Large Corpora Lutea Research Articles

Effects of dietary ferulic acid on reproductive function and metabolism of pre-pubertal hairbreed ewes during the anestrous season

This study aimed to evaluate the effects of dietary supplementation of ferulic acid (FA) on reproductive tract weight (RTW), ovarian activity and serum concentrations of some metabolites and thyroid hormones of pre-pubertal hairbreed ewe lambs during the natural anestrous season. Twenty Dorper x Pelibuey crossbred ewe lambs (initial body weight = 28.5 ± 0.5 kg and age = 5.0 mo) were fed a basal diet and assigned to two treatments (n = 10) under randomized complete block design: FA supplementation to dosages of 0 (control) or 300 mg of FA/d animal−1. Females were treated with FA during 34 d and then slaughtered to evaluate RTW and ovary. Blood samples were collected at days 1 and 34 to determine serum concentrations of metabolites (i.e. glucose, cholesterol, triglycerides, urea and total protein) and thyroid hormones. Final weight, dry matter intake, and serum concentrations of metabolites (except glucose) and thyroid hormones were unaltered by FA. Compared to control, FA-fed ewe lambs had lower (P = 0.05) serum glucose level but higher (P ≤ 0.05) RTW, ovarian mass, number of larger follicles and corpus luteum (CL). While percentage of ewe lambs with small follicles and number of small follicles per female did not vary, percentage of ewe lambs with large follicles and CL increased (P = 0.02) with FA. In conclusion, FA supplementation improves the reproductive tract development and ovarian activity of pre-pubertal hairbreed ewe lambs during the natural anestrous season. An improvement in functionality of the glucose-insulin system could be related with this benefic effect of FA.

XY (SRY-positive) Ovarian Disorder of Sex Development in Cattle

In mammals, the sex of the embryo depends on the SRY gene. In the presence of at least one intact and functional copy of this genetic factor (XY embryo) undifferentiated gonads will develop as testicles that subsequently determine the male phenotype. When this factor is not present, i.e., in subjects with 2 X chromosomes, an alternative pathway induces the development of ovaries, hence a female phenotype. In this case study, we describe a female cattle affected by a disorder of sex development (DSD). The subject, despite having a chromosomal XY constitution, did not develop testicles but ovaries, although they were underdeveloped. Moreover, genetic analysis highlighted the presence of the SRY gene with a normal coding region in both blood- and tissue-derived DNA. A chimeric condition was excluded in blood by sexing more than 350 cells and by allele profile investigation of 18 microsatellite markers. Array CGH analysis showed the presence of a not yet described 99-kb duplication (BTA18), but its relationship with the phenotype remains to be demonstrated. Gonadal histology demonstrated paired ovaries: the left one containing a large corpus luteum and the right one showing an underdeveloped aspect and very few early follicles. To our knowledge, we describe the first case of XY (SRY+) DSD in cattle with a normal SRY gene coding sequence.

Karayaka Koyunlarında Gonadotropin Salgılatıcı Hormon Uygulamasının (GnRH) Ovaryum Aktivitesi Üzerine Etkisi

