤0.05) higher in the post-LH surge group compared with the luteal or pre-LH surge groups. Overall mean expression of CD44 in the vaginal and mid regions was significantly (P≤0.001) higher than that in the uterine region, with no difference between the vaginal and mid regions of the cervix. Pattern of CD44 expression depended on the stage of the oestrous cycle. At the luteal stage, CD44 expression did not vary among epithelial, sub-epithelial, circular and longitudinal smooth muscle layers, whereas at the pre- and post-LH surge stages, the expression in the epithelial layer was significantly (P≤0.001) higher than that in the other three layers. In general, CD44 expression in the transverse smooth muscle layer was significantly (P≤0.05) lower than the expression in all the other layers at all the stages of the oestrous cycle. The results indicated that the HA varied with the steroid status. Higher HA values at a time when cervical relaxation is naturally higher may indicate its involvement in remodelling of the cervix at oestrus.

Associations between Neospora caninum infection and the reproductive performance of dairy heifers in their first and second pregnancy on 18 UK farms were examined. Six-month-old heifer calves were tested for N caninum-specific antibodies with a commercial ELISA, and were then monitored until their second calving. Random-effects regression analyses were performed on the data, using serological status as the explanatory variable. Of 460 heifers tested, 7.2 per cent were seropositive. Seropositive heifers were more likely to suffer gestational loss (late embryonic/early fetal loss and abortion) than seronegative heifers during their first (odds ratio [OR] 5.3, P0.01) and second (OR 6.0, P0.001) pregnancy. Seropositive heifers were also four times more likely to experience perinatal mortality (calf born dead or dying within 24 hours of parturition) at first (OR 3.9, P0.01) and second (OR 4.5, P0.1) calving. No significant association between seropositivity and the fertility parameters (age at first breeding and calving, days from calving to first service and conception, services per conception and calving interval) or conception failure was found. The results suggest that N caninum infection before pregnancy is a significant contributing factor to gestational loss and perinatal death, and thus serological screening of potential replacement heifers is recommended.

Leptin is an important regulator of fetal and placental growth. This study evaluated the association of single nucleotide polymorphisms (SNP) in the leptin gene with perinatal mortality (stillbirths and mortality within 24h of parturition) in 385 Holstein-Friesian heifers on 18 dairy farms in the United Kingdom. The 3 SNP evaluated were exon 2FB, UASMS1, and UASMS2. The mean age at first calving was 27.0±0.2 mo. Associations between each SNP and perinatal mortality (calf alive or dead) were tested using a generalized linear model that included herd-year-season, calf sex, age at first calving, and age and pedigree of the dam. The overall level of perinatal mortality in the population was 16.9%, with significant allelic substitution effects for exon 2FB and UASMS1. These 2 SNP were in close linkage disequilibrium with each other (r 2 =0.98) but not with UASMS2 (r 2 =0.10). For exon 2FB, perinatal mortality was similar between heifers carrying the CT and TT alleles (20%), but was higher than in heifers carrying the CC allele (11%). For UASMS1, mortality was 21% with the CC and CT alleles but only 10% with the TT allele. No associations of perinatal mortality with SNP were found in the UASMS2 data set, possibly influenced by the low frequency (2%) of the TT genotype. No significant effects of herd-year-season, age at first calving, or calf sex were found. In conclusion, polymorphisms in the leptin gene were associated with 2-fold differences in perinatal mortality in dairy heifers.

This study investigated the effect of growth parameters and metabolic indices during the rearing period on the fertility of nulliparous Holstein-Friesian heifers managed on 17 UK dairy farms. Growth parameters (body weight [BW], heart girth, height, and crown-rump length) and metabolic indices (insulin-like growth factor-I [IGF-I], insulin, glucose, and urea) were measured at approximately 30, 180, and 450 d of age. Fertility data collected included age at first breeding (AFB), number of services per conception, pregnancy rate to first artificial insemination (AI), and age at first calving (AFC). Of the heifers initially bred (n=428), 4% failed to conceive. The mean pregnancy rate to first AI for heifers that conceived and calved without suffering reproductive loss (n=392) was 67%, and 6% required2 inseminations. The mean AFB and AFC was 473+/-5 d (range, 357 to 936 d) and 791+/-6 d (range, 636 to 1529 d), respectively. Increased BW, girth, and IGF-I concentration (at 30, 180, and 450 d) and increased skeletal growth (at 180 and 450 d) was associated with a reduced AFB and AFC (P0.05 to 0.001). Heifers calving at775 d had a mean BW gain of 0.82+/-0.01kg from 30 to 180 d. Increased glucose concentration at 180 d was associated with a reduced AFB (P0.01), but no associations were found between insulin and urea concentrations and any of the fertility traits recorded (P0.1). Suboptimal growth associated with an increased AFC could be alleviated by improved monitoring of replacement heifers during the rearing period.

