Impact of endometritis on post-partum ovarian cyclicity in dairy cows.

Endometritis in dairy cows is a major global issue and has been associated with a decrease in reproductive performance. The aim of this study was to quantify the effect of endometritis (as defined by the presence of any abnormal vaginal discharge after 21 days post-partum) on post-partum ovarian cyclicity in dairy cows. Milk progesterone analysis was used to monitor reproductive cyclicity in 170 dairy cows across three different commercial herds. Associations between the occurrence of endometritis and the incidence risk of a variety of atypical cycle profiles during the calving to conception period were investigated to establish the importance of endometritis on post-partum ovarian activity. Endometritis increased the incidence odds of atypical ovarian profiles (P < 0.05) with prolonged luteal activity being the most affected (P < 0.05), but also showed prolonged time (3 days) to onset of luteal activity after parturition (P < 0.05). Using milk progesterone analysis, we found a relatively low incidence odds for reproductive cycle problems in healthy cows during the calving to conception period. However, the incidence odds of cycle problems, in particular prolonged luteal activity, were high in cows that had experienced endometritis, which would have significantly impaired reproductive function.

Novel gonadal characteristics in an aged bovine freemartin.

The gonads from a five-year-old freemartin Holstein animal were subjected to morphological analysis and to immunohistochemistry using antibodies against developmental and functional markers. We demonstrate, for the first time, the retention of anti-mullerian hormone (AMH) producing intratubular cells (Sertoli cells) in the context of abundant steroidogenic interstitial cells, and structures consistent with clusters of luteal cells. This novel report describes the clinical, gross and histological findings accompanying this newly described gonadal immunophenotype, and its implication in the understanding of freemartin development.

The progesterone receptor antagonist, onapristone has differential effects on the timing and control of the luteolytic mechanism depending on timing of administration in sheep.

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.

Angiogenesis and vascular function in the ovary.

Ovarian function is dependent on the establishment and continual remodelling of a complex vascular system. This enables the follicle and/or corpus luteum (CL) to receive the required supply of nutrients, oxygen and hormonal support as well as facilitating the release of steroids. Moreover, the inhibition of angiogenesis results in the attenuation of follicular growth, disruption of ovulation and drastic effects on the development and function of the CL. It appears that the production and action of vascular endothelial growth factor A (VEGFA) is necessary at all these stages of development. However, the expression of fibroblast growth factor 2 (FGF2) in the cow is more dynamic than that of VEGFA with a dramatic upregulation during the follicular-luteal transition. This upregulation is then likely to initiate intense angiogenesis in the presence of high VEGFA levels. Recently, we have developed a novel ovarian physiological angiogenesis culture system in which highly organised and intricate endothelial cell networks are formed. This system will enable us to elucidate the complex inter-play between FGF2 and VEGFA as well as other angiogenic factors in the regulation of luteal angiogenesis. Furthermore, recent evidence indicates that pericytes might play an active role in driving angiogenesis and highlights the importance of pericyte-endothelial interactions in this process. Finally, the targeted promotion of angiogenesis may lead to the development of novel strategies to alleviate luteal inadequacy and infertility.

Foraminiferal isotope evidence of reduced nitrogen fixation in the ice age Atlantic Ocean.

Fixed nitrogen (N) is a limiting nutrient for algae in the low-latitude ocean, and its oceanic inventory may have been higher during ice ages, thus helping to lower atmospheric CO2 during those intervals. In organic matter within planktonic foraminifera shells in Caribbean Sea sediments, we found that the 15N/14N ratio from the last ice age is higher than that from the current interglacial, indicating a higher nitrate 15N/14N ratio in the Caribbean thermocline. This change and other species-specific differences are best explained by less N fixation in the Atlantic during the last ice age. The fixation decrease was most likely a response to a known ice age reduction in ocean N loss, and it would have worked to balance the ocean N budget and to curb ice age-interglacial change in the N inventory.

Corpus luteum-endometrium-embryo interactions in the dairy cow: underlying mechanisms and clinical relevance.

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.

A novel physiological culture system that mimics luteal angiogenesis.