Bu çalışmanın amacı, Karayaka koyunlarında dönem içi gonadotropin salgılatıcı hormon (GnRH) uygulamasının kızgınlık gösterme oranı ve ovaryum aktivitesi üzerine etkisini belirlemektir. Çalışmada en az iki doğum yapmış ve benzer vücut ağırlığına sahip 20 baş Karayaka ırkı koyun hayvan materyali olarak kullanılmıştır. Deneme başında tüm koyunlara ovaryum üzerinde mevcut olabilecek olan korpus luteum (CL)‘un yıkımı için 1 ml PGF2α kas içi enjekte edilmiştir. PGF2α uygulamasından 9 gün sonra ise koyunlar rastgele iki eşit gruba ayrılmıştır. Birinci gruptaki koyunlara (n=10) 40 mg flugestone asetat içeren sünger vajina içine yerleştirilmiştir ve bu uygulamadan 14 gün sonra süngerler çıkartılarak, 1 ml PGF2α kas içi enjekte edilmiştir. PGF2α enjeksiyonunu takiben 36 saat sonra 1 ml GnRH kas içi enjekte edilmiştir. İkinci gruptaki koyunlara (n=10) ise mevcut olabilecek olan korpus luteum (CL)‘un yıkımı için 1 ml PGF2α kas içi enjeksiyonun dışında herhangi bir uygulama yapılmamış olup, bu grup çalışmamızın kontrol grubunu oluşturmuştur. Bu işlemleri takiben her iki gruptaki bütün koyunlar Karayaka koçuna verilmiş ve 72 saat boyunca kızgınlık gösteren koyunlar belirlenmiştir. Kızgınlık taramasının sonunda bütün koyunlar kesime sevk edilmiş ve her iki ovaryum üzerindeki küçük (1-3 mm), büyük (>3 mm) ve toplam folikül sayısı ve CL sayısı belirlenmiştir. GnRH uygulaması yapılan koyunlar (%100) kontrol grubundaki koyunlara (%70) göre daha yüksek oranda kızgınlık göstermişlerdir. GnRH uygulaması yapılan koyunların her iki ovaryumundaki küçük ve toplam folikül sayısının ve CL sayısının, kontrol grubundaki koyunlarınkinden daha yüksek olduğu tespit edilmiştir. Ancak büyük folikül sayısı bakımından deneme grupları arasında bir farklılık tespit edilmemiştir. Sonuç olarak bu çalışmada mevsim içi dönemde Karayaka koyunlarında sünger uygulaması sonrasındaki GnRH uygulamasının kızgınlık gösterme oranını yükselttiği ve ovaryum aktivitesini arttırdığı tespit edilmiştir.

MiR-29b affects the secretion of PROG and promotes the proliferation of bovine corpus luteum cells.

The regulatory role of miRNAs has been explored in ovarian cells, and their effects on gonadal development, apoptosis, ovulation, steroid production and corpus luteum (CL) development have been revealed. In this study, we analyzed the expression of miR-29b at different stages of bovine CL development and predicted the target genes of miR-29b. We confirmed that miR-29b reduces the expression of the oxytocin receptor (OXTR), affects progesterone (PROG) secretion and regulates the function of the CL. RT-PCR showed that the expression of miR-29b was significantly higher in functional CL phases than in the regressed CL phase. Immunohistochemistry showed that OXTR was expressed in both large and small CL cells and was mainly located in the cell membrane and cytoplasm of these cells. We analyzed the expression levels of OXTR and found that transfection with a miR-29b mimic decreased OXTR expression, but transfection with the inhibitor had a limited effect on the expression of the OXTR protein. At the same time, the secretion of PROG was significantly increased in the miR-29b mimic-transfected group. We also analyzed the effect of miR-29b on the apoptosis of CL cells. Finally, we found that miR-29b could promote the proliferation of bovine CL cells. In conclusion, we found that miR-29b reduces the expression of OXTR and can promote PROG secretion and the proliferation of CL cells via OXTR.

Large corpus luteum cyst associated with pregnancy: a case report

A gestational corpus luteum cyst is the most common adnexal mass in early pregnancy and rarely exceeds five cm in diameter. Thus, surgery is rarely indicated. The present study reports the case of a 24-year-old pregnant woman who presented with a large adnexal mass that manifested as a large corpus luteum cyst at 32 weeks of pregnancy which required surgical intervention due to significant abdominal discomfort reported by the patient.

Pairing ultrasonography with endocrinology to elucidate underlying mechanisms of successful pregnancy in the northern fur seal (Callorhinus ursinus)