The incidence of mortality and culling in Holstein-Friesian heifers from birth through first calving was determined on 19 dairy farms selected from across southern England. The outcome of 1097 calvings was determined. Size (BW, heart girth, crown–rump length and height at withers) and insulin-like growth factor-I concentration of live heifer calves were measured at a mean age of 26 ± 0.7 days (n = 506). Associations between the heifer-level variables and mortality were determined using clustered binary logistic regression. Perinatal mortality (stillbirths and mortality within the first 24 h of birth) of male and female calves was 7.9%. This figure was significantly higher in cases where calving assistance was required (19.1% v. 5.6%, P < 0.001) and in twin births (18.5% v. 7.0%, P < 0.05), and was lower in pluriparous v. primiparous dams (5.6% v. 12.1%, P < 0.01). On average, 6.8% of heifers died or were culled between 1 day and 6 months of age. Low BW at 1 month was associated with reduced subsequent survival up to 6 months. Between 6 months and first calving, a further 7.7% of heifers either died (42%) or were culled (58%); accidents and infectious disease accounted for the majority of calf deaths between 6 and 15 months, whereas infertility (16/450 animals served, 3.5%) was the main reason for culling following the start of the first breeding period. In total, 11 heifers (2.2%) were culled as freemartins; eight at birth and three around service. Overall, 14.5% of liveborn potential replacement heifers died or were culled before first calving.

Insulin-like growth factor-I (IGF-I) plays a pivotal role in cattle fertility, acting as a monitoring signal that allows reproductive events to occur when nutritional conditions for successful reproduction are reached. However, endocrine IGF-I is not a predictor of reproductive events, but rather an indirect estimator of the suitability of the animal to achieve the reproductive event in question. Although measuring circulating IGF-I concentrations might not have any clinical application in the cattle industry, endocrine IGF-I screening will continue to be important for the study of interactions between nutrition and reproduction. In addition, endocrine IGF-I screening could be used as an ancillary test for the selection of cattle for high reproductive potential, especially in herds of high genetic merit for milk production, in which a decline in fertility has been identified.

This study related growth and metabolic traits in Holstein-Friesian calves (n = 45) to subsequent performance and longevity. Animals were measured at birth, 3, 6 and 9 months (weight, crown rump length (CRL), heart girth and height). Endocrine and metabolic traits were assessed at 6 months of age. These traits were not influenced by sire (n = 5). Milk production and mortality records were obtained through 3 lactations. Seven heifers failed to achieve a lactation, 6 were culled after one, 17 after two and 15 (33%) survived ≥3 lactations. Birth weight and girth but not skeletal measures were highly positively correlated with the repeated size measurements at 3–9 months. Calves with higher cortisol at 6 months were smaller in weight and girth at 3 months, with reduced 3–6 months skeletal growth. GH secretion was related to height, weight and growth, whereas urea was most strongly related to CRL. Calves not achieving a single lactation had lower IGF-I. Calves with higher glucose or BHB were more likely to be culled after 2 lactations, an effect which was independent of yield. Size and metabolic measurements of growing calves may therefore prove useful in predicting longevity.

Reproductively normal crossbred beef heifers were individually offered a diet of barley straw and concentrate supplemented with one of four levels of a fish oil (FO) enriched supplement. Following oestrous cycle synchronisation, blood samples were collected at appropriate intervals for the measurement of progesterone (P(4)), oestradiol (E(2)), fatty acids, insulin-like growth factor 1 (IGF-1) and metabolites. On days 15 and 16 of the cycle, oxytocin was administered intravenously and the prostaglandin F(2alpha) (PGF(2alpha)) response was measured as venous concentrations of 13,14-dihydro-15-keto PGF(2alpha) (PGFM). The heifers were slaughtered on days 17 or 18 of the oestrous cycle and endometrial tissue, rumen fluid and follicular fluid were collected for determination of fatty acid concentrations. In general there was no effect (P0.05) of diet on plasma P(4) or E(2) concentrations. Increasing FO supplementation increased CL diameter on day 7 post-oestrus (P0.0001) but had no effect on diameter on day of slaughter (P0.05). On day 15, PGFM concentration was greater on the highest level of FO supplementation compared to controls (P0.05), however, there were no differences between other diet comparisons (P0.05). There was no effect of diet on PGFM concentration on day 16 (P0.05). There was a strong positive relationship between plasma and uterine endometrial concentrations of both EPA (R(2)=0.86; P0.0001) and total n-3 PUFA (R(2)=0.77; P0.0001). IGF-1 concentrations increased on all diets and were greatest at the highest level of n-3 PUFA supplementation (P0.05).

Recent work has suggested that the recommended intakes of vitamin E for dairy cattle need to be increased, particularly in dry cows. However, these suggestions are based on data from cattle in the USA, which may have significantly different oxidative stresses than European cattle. This study, which involved 594 cattle on three dairy farms, was designed to determine the effect of increased vitamin E supplementation on the health and fertility of UK dairy cows. Cattle were randomly allocated to receive either two intramuscular injections of 2100 mg of vitamin E (and 7 g of sodium selenite) 2 weeks before calving and on the day of calving, or no additional vitamin E supplementation. Although supplementation had no effect on milk yield, reproductive efficiency, or incidence of uterine infections, supplemented cattle had a lower risk of culling and a lower rate of mastitis. These figures were economically significant but not statistically significant at the 10% level. Supplementation reduced the incidence of retained fetal membranes from 6.5% to 3%, an effect which was almost significant at the 5% level. If these data are representative they suggest that vitamin E recommendations for UK cattle should be reassessed.