Luteal inadequacy is a major cause of poor embryo development and infertility. Angiogenesis, the formation of new blood vessels, is an essential process underpinning corpus luteum (CL) development and progesterone production. Thus, understanding the factors that regulate angiogenesis during this critical time is essential for the development of novel strategies to alleviate luteal inadequacy and infertility. This study demonstrates the development of a physiologically relevant primary culture system that mimics luteal angiogenesis. This system incorporates all luteal cell types (e.g. endothelial, steroidogenic cells, fibroblasts and pericytes). Using this approach, endothelial cells, identified by the specific marker von Willebrand factor (VWF), start to form clusters on day 2, which then proliferate and develop thread-like structures. After 9 days in culture, these tubule-like structures lengthen, thicken and form highly organized intricate networks resembling a capillary bed. Development of the vasculature was promoted by coating wells with fibronectin, as determined by image analysis (P<0.001). Progesterone production increased with time and was stimulated by LH re-enforcing the physiological relevance of the model in mimicking in vivo luteal function. LH also increased the area stained positively for VWF by twofold (P<0.05). Development of this endothelial cell network was stimulated by fibroblast growth factor 2 and vascular endothelial growth factor A, which increased total area of VWF positive staining on day 9, both independently (three- to fourfold; P<0.01) and in combination (tenfold; P<0.001). In conclusion, the successful development of endothelial cell networks in vitro provides a new opportunity to elucidate the physiological control of the angiogenic process in the developing CL.

Quantitative analysis of changes in endometrial gland morphology during the bovine oestrous cycle and their association with progesterone levels.

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

Corpus luteum size and function following single and double ovulations in non-lactating dairy cows.

Data was collated from a number of studies on various aspects of luteal function in non-lactating dairy cows to allow comparisons to be made between single and double ovulating animals. In these studies, estrous cycles had been synchronized and animals slaughtered on day 5 or 8. The overall incidence of double ovulations was 28.3%. Double ovulation was associated with smaller individual corpora lutea but no difference in total weight of luteal tissue or any aspect of luteal tissue function or plasma concentrations of progesterone. Furthermore, in a sub set of animals, there was no difference in preovulatory follicle characteristics or plasma concentrations of estradiol around ovulation. These results demonstrated a high incidence of double ovulation in non-lactating cows that had no influence on circulating progesterone concentrations.

Fibroblast growth factor 2 is more dynamic than vascular endothelial growth factor A during the follicle-luteal transition in the cow.

Luteal inadequacy is a major cause of infertility in a number of species. During the early luteal phase, progesterone production requires the rapid growth of the corpus luteum (CL), which is in turn dependent on angiogenesis. In the present study, we examined the temporal changes in vascular endothelial growth factor A (VEGFA), fibroblast growth factor 2 (FGF2) and secreted protein, acidic, cysteine-rich (osteonectin) (SPARC) during the follicular-luteal transition and CL development in the cow. Luteal VEGFA concentrations increased as the CL developed but were lower in the regressing CL. Conversely, luteal FGF2 concentrations were highest immediately postovulation in the collapsed follicle and declined as the CL developed. Furthermore, three FGF2 isoforms were present in the collapsed follicle, but only one isoform was detected in older CL. Interestingly, FGF2 concentrations increased in the regressing CL. Western blot analysis for SPARC showed the presence of two isoforms, which were constitutively expressed throughout CL development. Further studies investigated the regulation of FGF2 by LH, which showed that FGF2 concentrations in preovulatory follicular fluid were higher in those animals that had experienced an LH surge. Moreover, LH stimulated FGF2 production in dispersed luteal cells. Conversely, the LH surge had no effect on follicular fluid VEGFA concentrations. In conclusion, FGF2 was more dynamic than VEGFA and SPARC during the follicular-luteal transition, which suggests that FGF2 plays a key role in the initiation of angiogenesis at this time. Furthermore, it is likely that this is stimulated by the LH surge. The results also suggest that VEGFA and SPARC have a more constitutive, but essential, role in the development of the CL vasculature.

Leptin infusion during the early luteal phase in ewes does not affect progesterone production.

Infusion of leptin during the ovine follicular phase has been shown to increase progesterone secretion during the subsequent luteal phase. In this study, we have assessed the effects of infusing leptin during the early luteal phase. Infusion of leptin (2.5 microg/h) into the ovarian artery of ewes with ovarian autotransplants (n=5) on day 3 of the luteal phase for 12h did not affect progesterone estradiol or LH concentrations compared to control ewes (n=5). These results suggest no direct effect of leptin on ovarian function at this stage of the estrous cycle.

Leptin in the bovine corpus luteum: receptor expression and effects on progesterone production.

In cattle, leptin has been implicated in the control of ovarian function and has been shown to modulate steroid production by theca and granulosa cells in a number of species. However, a direct effect of leptin on bovine luteal function has not been demonstrated. This study was conducted to determine if the leptin receptor (OB-R) is expressed in the bovine corpus luteum (CL), and to examine the effects of leptin on progesterone production by dispersed luteal cells in vitro. RT-PCR was used to detect the presence of OB-R and, more specifically, the long, biologically active isoform (OB-Rb), in CL, collected on days 2-18 of the oestrous cycle (n=18). The effects of leptin on progesterone production were investigated in dispersed luteal cells prepared from CL collected on days 5 and 8 (n=14) of the cycle. The dispersed luteal cells were cultured for 24 hr with recombinant human leptin and/or LR3-IGF-1 and/or LH. OB-Rs, in particular, OB-Rb, were expressed in the CL at all stages of development. Progesterone production by luteal cells was increased (P<0.001) by treatment with LH (10 ng/ml) but treatment with leptin alone had no effect. However, in the presence of IGF-1 (100 ng/ml), leptin (10 ng/ml) caused a significant (P<0.005) increase in progesterone production. In conclusion, we have shown that the leptin receptor is expressed in the bovine CL and have demonstrated a modulatory effect of leptin on luteal progesterone production in vitro.