Reproductive success is one of the central tenets of conservation management programs, yet the inability to study underlying physiological processes in a minimally-invasive manner and the unpredictable nature of wild animal populations leaves large gaps in our knowledge of factors critical to successful reproduction in wild species. This study integrated ultrasonography of the reproductive tract and analysis of reproductive hormones in 172 northern fur seals (Callorhinus ursinus) to identify intrinsic factors associated with reinitiating embryonic growth at the end of diapause. Within the first 3–4 weeks of active gestation, pregnant fur seals (n = 126) had a larger corpus luteum and fewer antral follicles than non-pregnant fur seals, or those still in diapause (n = 46). This suggests that the conceptus drives changes in ovarian status to convey its presence to the female. Morphological changes in the reproductive tract associated with pregnancy were not reflected in differences in endocrine profiles (estradiol, estrone, progesterone, and relaxin) between pregnant and non-pregnant individuals. Hormone concentrations correlated more strongly with calendar date than with the presence or size of the conceptus, demonstrating that none of these reproductive hormones were reliable markers for early pregnancy diagnosis. Instead, the northern fur seal’s long diestrus may serve to reduce the probability of a temporal mismatch between corpus luteum regression and embryo implantation. Indeed, conception rates were high and confirmed rates of pregnancy loss were relatively low (11%). In this study, minimally-invasive ultrasonography was used in wild pinnipeds to detect very early pregnancy (embryonic vesicles >2 mm) in combination with ovarian and endocrine dynamics at the time of embryo implantation, shedding light on mechanisms for maternal recognition of pregnancy. This study is also the first to track whether these same animals carried the embryo to term, by observing fur seals during the birthing season the following year. Data do not support the notion that decreased pregnancy rates or higher pregnancy loss rates are major contributing factors to the northern fur seal’s population decline.

Supplementation of dairy cows with docosahexaenoic acid did not affect ovarian activity

The effect of docosahexaenoic acid (DHA) on ovarian activity of dairy cows was determined. Experimental cows (n = 25) were fed a total mixed ration supplemented daily with 100 g/cow of an algae product All-G-Rich (Alltech, Ireland) containing 10% DHA divided into 2 doses for 52 days. Determination of DHA from milk samples taken from all cows was performed before the All-G-Rich supplementation (on Day 0, D0), and on D21 and D42 of algae supplementation. Cows were synchronized to be in oestrus on D21 and D42 of the experimental period. Monitoring of ovarian activity was performed by transrectal ultrasonography. Examinations were performed at 2–3-day intervals from D0 until D52 of the experimental period. Plasma concentrations of progesterone, oestradiol, insulin, NEFA, and cholesterol were determined. Control cows (n = 25) were examined in the same way as the experimental cows. Milk DHA concentrations on D21 and D42 were significantly higher in treated cows (D21 1.38 vs 0.28, P < 0.0001; D42 1.34 vs 0.20, P < 0.0001). There were neither important effects of DHA on ovarian structures, nor on evaluated variables in plasma. Cows in the experimental group tended to have larger corpora lutea and higher cholesterol concentrations, but differences were not significant.

Sex steroids modulate morphological and functional features of the bovine oviduct.

In cattle, the oviduct plays a major role in the reproductive process; however, molecular control of oviduct receptivity to the embryo is poorly understood. A model for receptivity based on size of the pre-ovulatory follicle (POF) was used to compare oviductal morphology, cellular proliferation, and candidate transcript abundance. Growth of the POF of Nelore (Bos indicus) cows was manipulated to produce two groups: a large POF-large corpus luteum (CL) group (LF-LCL; greater receptivity) and a small POF-small CL group (SF-SCL). Samples of the ampulla and isthmus ipsilateral and contralateral to CL were collected 4days after GnRH-induced ovulation. Tissues were either embedded in paraffin for Harris-Hematoxylin and Eosin and periodic acid-Schiff staining and KI67 immunostaining, followed by morphological analyses, or stored at -80°C for RNA extraction, cDNA synthesis, and qPCR analyses. The effects of group (LF-LCL and SF-SCL), region (ampulla and isthmus), and side (ipsilateral and contralateral) were analyzed using three-way nested ANOVA. The ipsilateral ampulla of the LF-LCL group presented more primary mucosal folds, a greater mucosal-folding grade and luminal perimeter, and more secretory cells and proliferating cells when compared with the ampulla of the SF-SCL group and with the contralateral ampulla of both groups. There were no morphological differences in the isthmus between groups and sides. Changes in transcript abundance are suggestive of LF-LCL-stimulated secretory activity. In summary, ovulation of a larger POF generates a periovulatory endocrine milieu that modulates morphological and functional features of the bovine oviduct which may support embryo survival and development.