Conception rates of dairy cows are currently declining at an estimated 1% every year. Approximately, 35% of embryos fail to prevent luteolysis during the first three weeks of gestation. Interactions between the corpus luteum, endometrium and embryo are critical to the successful establishment of pregnancy and inadequacies will result in the mortality of the embryo. For example, as little as a one day delay in the post-ovulatory rise of progesterone has serious consequences for embryo development and survival. Recently, we found that LH support, degree of vascularization and luteal cell steroidogenic capacity were not the major factors responsible for this luteal inadequacy, but are nevertheless essential for luteal development and function. Progesterone acting on its receptor in the endometrium stimulates the production of endometrial secretions on which the free-living embryo is dependent. However, their exact composition and effects of inadequate progesterone remains to be determined. The embryo is recognized through its secretion of interferon tau (IFNT), which suppresses luteolytic pulses of prostaglandin F(2 alpha). In the cow, it is most likely that IFNT inhibits oxytocin receptor up-regulation directly and does not require the prior inhibition of oestrogen receptor alpha (ESR1). Unravelling the precise luteal-endometrium and embryo interactions is essential for us to understand pregnancy establishment and development of strategies to reverse the declining fertility of dairy cows.

Lifetime records of 442 Holstein cows on one farm in North Queensland, Australia, were analysed to evaluate the effects of age at first calving (AFC) and first lactation milk yield on parities per lifetime, longevity, milk yield in subsequent lactations, lifetime milk production and the longevity index (LI), that is the proportion of a cow's life spent in active milk production. The mean daily yield in the first lactation was a reliable indicator of milk yield in subsequent lactations, estimated lifetime production, longevity and LI. The afc had no effect on lifetime days in milk. Neither the AFC nor the milk yield in the first lactation significantly affected the number of parities per lifetime. For the majority of cows, which produced less than 30 l/day in the first lactation, there was a significant positive relationship between longevity and AFC. However, none of the cows that produced more than 30 l/day in the first lactation survived for more than two lactations. The optimal AFC was thus two to two-and-a-half years, as these cows had the highest first lactation yield, estimated lifetime production and LI. The optimal mean daily yield in the first lactation was 20 to 30 l/day, as these cows lived longer and had a higher LI and lifetime production.

During the early postpartum period dairy cows mobilize fat and muscle to support lactation. This is associated with alterations in blood metabolite and hormone profiles which in turn influence milk yield and fertility. This study developed models to determine how metabolic traits, milk yield and body condition score were inter-related at different times in the periparturient period and to compare these relationships in primiparous (PP, n=188) and multiparous (MP, n=312) cows. Data from four previous studies which included information on blood metabolic parameters, parity, milk yield, body condition score and diet were collated into a single dataset. Coefficients of polynomial equations were calculated for each trait between -1 week pre-calving and week +7 postpartum using residual maximum likelihood modelling. The completed dataset was used in a multiple correlation model to determine how the best fit curves were related to each other over time. PP cows had higher concentrations of insulin-like growth factor-I and lower beta-hydroxybutyrate concentrations throughout, higher leptin concentrations pre-partum and both the peak in non-esterified fatty acids and the nadir in urea concentration occurred earlier after calving. These differences were associated with significantly lower milk production. Leptin concentrations fell at calving and were related to body condition score. Insulin was negatively correlated with yield in MP cows only. In MP cows the relationship between insulin-like growth factor-I and yield switched from negative to positive between weeks +4 and +7. Both beta-hydroxybutyrate and urea were positively related to yield in PP cows. In contrast, in MP cows beta-hydroxybutyrate was negatively correlated with yield and urea was strongly related to body condition score but not yield. These results suggest that there are differences in the control of tissue mobilization between PP and MP cows which may promote nutrient partitioning into growth as well as milk during the first lactation.

A meta-analysis was performed to consolidate the results of studies which have evaluated the effects of Vitamin E supplementation during the dry period on the risk of retained foetal membranes (RFM) in the dairy cow. Twenty studies demonstrated a beneficial response to Vitamin E whilst 21 found no benefit and 3 reported an increase in the incidence of RFM in treated cows. The odds ratios (OR) of the available studies exhibited significant heterogeneity, so multivariable logistic regression analysis was performed to enable the identification of factors associated with the response to Vitamin E supplementation. Our multivariable analysis included parity and Vitamin E supplementation (control/treated) in the model, because all other factors were co-linear. Results indicated that Vitamin E supplementation led to a reduction in the incidence of RFM. A second multivariable analysis was undertaken on a subset of the data including only supplemented cows to determine the influence of supplementation factors on the risk of RFM. All factors were co-linear with each other, therefore, only type of Vitamin E supplementation was included in this analysis. The regression model demonstrated that administration of the synthetic Vitamin E alpha-tocopheryl acetate was associated with a lower risk of RFM than treatment with natural Vitamin E (alpha-tocopherol) (P=0.047, OR=0.49), whereas the difference between the synthetic Vitamin E alpha-tocopherol acetate and natural Vitamin E just failed to attain statistical significance (P=0.059, OR=0.53). Overall the analyses indicate that Vitamin E supplementation during the dry period is associated with a reduced risk of RFM, and that the synthetic forms of Vitamin E are more effective than the natural compound.

