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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

Rearing quality dairy heifers is essential to maintain herds by replacing culled cows. Information on the key factors influencing the cost of rearing under different management systems is, however, limited and many farmers are unaware of their true costs. This study determined the cost of rearing heifers from birth to first calving in Great Britain including the cost of mortality, investigated the main factors influencing these costs across differing farming systems and estimated how long it took heifers to repay the cost of rearing on individual farms. Primary data on heifer management from birth to calving was collected through a survey of 101 dairy farms during 2013. Univariate followed by multivariable linear regression was used to analyse the influence of farm factors and key rearing events on costs. An Excel spreadsheet model was developed to determine the time it took for heifers to repay the rearing cost. The mean +/- SD ages at weaning, conception and calving were 62 +/- 13, 509 +/- 60 and 784 +/- 60 days. The mean total cost of rearing was 1819 pound +/- 387/heifer with a mean daily cost of 2.31 pound +/- 0.41. This included the opportunity cost of the heifer and the mean cost of mortality, which ranged from 103.49 pound to 146.19 pound/surviving heifer. The multivariable model predicted an increase in mean cost of rearing of 2.87 pound for each extra day of age at first calving and a decrease in mean cost of 6.06 pound for each percentile increase in time spent at grass. The model also predicted a decrease in the mean cost of rearing in autumn and spring calving herds of 273.20 pound and 288.56 pound, respectively, compared with that in all-year-round calving herds. Farms with herd sizes100 had lower mean costs of between 301.75 pound and 407.83 pound compared with farms with

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.

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.

Housed pigs are often exposed to elevated concentrations of atmospheric ammonia. This aerial pollutant is widely considered to be an environmental stressor that also predisposes to reduced growth rates and poor health, although evidence to support this view is limited. Hepatic gene expression is very responsive to stress and metabolic effects. Two batches of growing pigs were therefore exposed to a nominal concentration of atmospheric ammonia of either 5 ppm (low) or 20 ppm (high) from 4 weeks of age for 15 weeks. Growth rates were monitored. Samples of liver were taken after slaughter (at ∼19 weeks of age). Samples from the second batch were analysed for global gene expression using 23 K Affymetrix GeneChip porcine genome arrays. Samples from both batches were subsequently tested for five candidate genes using quantitative real-time PCR (qPCR). The array analysis failed to detect any significant changes in hepatic gene expression following chronic exposure to atmospheric ammonia. Animals clustered into two main groups but this was not related to the experimental treatment. There was also no difference in growth rates between groups. The qPCR analyses validated the array results by showing similar fold changes in gene expression to the arrays. They revealed a significant batch effect in expression of lipin 1 (LPIN1), Chemokine (C-X-C motif) ligand 14 (CXCL14), serine dehydratase (SDS) and hepcidin antimicrobial peptide (HAMP). Only CXCL14, a chemotactic cytokine for monocytes, was significantly down-regulated in response to ammonia. As chronic exposure to atmospheric ammonia did not have a clear influence on hepatic gene expression, this finding implies that 20 ppm of atmospheric ammonia did not pose a significant material risk to the health or metabolism of housed pigs.

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.

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.

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.

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).

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.

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.

Previous studies have shown that 35 — 40 % ofmodern dairy cows experience ovarian disturbances during early lactation. Even though negative energy balance (NEB) has been implicated as a regulator of ovarian function, the exact metabolite(s) or hormone(s), which mediate this effect is still not clear. In this study, we investigated the relationship between blood metabolites (NEFA, BHB and glucose) and plasma insulin like growth factor-I (IGF-I) and ovarian dysfunction. Thirty three Holstein-Friesian cows were fed a lactational ration ad libtium and were inseminated at observed oestrus starting from 56 days post calving. Three times weekly milk samples and weekly blood samples were collected from cows from calving until confirmed pregnant. Cows were placed into “NORMAL” or “ABNORMAL” categories of ovarian activity based on their milk progesterone profiles. The days to first service were 71 ± 2 in “NORMAL” and 78 ± 6 in “ABNORMAL” animals. The calving to conception interval (days open) was shorter in the “NORMAL “ than the “ABNORMAL” group (90 ± 8.7 vs 119 ± 15.2 days). IGF-I levels were significantly lower in the “ABNORMAL” group from 2 until 7 weeks after calving (P

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.