Reproductive outcomes of anestrous goats supplemented with spineless Opuntia megacantha Salm-Dyck protein-enriched cladodes and exposed to the male effect.

The possible influence of the "male effect" upon reproductive outcomes of adult anestrous goats under marginal rangeland conditions and supplemented with protein-enriched Opuntia megacantha Salm-Dyck was evaluated. Reproductive variables included: estrus percentage (EST, %), estrus latency (ESL, hours), ovulation percentage (OP, %), ovulation rate (OR, units), average largest follicle at ovulation (LFO, mm), largest corpus luteum (LCL, mm), embryo number (EBN, units), and embryo implantation percentage (EIP, %). During early May, anestrous mix-breed adult goats (Criollo x Alpine-Saanen-Nubian; n=38, 26° N) were randomly distributed to (1) Control (CC; n=12), (2), Non-enriched Opuntia (NEO; n=14), and (3) Protein-enriched Opuntia (PEO; n=12). Neither LW (P>0.05) nor BCS (P>0.05) or any of the evaluated ovarian variables differed (P>0.05) among treatments; EST=89.66%, ESL=53.66 h, OP=70.33%, OR=1.07 units, LFO=4.5 mm, LCL=9.6 mm, EBN=0.94 embryos, and EIP=48.66%. Irrespective of nutritional supplementation regime, all goats denoted an increased response to the male effect just in the middle of the anestrous season and managed under marginal grazing conditions during the dry season (May to June; 26° N). The use of the male effect successfully invoked neurophysiological pathways to re-activate ovarian follicular and luteal pathways during the natural anestrous season in the female goat. Yet, such successful physiological scenario was not equally exerted to promote an increased embryo implantation rate; this issue claims further consideration. Therefore, it is essential to align not only the peri-conceptional but also the peri-implantation stages to the best suited environmental conditions in the rangeland, in order to increase both reproductive and economic efficiency while promoting sustainability in those rangeland-based marginal goat production systems.

Identification and expression of X-linked inhibitor of apoptosis protein during follicular development in goat ovary

X-linked inhibitor of apoptosis protein (XIAP), an endogenous of inhibitor of caspases, plays crucial roles in regulating ovarian granulosa cell apoptosis during follicular atresia. The aim of the present study was to determine the presence and localization of XIAP in the goat ovary and its expression level during follicular development. The full length cDNA of XIAP from goat ovary cells was cloned using reverse transcription PCR. A total of 497 amino acid residues were encoded by open reading frame and had high identity with homologous sequences from other mammals. XIAP was widely expressed in adult goat tissues as determined by real-time PCR and it demonstrated higher expression in propagative organs. High level of XIAP was detected in large healthy follicles and corpus luteum in comparison with that in small antral follicles, which was in accordance with the immunohistochemistry results and atretic follicles had very low expression. XIAP was localized in both granulosa and theca cells in antral follicles but not in primordial follicles. Furthermore, luteinizing hormone stimulated the proliferation of mRNA encoding XIAP in granulosa cells in vitro. The present study demonstrated that XIAP was expressed in a follicular-stage–dependent manner in goat ovaries.

13 EFFECTS OF NERVE GROWTH FACTOR-β, PURIFIED FROM BULL SEMINAL PLASMA, ON CORPUS LUTEUM FUNCTION AND CONCEPTUS DEVELOPMENT IN COWS