Good management of the pregnant heifer is crucial to ensure that she is well grown and healthy and calves down easily before joining the milking herd. This study collected primary data on all aspects of heifer management on 101 UK farms during heifer pregnancy from conception to calving including farm factors and associated costs of system inputs. A cost analysis workbook was developed to calculate the cost of rearing per heifer for each of the study farms. Associations between cost of rearing and farms factors were determined using linear regression and analysis of variance. Heifers had a mean age of conception of 509 d (range 365 - 700 d) and an age at first calving of 784 d (range 639 - 973 d). The mean total cost of rearing during pregnancy was ￡450.36 (range ￡153.11 to ￡784.00) with a mean daily cost of ￡1.64 (range ￡0.56 to ￡2.86). The inputs contributing the most to cost were feed (32.7%), labour (23.8%) and slurry disposal (11.2%). Total purchased and homegrown feed and grazing contributed between 25.5% and 65.4% of total costs with a mean contribution of 43.6%. The cost of rearing was lowest in spring calving herds and highest in all year round calving herds with intermediate values in autumn and multi block calving herds. The main variables influencing the cost were the number of days spent at grass, age at first calving, calving pattern, breed, herd size and region. Each extra day in age at first calving increased the mean cost of rearing during pregnancy by ￡0.33/d whereas every extra day at grass reduced the cost by ￡1.75/d.

Simple Summary Dairy cows fed high levels of protein to increase milk yield tend to have reduced fertility but the reasons behind this are unclear. Differing dietary protein levels are reflected in altered urea concentrations in both blood and other tissues including the uterus. We showed that the circulating urea concentration was highly correlated to changed expression levels of many genes in the endometrium shortly after calving. These were predominantly associated with tissue repair, innate immunity and lipid metabolism. A subsequent study found no effect of altered urea concentration on endometrial gene expression in vitro implying that the dietary influence is indirect. Abstract Both high and low circulating urea concentrations, a product of protein metabolism, are associated with decreased fertility in dairy cows through poorly defined mechanisms. The rate of involution and the endometrial ability to mount an adequate innate immune response after calving are both critical for subsequent fertility. Study 1 used microarray analysis to identify genes whose endometrial expression 2 weeks postpartum correlated significantly with the mean plasma urea per cow, ranging from 3.2 to 6.6 mmol/L. The biological functions of 781 mapped genes were analysed using Ingenuity Pathway Analysis. These were predominantly associated with tissue turnover (e.g., BRINP1, FOXG1), immune function (e.g., IL17RB, CRISPLD2), inflammation (e.g., C3, SERPINF1, SERPINF2) and lipid metabolism (e.g., SCAP, ACBD5, SLC10A). Study 2 investigated the relationship between urea concentration and expression of 6 candidate genes (S100A8, HSP5A, IGF1R, IL17RB, BRINP1, CRISPLD2) in bovine endometrial cell culture. These were treated with 0, 2.5, 5.0 or 7.5 mmol/L urea, equivalent to low, medium and high circulating values with or without challenge by bacterial lipopolysaccharide (LPS). LPS increased S100A8 expression as expected but urea treatment had no effect on expression of any tested gene. Examination of the genes/pathways involved suggests that plasma urea levels may reflect variations in lipid metabolism. Our results suggest that it is the effects of lipid metabolism rather than the urea concentration which probably alter the rate of involution and innate immune response, in turn influencing subsequent fertility.

Elevated circulating non-esterified fatty acids including oleic acid (OA) are associated with many pregnancy related complications. Prostaglandins (PGs) play crucial roles during parturition. We investigated the effect of OA supplementation on PG production using an in vitro model of ovine placenta.Maternal endometrium (ME) and fetal allantochorion (FC) were collected in late pregnancy (day 135). Confluent cells were cultured in serum-free medium supplemented with 0, 20 or 100 μM OA and challenged with control medium, oxytocin (OT, 250 nM), lipopolysaccharide (LPS, 0.1 μg/ml) or dexamethasone (DEX, 5 μM). Spent medium was harvested at 2 and 24 h after challenge for quantifying PGs.In ME cells OA increased PGE2 production moderately but attenuated PGF2α production leading to a doubling of the PGE2:PGF2α ratio (E:F) (P 0.01). Without OA, both OT and LPS stimulated PG production for about 3-fold (P 0.01) without changing the E:F ratio. In the ME cells challenged with OT, OA decreased both PGE2 and PGF2α production by up to 70% (P 0.01) whereas in LPS treated cells OA increased the E:F ratio. In FC cells PGE2 production at 2 h was stimulated by 100 μM OA (P 0.05). In these cells LPS caused a 3-fold increase in PGE2 (P 0.01), an effect which was completely inhibited by DEX.OA supplementation favours basal PGE2 production in both ME and FC. In ME OA increased E:F ratios and antagonized the stimulatory effect of OT on PG production. This suggests that raised circulating OA may affect both the initiation and progression of parturition.