Identification of elevated concentrations of estradiol in bovine uterine endometrium.

We have investigated the estradiol content of bovine endometrium and related this to circulating plasma estradiol content. In 9 heifers, mean+/-S.E.M. plasma estradiol concentration was 0.64+/-0.25 pg/ml while the mean+/-S.E.M. endometrial estradiol content was 43.0+/-14.7 pg/g tissue; there was a close relationship between plasma and tissue estradiol levels (R(2)=0.81; P<0.001). During culture of endometrial tissue there was a progressive transfer of estradiol from tissue to culture media but no change in total estradiol. Culture of endometrium from 4 heifers with 5 ng/ml testosterone for 72 h resulted in no increase in estradiol. Furthermore, immunohistochemistry revealed no aromatase protein in uterine endometrium. These results confirm high stored tissue concentrations of estradiol in bovine endometrium while providing no evidence for estradiol synthesis by this tissue. The mechanism(s) through which this sequestration of estradiol into uterine tissue occurs remains to be determined.

In vivo expression of interferon tau mRNA by the embryonic trophoblast and uterine concentrations of interferon tau protein during early pregnancy in the cow.

In this study, we have measured uterine concentrations of interferon tau and intensity of embryonic interferon tau mRNA expression between day 14 and 18 in cows. While interferon tau concentrations rose dramatically (P < 0.001) from day 14 to 18, there was no significant increase in the intensity of expression of interferon tau mRNA by the trophoblast. When results were analyzed on the basis of embryo size, well elongated embryos (>10 cm) produced significantly (P < 0.001) more interferon tau than smaller embryos but showed similar levels of interferon tau mRNA expression. These results demonstrate that the increase in interferon tau concentrations responsible for the maternal recognition of pregnancy results from the increase in embryo size during elongation and not from any upregulation of mRNA expression.

Endocrine and cellular characteristics of corpora lutea from cows with a delayed post-ovulatory progesterone rise.

The timing of the post-ovulatory progesterone rise is critical to the embryonic development and survival. The aim of this study was to determine the underlying causes of delayed post-ovulatory progesterone rises. Two groups of non-lactating dairy cows with early (n = 11) or late (n = 9) post-ovulatory progesterone rises were created by inducing luteolysis in the presence of either a large (> 10 mm) or small (< 10 mm) follicle, respectively. LH pulses were measured on days 4 (all cows) and 7 (n = 7, early; n = 5, late) (day 1= ovulation). The cows were slaughtered on day 5 (n = 4 each group) or 8 (n = 7, early; n = 5, late). Immunohistochemical analysis for endothelial cells (von Willebrand Factor, VWF), steroidogenic cells (3beta-HSD) and proliferation marker (Ki67) were performed. The basal progesterone production and LH responsiveness (0.001-100 ng/ml) of dispersed luteal cells was investigated. The luteal concentrations of FGF-2 and VEGF were measured by ELISA and RIA, respectively. There were no differences in LH pulse characteristics, area of VWF staining, proliferation index, steroidogenic cell characteristics, basal or LH-stimulated progesterone production by luteal cells between cows with an early or late progesterone rise (P > 0.10). However, the area of VWF staining increased from days 5 to 8, while the proliferation index decreased (P < 0.05). Furthermore, the luteal cells were more responsive to LH on day 8 (P < 0.01). Luteal concentrations of FGF-2 were higher on day 5 (P = 0.05), while VEGF was greater on day 8 (P < 0.01). In conclusion, we have clearly shown that LH support, degree of vascularization or luteal cell steroidogenic capacity were not the major factors responsible for inadequate secretion of progesterone by the developing bovine CL.

The induction of a delayed post-ovulatory progesterone rise in dairy cows: a novel model.