An effective method for enhancing milk production efficiency in dairy cows is to increase milk yield and significant progress has been achieved through intense selection, assisted by the application of new reproductive techniques. However this increased milk yield has been accompanied by a slow but steady decline in dairy cow fertility. The two main reasons for this reducing level of fertility appear to be selection for increased milk yield and large herd sizes, although the affect of the introduction of Holstein genes needs to be investigated. In addition, other negative consequences such as an increase in the incidence of metabolic diseases and lameness have been observed. This has given rise to public concern that the high-yielding dairy cow may be under a state of metabolic stress during peak lactation and therefore the welfare and performance of other body functions are compromised.The reason for this decline in fertility is not well understood, although a nutritional influence on the initiation of oestrous cycles, follicular growth, oocyte quality and early embryonic development has been implicated. In early lactation dietary intake is unable to meet the demands of milk production and most cows enter a period of negative energy balance. Negative energy balance has a broadly similar effect to undernutrition leading to a mobilization of body reserves. Furthermore diets high in rumen degradable protein lead to an excess of rumen ammonia, which before it is converted to urea by the liver and excreted in the urine, may cause an alteration in the reproductive tract environment reducing embryo survival. Such major changes in the metabolic and endocrine systems can therefore influence fertility at a number of key points.Possible reproductive sites where inadequate nutrition may have detrimental effects include: (i) the hypothalamic/pituitary gland where gonadotropin release may be impaired; (ii) a direct effect on the ovaries, where both follicular growth patterns and corpus luteum function may be directly influenced; (iii) the quality of the oocyte prior to ovulation may be reduced and coupled with an inadequate uterine environment will result in reduced embryo survival and (iv) there may be effects on subsequent embryo development. The initiation of normal oestrous cycles post partum is usually delayed in dairy cows with a higher genetic merit for milk production, confirming that intense selection towards high milk yield can compromise reproductive function. In addition, the effects of increased milk yield may include changes in circulating GH and insulin concentrations, which in turn alter both insulin-like growth factor (IGF) and IGF binding protein production. Nutrition has recently been shown to have a direct effect at the level of both the ovaries and the uterus to alter the expression of these growth factors.In conclusion, further knowledge is required to determine how the metabolic changes associated with high milk output reduce fertility. Identification and understanding of the mechanisms involved and the key sites of action responsible for compromised reproductive function, will enable the identification of possible indices for future multiple-trait selection programmes.

Changes in perineal odor as estrus is approached could form the basis of a new method of estrus detection. Perineal odor of cyclic cows was monitored. Estrus was identified using ovarian ultrasound, behavioral observations, and plasma assay for progesterone and estradiol. Samples were taken from the dorsal lateral perineal (perivulval) area using cotton bud swabs and presented to an electronic nose. Twelve conducting polymer sensors were used to quantify odor in terms of a change in sensor resistance. Preliminary data (Experiment 1) indicate that the odor signals between the luteal phase and estrus could be distinguished for a group of five cows. In Experiment 2, samples were obtained daily from eight cows during the midluteal phase and from d -2 to d 8 of the cycle (d 0 = day of estrus, induced using cloprostenol). Seven of the eight cows cycled normally. Of the 12 sensors, 7 showed a significant change in resistance that was dependent on the day of the estrous cycle. Basal values were those taken in the luteal phase; values peaked on d -1, rose transiently on d 3, and returned to baseline on d 5 to 6. This pattern is strongly correlated with plasma estradiol concentration. The use of artificial olfaction could enable more accurate detection of estrus and has the potential to increase fertility in cows.