This study investigated possible relationships between measurements of the somatotrophic axis in pre-pubertal dairy calves and subsequent milk yields. Endogenous growth hormone (GH) release was measured through a fed and fasted period in fifty 6-month-old Holstein-Friesian heifers and they were then challenged with growth hormone-releasing factor (GRF) to assess their GH release pattern. Insulin-like growth factor-I (IGF-I), insulin and glucose concentrations were measured in relation to time of feeding. Cows were subsequently monitored through their first three lactations to record peak and 305-day milk yields. In the first lactation, milk energy output for the first 120 days of lactation was also calculated. The mean 305-day milk yield increased from 7417 +/- 191 kg in the first lactation (n = 37) to 8749 +/- 252 kg in the third (n = 25). There were no significant relationships between any measures of GH secretion and peak or 305-day yield in any lactation. A highly significant positive relationship was established between the GH peak measured 10 min post-GRF challenge and 120-day milk energy values in the first lactation. This relationship was, however, only present in the subpopulation of 12 cows culled after one or two lactations and was absent in the 25 animals remaining for the third lactation. There were no significant relationships between pre-pubertal IGF-I and fed or fasted insulin or glucose concentrations and any subsequent measurement of yield. The usefulness of GH secretagogue challenges in calves as a predictive test for future milk production is thus limited but may have some bearing on nutrient partitioning and longevity. (c) 2005 Elsevier Inc. All rights reserved.

Preterm labour is the major cause of perinatal morbidity and mortality in humans. The incidence is around 10% and the causes are often unknown. Consumption of dietary n-6 polyunsaturated fatty acids (PUFAs) in western societies is increasing. These are metabolized to arachidonic acid, the precursor for 2-series prostaglandins (PGs), major signalling molecules during labour. This study investigated the effect of dietary supplementation with linoleic acid (LA, 18: 2, n-6) on parturition. Ewes were fed a control or LA-supplemented diet from 100 days gestation. Labour was induced using a standardized glucocorticoid challenge (dexamethasone, Dex) to the fetus, starting on day 139. Electromyographic (EMG) activity and fetal and maternal circulating PG concentrations were monitored. One third of LA-fed ewes delivered early (pre-Dex) although basal uterine EMG activity preceding Dex was higher in control ewes (P < 0.05). A steep increase in EMG activity occurred 18–38 h after the start of Dex infusion. Twice basal EMG activity (defined as established labour) occurred on average 7 h earlier in the LA-supplemented ewes (P < 0.05). The basal concentrations of maternal and fetal PGFM and fetal PGE2 were approximately doubled in LA-supplemented ewes before the start of Dex infusion (P < 0.01). The rise in fetal PGE2 and maternal oestradiol concentrations post-Dex occurred earlier in the LA-supplemented ewes. All PG measurements remained significantly higher in the LA-supplemented ewes during labour onset. This study suggests that consumption of a high LA diet in late pregnancy can enhance placental PG production and may thus increase the risk of preterm labour.

We investigated the effect of n-6 polyunsaturated fatty acids (PUFAs) on prostaglandin (PG) production by the uterus. A mixed population of endometrial cells (epthelium and stroma) from late-gestation ewes were cultured in defined medium containing linoleic acid (LA, 18:2, n-6), gamma-linolenic acid (GLA, 18:3, n-6) or arachidonic acid (AA, 20:4, n-6) in concentrations of 0 (control), 20 or 100 microM. After 45 h in test medium with or without added PUFAs, cells were challenged with control medium (CM), oxytocin (OT, 250 nM), lipopolysaccharide (LPS, 0.1 micro g/ml) or dexamethasone (DEX, 5 microM) for 22 h in the continued presence of the same concentration of PUFA and the medium was collected for measurement of PGF(2alpha) and PGE(2). Supplementation with LA inhibited the production of PGF(2alpha) but did not alter PGE(2), whereas GLA and AA increased production of both PGs. All PUFA supplements thus increased the ratio of PGE(2) to PGF(2alpha) (E:F ratio) two- to threefold. In control cells, OT and LPS challenges stimulated the production of PGF(2alpha) and PGE(2). In all challenge groups, the concentrations of PGF(2alpha) in response to PUFAs followed the same pattern - LA

This study has localised oxytocin receptor (OTR) mRNA expression within the cervix of non-pregnant ewes and related this to changes in the sensitivity of the cervical musculature to administered oxytocin (OT) during the oestrous cycle by recording electromyographic (EMG) activity. Cervices were collected from 34 ewes at specified time points throughout the cycle. OTR mRNA expression was localised by in situ hybridisation and results were expressed as optical density measurements from autoradiographs in each of four different cervical regions. EMG recordings were made for up to 8 h per day from four non-pregnant ewes undergoing seasonal oestrous cycles between Days -3 and +3 relative to oestrus (Day 0). The highest concentrations of OTR mRNA were detectable within the luminal epithelium (LE) of the cervix, with values increasing from Day 15 of the cycle, peaking during the follicular phase (P0.001, compared to the mid-luteal phase) and returning to basal by Day 2. There was a small but significant increase in OTR mRNA hybridisation (above basal/luteal phase values) within the stromal cells (STR) adjacent to the lumen (P0.05) during the same time period, but no differences from basal values were detectable in the dense collagenous annular ring or in tissue superficial to this. Analysis of pooled EMG activity recorded daily from the cervix indicated that endogenous contractile activity was higher on Day 0 than on the Days +1 (P0.05), -2, +2 and +3 (P0.001). The response to bolus intravenous (i.v.) injections of 25 mU OT (25 mU) varied with day of the cycle. This dose produced a measurable and significant response on Days 0 (P0.001) and +1 (P0.001), but not on any of the other days, indicating that the sensitivity of the cervical musculature to OT peaked on these days. These data show that the cervix is highly responsive to OT at oestrus. This coincides with an increase in OTR mRNA expression in the luminal epithelial cells, suggesting the likely production of an intermediary messenger between the epithelial and smooth muscle cells.