A delayed rise in post-ovulatory progesterone is associated with poor embryo development in the cow, although the underlying cause of this aberrant luteal function is poorly understood. The objective of this study was to develop a novel model, in which a delayed progesterone rise could be induced by manipulating the dynamics of the follicular phase. Luteolysis was induced in 20 dairy cows in the presence of either a larger follicle > 10 mm (LF, n = 11) or a smaller follicle < 10 mm (SF, n = 9) and transrectal ultrasonography was performed to determine follicle and CL growth and timing of ovulation. Plasma progesterone and oestradiol were analysed 3x daily. Cows were slaughtered on either day 4 (n = 4 per group) or day 7 (SF, n = 5; LF, n = 7) after ovulation. The pre-ovulatory follicle was larger in the LF group than the SF group at luteolysis (13.5 +/- 0.4 mm versus 6.7 +/- 0.7 mm, P < 0.001) and ovulation (16.7 +/- 0.3 mm versus 13.6 +/- 0.6 mm, P < 0.001). The LF group experienced a shorter follicular phase and ovulated 36 h earlier than the SF group (P < 0.001). At luteolysis, plasma oestradiol concentrations were greater in the LF group (P < 0.001), although peak concentrations were not different (P > 0.05). Moreover, higher progesterone concentrations were observed in the LF group during the early luteal phase (P < 0.05). Luteal weights were positively correlated with plasma progesterone concentrations on day 5 (P < 0.05) but not day 8. In conclusion, a model has been developed which has shown that the dynamics of follicle development during the pre-ovulatory period is an important determinant of subsequent CL development and function.

Endocrine and paracrine control of follicular development and ovulation rate in farm species.

Productivity in farm species is controlled by many factors, including ovulation rate. In cattle, single ovulations occur most frequently and in sheep (and goats) the number of ova released can range from one to many depending upon the breed, whilst the pig is polyovular. The processes of recruitment and selection determine the number of ovulatory follicles in all these species with FSH and subsequently LH playing major roles. GnRH-agonist models in which endogenous gonadotrophin secretion is suppressed and exogenous LH and/or FSH are administered at specific concentrations in defined patterns, are useful in all three species for elucidating the precise roles of specific hormones in stimulating follicular development. Differences in the hypothalamic-pituitary-ovarian feedback response lead to the differences in the number of ovulatory follicles, as does the pool of antral follicles from which the ovulatory ones are selected. Precocious development of follicles is also associated with more ovulations, as is the case with the Booroola due to the single gene acting through bone morphogenetic proteins (BMPs). It is well established that ovulation rate can also be influenced by exogenous hormone administration and by environmental factors such as nutrition. It has become apparent that these nuritional effects are mediated by a direct action at the level of the ovary, involving insulin, insulin-like growth factors (IGF) I and II and their binding proteins among other factors. These factors can also affect the quality of the oocyte and consequently embryo development and survival. Recently, the regulation of follicular angiogenesis has been shown to be important for the development of ovulatory follicles, particularly vascular endothelial growth factor (VEGF) which is produced primarily by the granulosa cells within the ovary and can be stimulated by gonadotrophins. Administration of VEGF has been shown to stimulate pre-antral follicular growth and increase the number of pre-ovulatory follicles. In summary both extra- and intra-ovarian factors are involved in the control of ovulation rate. Manipulation of the angiogenic process may also provide new opportunities for regulating the quality and number of follicles that ovulate.

Effects of dietary polyunsaturated fatty acids on ovarian and uterine function in lactating dairy cows.

Alteration of the polyunsaturated fatty acid (PUFA) composition of milk by dietary supplementation of cows may be beneficial to human health. However, dietary PUFAs may influence synthesis of both prostaglandins and steroid hormones. This study examined the effects of dietary PUFAs on reproductive parameters in lactating cows. Cows were fed an isoenergetic control ration (n = 8) or a diet supplemented with LinPreme (n = 7) or SoyPreme (n = 8). These proprietary feeds are derived from linseed or soybeans and contain high concentrations of linolenic acid (LNA, n-3) or linoleic acid (LA, n-6) protected PUFA, respectively. Both PUFA-supplemented diets reduced plasma progesterone, particularly in the early luteal phase, and increased the number of medium-sized (5-10 mm in diameter) follicles. The diameter of the first dominant follicle, insulin-like growth factor I (IGF-I) concentrations at oestrus and cholesterol concentrations were all higher in cows fed a diet supplemented with LA (n-6) than in cows that did not receive this supplement. In cows fed a diet supplemented with LNA (n-3), there was an increase in oestradiol during the follicular phase. Diet had no effect on non-esterified fatty acid or insulin concentrations, or on the duration of the oestrous cycle. The plasma concentration of 13,14,dihydro-15 keto PGF(2alpha) after administration of 50 iu oxytocin was unaffected by diet on day 15 and day 16 of the oestrous cycle, but showed a greater response on day 17 in the LA (n-6) supplemented group. Therefore, the PUFA content of the diet can influence both ovarian and uterine function in cows. However, further studies using larger numbers of cows are required to test whether fertility is also affected by such diets.

Expression of the insulin-like growth factor (IGF) system in the bovine oviduct at oestrus and during early pregnancy.

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.

Expression of oxytocin, oestrogen and progesterone receptors in uterine biopsy samples throughout the oestrous cycle and early pregnancy in cows.

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.