The regulation of oxytocin, oestradiol and progesterone receptors in different uterine cell types was studied in ovariectomized ewes. Animals were pretreated with a progestogen sponge for 10 days followed by 2 days of high-dose oestradiol to simulate oestrus. They then received either low-dose oestradiol (Group E), low-dose oestradiol plus progesterone (Group P) or low-dose oestradiol, progesterone and oxytocin (via osmotic minipump; Group OT). Animals (three to six per time-point) were killed following ovariectomy (Group OVX), at oestrus (Group O) or following 8, 10, 12 or 14 days of E, P or OT treatment. In a final group, oxytocin was withdrawn on day 12 and ewes were killed on day 14 (Group OTW). Oxytocin receptor concentrations and localization in the endometrium and myometrium were measured by radioreceptor assay, in situ hybridization and autoradiography with the iodinated oxytocin receptor antagonist d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH29]-vasotocin. Oestradiol and progesterone receptors were localized by immunocytochemistry. Oxytocin receptors were present in the luminal epithelium and superficial glands of ovariectomized ewes. In Group O, endometrial oxytocin receptor concentrations were high (1346 ± 379 fmol [3H]oxytocin bound mg protein−1) and receptors were also located in the deep glands and caruncular stroma in a pattern resembling that found at natural oestrus. Continuing low-dose oestradiol was unable to sustain high endometrial oxytocin receptor concentrations with values decreasing significantly to 140 ± 20 fmol mg protein−1 (P−1. Oxytocin treatment caused a small increase in oxytocin receptor concentration in the luminal epithelium on days 8 and 10 (20 ± 4 in Group P and 107 ± 35 fmol mg protein−1 in Group OT, P−1 in Group OTW). In contrast, oestradiol treatment was able to sustain myometrial oxytocin receptors (635 ± 277 fmol mg protein−1 in Group O and 255 ± 36 in Group E) and there was no increase over time in Groups P, OT and OTW with values of 61 ± 18, 88 ± 53 and 114 ± 76 fmol mg protein−1 respectively (combined values for days 8–14). Oestradiol receptor concentrations were high in all uterine regions in Group O. This pattern and concentration was maintained in Group E. In all progesterone-treated ewes, oestradiol receptor concentrations were lower in all regions at all time-points. The only time-related change occurred in the luminal epithelium in which oestradiol receptors were undetectable on day 8 but developed by day 10 of progesterone treatment. Progesterone receptors were present at moderate concentrations in the deep glands, caruncular stroma, deep stroma and myometrium in Group O. Oestradiol increased progesterone receptors in the luminal epithelium, superficial glands, deep stroma and myometrium. Progesterone caused the loss of its own receptor from the luminal epithelium and superficial glands and decreased its receptor concentration in the deep stroma and myometrium at all time-points. There was a time-related loss of progesterone receptors from the deep glands of progesterone-treated ewes between days 8 and 14. These results show differences in the regulation of receptors between uterine regions. In particular, loss of the negative inhibition by progesterone on the oxytocin receptor by day 14 occurred only in the luminal epithelium, but is unlikely to be a direct effect of progesterone as no progesterone receptors were present on luminal epithelial cells between days 8 and 14. The presence of oxytocin receptors in the luminal epithelium of ovariectomized ewes suggests that oestradiol is not essential for oxytocin receptor synthesis at this site. Oestradiol was able to sustain its own receptor at all sites, but high circulating progesterone was always inhibitory to oestradiol receptors. In general, oestradiol stimulated progesterone receptors in epithelial cells whereas progesterone abolished its own receptor from epithelial cells over a period of time, but had a lesser effect on stromal cells. The concentration of all three receptors is therefore differentially regulated between different uterine cell types, suggesting the importance of paracrine effects which remain to be elucidated. Journal of Endocrinology (1996) 151, 375–393

The oviduct is the site of fertilization, and factors present in the oviductal fluid appear to be crucial to the future success of conceptus development. The spatial and temporal localization of mRNA encoding components of the insulin-like growth factor (IGF) system (IGF-I, IGF-II, the type 1 IGF receptor, and IGF-binding proteins -2, -3 and -4) in the ovine oviduct were examined in tissue samples taken during the early and late stages of follicular development, and the early, mid-, and late luteal phases using in situ hybridization. Expression of mRNA encoding IGF-I showed a cyclical pattern, increasing sharply in the mucosa and muscularis during the late follicular phase (P < or = 0.001), then declining. In the muscularis, mRNA encoding IGF-II exhibited no temporal changes, but concentrations in the mucosa increased from the late follicular stage to the early luteal phase. mRNA encoding the type 1 IGF receptor was present throughout the oviduct. Concentrations increased during the follicular phase to peak in the early luteal phase in both the mucosa and muscularis (P < 0.001). IGFBP-2 gene transcripts were undetectable at all time points examined. mRNAs encoding IGFBP-3 and IGFBP-4 were localized primarily in the stromal region. IGFBP-3 expression peaked in the late follicular stage of the cycle P < or = 0.001). The concentration of mRNA encoding IGFBP-4 increased in the follicular phase and was maintained at a significantly higher concentration during the early and mid-luteal stages (P < 0.001). The co-ordinate maximum expression of mRNA for both IGF-I and IGF-II, the type 1 IGF receptor and IGFBP-3 during the period when the gametes and embryo are in transit suggests a role for IGF-I and possibly IGF-II peptides in providing an oviductal environment propitious to conception and early embryonic growth and metabolism.