SummaryThe high yielding dairy cow is expected to produce a substantial milk output every year and at the same time to conceive and maintain a pregnancy to term. To fulfil lifetime production potential a balance between yield, fertility and other influential factors has to be achieved. Any inability on the part of the management system to identify and rectify problems or on the part of the cow to cope with metabolic demands invariably results in economic or welfare issues. Our studies of high yielding dairy cows have revealed that some animals are capable of normal reproductive function whilst others are classic repeat breeders (requiring 3+ services per conception) or simply fail to rebreed. It is well established that the somatotrophic axis (growth hormone and insulin-like growth factors) drives lactation in ruminants but it is also intimately involved in reproductive processes. An awareness of metabolic adaptations to lactation that impact on reproduction in dairy cows is needed for appropriate management.The objective of our studies was to explore the metabolic profiles of high yielding dairy cows to identify factors influencing their ovarian function and fertility, hence to characterise the physiological adaptations involved. Our studies revealed different relationships between progesterone profile categories and metabolic status post partum. Delayed ovulation (DOV) or persistent corpora lutea (PCL) may be an appropriate response to a nutritional state or physiological situation and it may therefore be inaccurate to refer to these as ‘abnormal’. Whilst associated with high milk yields, not all profile categories detrimentally affected fertility parameters. Delayed ovulation postcalving (DOV1) was identified as the most prevalent abnormal profile encountered in first lactation high yielding cows. This may have occurred because the cows were not yet physically mature and unable to sustain both milk production and growth. The condition lasted long enough (71 ± 8.3 days from calving) to have a detrimental impact on their overall fertility parameters and was associated with significant physiological changes, representative of tissue mobilisation. Although the incidence of persistent luteal phases (PCL1 and PCL2) in dairy cows is increasing, this condition was not found to have any substantial detrimental effects on fertility or production parameters of the primiparous or multiparous cows in these studies. The main reproductive problems in our high yielding primiparous and multiparous cows appeared to be a failure to ovulate and conceive at the expected time or to maintain a pregnancy. These situations were associated predominantly with high milk yields and low concentrations of plasma IGF-I. A failure to ovulate appears to occur when body reserves are mobilised to maintain milk yield at the expense of reproduction and seems most likely to occur in primiparous high yielding cows or those experiencing GH-resistance (low IGF-I) due to excessive body condition loss, reduced feed intakes and factors such as stress and disease. More detailed investigations of dietary means of increasing IGF-I and optimising insulin concentrations, targeted at important reproductive times, are required in high yielding dairy cows, to aid in their management.

Early mammalian embryo development in vitro can be enhanced by co-culture with oviductal cells and by the addition of insulin-like growth factors (IGFs). This study examined the expression patterns of the oviductal IGF system in cattle in relation to the number of days after oestrus and the presence or absence of embryos. Oviducts were collected from: (i) 66 nulliparous heifers on day 3, day 6 or day 16 after insemination and from (ii) ten non-pregnant, lactating cows on day 0 or day 1 of the oestrous cycle. Oviducts were coiled, frozen whole and sectioned for in situ hybridization. Expression patterns of mRNAs encoding IGF-I, IGF-II, type 1 IGF receptor (IGF-1R), and the IFG binding proteins (IGFBP)-1, -3 and -5 were determined from autoradiographs. Separate measurements were made for the mucosa and muscle layers of the infundibulum, ampulla and isthmus. None of the parameters measured differed between heifers with or without the presence of an embryo. mRNAs encoding IGF-I and IGF-1R were present in the mucosa and muscle of all three oviductal regions, and the highest value of IGF-I mRNA was measured in heifers on day 3. IGF-II mRNA was expressed predominantly in the muscle wall. IGFBP-1 mRNA was not detectable, whereas mRNAs encoding IGFBP-3 and -5 were expressed in both the muscle and mucosa. IGFBP-3 expression was higher in cows on day 0 and day 1 of the oestrous cycle than in heifers on day 3, day 6 and day 16 after insemination. A peak of IGFBP-5 expression was reached on day 6. Locally or systemically produced IGFs, regulated by IGFBPs, may act directly on the embryo or indirectly via modulation of oviductal secretions and muscular activity to influence the success of early embryo development.

Left displacement of the abomasum (LDA) is an important periparturient disorder of dairy cows. This study evaluated differences in metabolic parameters between case-control pairs of cows (n=67) from 24 farms, and related these to outcomes in fertility and production. Cows with an assisted delivery were ×3 more likely to develop LDA, and affected cows tended to have had a longer dry period. At recruitment, cows with LDA tended to be in lower body condition accompanied by significantly higher circulating concentrations of β-hydroxybutyrate (BHB), non-esterified fatty acid (NEFA) and glucose and lower IGF1. Overall culling rate for all cows in the subsequent lactation was 22.5 per cent. Cows with LDA were not at increased odds of being culled but they produced, on average, 2272 l less milk and tended to have longer intervals to conception. Considering all cows irrespective of LDA status, the mean IGF1 level at recruitment was the only measured parameter associated with subsequent risk of culling (culled 11.7 ng/ml, not culled 23.5 ng/ml; P=0.005). Our findings support previous work indicating that poor insulin sensitivity through an uncoupling of the somatotrophic axis may be an important factor associated with LDA. Improved nutritional management of dry cows should reduce the incidence of both LDA and culling.

This study examined the expression patterns of oxytocin and steroid receptors in the bovine endometrium during the oestrous cycle and early pregnancy to elucidate their respective roles in the regulation of luteolysis and the maternal recognition of pregnancy. In Expt 1, uterine biopsies were collected from four cows throughout three oestrous cycles each, to provide daily samples. In Expt 2, uterine tissue was collected on days 12, 14, 16 and 18 of the oestrous cycle (n = 20) or early pregnancy (n = 16). Oxytocin receptor, oestrogen receptor alpha and progesterone receptor mRNAs were localized by in situ hybridization, and localization of oestrogen receptor and progesterone receptor was confirmed by immunocytochemistry. All three receptors showed time- and cell-specific expression patterns. Oestrogen receptor alpha increased in all regions at oestrus but high concentrations were also found in the luminal epithelium during the mid-luteal phase and in the deep glands throughout the oestrous cycle. Progesterone receptor expression was higher in the stroma than it was in the types of epithelial cell, and increased expression was observed at oestrus and during the early luteal phase. The cyclical upregulation of oxytocin receptors in the luminal epithelium on about day 16 was not related to preceding changes in the endometrial expression of either oestradiol alpha or progesterone receptors. During early pregnancy, oxytocin receptor expression was suppressed. Oestrogen receptor a concentrations increased in the non-pregnant cows and decreased in the pregnant cows between days 16 and 18, but these changes followed rather than preceded the upregulation of oxytocin receptors in the non-pregnant cows. It is concluded that the initial upregulation of oxytocin receptors in the luminal epithelium, which triggers luteolysis, is not associated directly with changes in expression of oestrogen receptor alpha.

In the cow, the embryo during the first three weeks of pregnancy is free living in the uterine lumen and is dependent on the maternal glandular secretions for its nutritional support. If the environment is appropriate, the embryo will develop sufficiently to prevent luteolysis. The aim of this study was to investigate the regulation of factors involved in embryonic-endometrial interactions during early pregnancy. Uterine horn sections were collected from 17 pregnant (PREG), 9 inseminated but no embryo present and 10 uninseminated cyclic control cows on days 12, 14, 16 and 18 after natural oestrus. The latter two groups were combined to form a single non-pregnant (NP) group. Trophoblast sections were also collected from the day 14, 16 and 18 embryos. The mRNA for interferon tau (IFNτ), oxytocin receptor (OTR), oestrogen receptor a (ER), prostaglandin G/H synthase -2 (PGHS-2), insulin-like growth factor (IGF) -I and IGF binding protein -1 (IGFBP-1) was determined by in situ hybridisation using 45 mer oligonucleotide probes end-labelled with35 S. The optical density (OD) readings were measured from the resulting autoradiographs. The expression of IFNτ mRNA in the trophodectoderm did not vary with embryo age. The expression of OTR mRNA in the luminal epithelium was first detectable on day 14 in 2 out of 5 NP cows and increased thereafter. Conversely, OTR mRNA was undetectable in all PREG cows except for one day 18 cow. In the NP cows, the first significant increase in ER mRNA concentrations in the luminal epithelium was observed on day 16. The pregnancy had no effect on ER mRNA concentrations in the luminal epithelium on days 12 and 14, but was significantly reduced on day 16 and was undetectable by day 18. On day 18, PGHS-2 mRNA was detectable in the luminal epithelium of all cows, but was unaffected by pregnancy status. The expression of IGF-I mRNA in the subepithelial stroma was maintained from days 12 to 18, but was reduced in the day 18 NP cows. IGFBP-1 mRNA concentrations in the luminal epithelium peaked on day 14 in both NP and PREG cows. Thereafter, concentrations declined in the NP group but were maintained in the PREG animals. In conclusion, the suppression of OTR mRNA expression by the embryo does not appear to require the prior suppression of ER mRNA. The continued expression of IGF-I and IGFBP-1 mRNA is likely to play an important role in the establishment of early pregnancy in the cow.

Circulating concentrations of insulin-like growth factor-I (IGF-I) are reduced in juvenile sheep during nutritional growth restriction and the associated delay in puberty. Since exogenous IGF-I has been shown to stimulate luteinizing hormone (LH) secretion, it is postulated that endogenous IGF-I may act as a stimulatory metabolic signal to the pubertal ovine hypothalamo-pituitary axis, yet its site of action is unknown. Using coronal hypothalamic and pituitary sections from pubertal ewe lambs, in vitro autoradiography was used to localise 125I-labelled IGF-I binding, and gene expression for components of the IGF system was localised by in situ hybridisation using oligonucleotide probes. High concentrations of 125I-IGF-I binding were seen in the pars tuberalis (PT) and pars distalis (PD) of the pituitary, and relatively little in the hypothalamus; binding in the PT but not the PD was displaced by excess unlabelled IGF-I. Large amounts of mRNA were detected for the type-1 receptor (IGF-1R) and for IGF-binding protein (IGFBP)-5, localised to the PT and PD, and less intense specific hybridisation signals were obtained with mRNAs for IGF-II, type-2 receptor (IGF-2R) and IGFBP-3. There was some evidence for specific hybridisation to IGFBP-4 mRNA in the PT. IGF-I, IGFBP-1 and IGFBP-2 mRNAs were not detected in PT and PD. None of the genes were expressed in hypothalamic tissue. Western-ligand binding on PD extracts from male castrates revealed by their molecular weights the likely presence of IGFBPs-2, -3, and -5. Finally, cultured PD cells from abattoir-killed sheep were challenged with IGF-I (0.1, 1, 10 or 30 nM) or luteinizing hormone-releasing hormone (LHRH, 10 nM) alone, or both together. Basal LH output was stimulated by 10 nM IGF-I (120+/-11.2%, P>0.05), 30 nM IGF-I (148+/-12.8%, P

Oestradiol treatment can increase uterine oxytocin receptor expression in vivo. The actions of oestrogen are usually mediated via its receptor, but it also has direct non-genomic effects in some cells. This study investigated the effect of oestradiol and the role of the oestradiol receptor in regulating endometrial oxytocin receptor expression in the bovine uterus. Explant cultures (in triplicate) from late luteal phase non-pregnant endometrium received the following treatments: control (serum-free medium), oestradiol (0. 1 and 0.01 micromol l(-1)), oestradiol (0.1 micromol l(-1)) with the oestradiol receptor antagonist ICI 182780 (0.5 micromol l(-1)), and ICI 182780 (0.5 micromol l(-1)) alone. Explants were collected 12, 24 and 48 h after the start of culture. Oxytocin receptor mRNA expression in the explants was measured by in situ hybridization and oxytocin protein concentrations were measured by autoradiography with the iodinated oxytocin receptor antagonist d(CH(2))(5) [Tyr (Me)(2) Thr(4) Tyr NH(2)(9)]-vasotocin ((125)I-labelled oxytocin receptor antagonist). Oxytocin receptor mRNA and protein expression were initially low but spontaneous upregulation occurred in the luminal epithelium between 24 and 48 h (P < 0.01). Oestradiol increased oxytocin receptor mRNA upregulation in the first 24 h (P < 0.05) but the effect on 125 I-labelled oxytocin receptor antagonist binding was not significant. ICI 182780 inhibited the oestrogenic effect but had no significant effect on oxytocin receptor mRNA expression when given alone. In conclusion, the results showed that oestradiol exerts its effect via the oestradiol receptor. Oestradiol facilitates oxytocin receptor gene transcription by increasing it more rapidly, but spontaneous upregulation of endometrial oxytocin receptor still occurs in the absence of oestradiol.

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 aim of the present study was to investigate the effects of administering a high plane diet during early to mid-gestation on the uterine and placental insulin-like growth factor (IGF) system and on systemic IGF-I concentrations in pregnant adolescent ewes with restricted placental growth. Embryos recovered from superovulated ewes inseminated by a single sire were transferred in singleton to the uterus of adolescent recipients. After transfer ewes were offered a high (H) or moderate (M) amount of a complete diet calculated to promote rapid or normal maternal growth rates, respectively. Five ewes from each group were switched from either M to H or H to M diets at day 52 of gestation. Maternal and fetal blood samples and placental tissues were collected from all animals at day 104. Ewes on the high plane diet from mid-gestation (HH, MH groups) had restricted placental mass (P < 0.01) and tended to have smaller fetuses. This was associated with increased maternal plasma IGF-I concentrations (P < 0.001). The pattern of expression of components of the IGF system in the uterus and placenta was studied by in situ hybridization. IGF-I mRNA concentrations were below the limit of detection. IGF-II mRNA expression was high in the fetal mesoderm and present in maternal stroma, but was not influenced by nutritional treatment. In contrast, IGF binding protein 1 (IGFBP-1) mRNA expression was higher (P < 0.05) and IGFBP-3 mRNA expression was lower (P < 0.05) in the endometrial glands of ewes in HH and MH groups. In the fetal trophoblast, IGFBP-3 mRNA expression was higher in the MH group. Type 1 IGF receptor expression was increased (P < 0.01) in the luminal epithelium of the HM group and IGFBP-2 mRNA expression was highest in the placentome capsule of ewes in the HH group. Together, these results indicate that reprogramming of the uterine and placental IGF axis by maternal nutrition could contribute to placental growth retardation in growing adolescent sheep.