Nerve growth factor-β (NGF) has been identified in the seminal plasma of livestock and is required for the induction of ovulation in camelids. Until recently, it was thought to play a negligible role in species with spontaneous ovulation. On the contrary, recent studies have shown that systemic administration of NGF, purified from llama seminal plasma, was associated with larger corpus luteum (CL) diameter and higher concentrations of progesterone (P) following ovulation. The objective of the current project was to determine if systemic administration of NGF, purified from bovine seminal plasma, would improve CL formation and enhance embryonic development. Our hypothesis was that systemic administration of NGF at the time of artificial insemination in cows would lead to increased CL volume, increased P secretion, and improved expression of markers of conceptus development and maternal recognition of pregnancy. Seminal plasma was harvested from semen collected by electroejaculation in Angus cross bulls. Purification of NGF was performed using a combination of anion- and cation-exchange chromatography and gradient elution. Beef cows were randomly assigned to CONT (n = 30) or NGF (n = 30) groups and synchronized using a 7-day Co-Synch + CIDR program. At time of insemination (day 0), NGF cows received 296 µg of purified NGF, reconstituted in 12 mL of PBS, and CONT cows received 12 mL of PBS intramuscularly. Blood samples were collected from the coccygeal vein of each cow at days 0, 3, 7, 10, 14, 19, 21, 28, 31, 38, 45, and 66. Ultrasound was performed at each time point for determination of ovarian structures (day 0), corpus luteum volume (all time points), and fetus detection (day 28). Statistical analysis was performed using analysis of variance with repeated-measures in R (R version 3.2.2; https://www.r-project.org/). At day 28, 17/30 (57%) CONT cows and 21/30 (70%) NGF cows were diagnosed as pregnant (P = 0.15). At day 0, cows that later became pregnant had a larger follicular diameter (1.46 cm) than those diagnosed as open (1.16 cm; P < 0.01). Follicular diameter at day 0 did not differ significantly between CONT and NGF groups for either open (P = 0.35) or pregnant (P = 0.90) cows. CL volume in open cows was affected by day (P < 0.001) with no treatment (P = 0.84) or treatment by day (P = 0.42) interaction. CL volume in pregnant cows was affected by both day (P < 0.001) and treatment (P < 0.001), with CONT cows having a higher CL volume than NGF cows. Though NGF appeared to have an inhibitory effect on CL volume in pregnant cows, results are still pending for quantification of P, insulin-like growth factor 1, pregnancy-specific protein B, and interferon-stimulated genes, which will more accurately assess the effects that NGF may have on conceptus development. The results of this study will help us to better understand the role of the seminal plasma protein, NGF, at ovulation and determine if it can be utilised to enhance insemination programs in cattle.

Size of the Ovulatory Follicle Dictates Spatial Differences in the Oviductal Transcriptome in Cattle.

In cattle, molecular control of oviduct receptivity to the embryo is poorly understood. Here, we used a bovine model for receptivity based on size of the pre-ovulatory follicle to compare oviductal global and candidate gene transcript abundance on day 4 of the estrous cycle. Growth of the pre-ovulatory follicle (POF) of Nelore (Bos indicus) cows was manipulated to produce two groups: large POF large corpus luteum (CL) group (LF-LCL; greater receptivity) and small POF-small CL group (SF-SCL). Oviductal samples were collected four days after GnRH-induced ovulation. Ampulla and isthmus transcriptome was obtained by RNA-seq, regional gene expression was assessed by qPCR, and PGR and ERa protein distribution was evaluated by immunohistochemistry. There was a greater abundance of PGR and ERa in the oviduct of LF-LCL animals thus indicating a greater availability of receptors and possibly sex steroids stimulated signaling in both regions. Transcriptomic profiles indicated a series of genes associated with functional characteristics of the oviduct that are regulated by the periovulatory sex steroid milieu and that potentially affect oviductal receptivity and early embryo development. They include tissue morphology changes (extra cellular matrix remodeling), cellular changes (proliferation), and secretion changes (growth factors, ions and metal transporters), and were enriched for the genes with increased expression in the LF-LCL group. In conclusion, differences in the periovulatory sex steroid milieu lead to different oviductal gene expression profiles that could modify the oviductal environment to affect embryo survival and development.

Molecular characterization and expression profile of the melatonin receptor MT1 in the ovary of Tianzhu white yak (Bos grunniens)

Melatonin plays crucial roles in a wide range of ovarian physiological functions via the melatonin receptors (MRs). Structure and function of MRs have been well studied in sheep, cattle, and humans, but little information exists on the genetic characterization and function of these receptors in the ovary of the white yak. In the present study, the melatonin receptor MT1 was cloned by RT-PCR in the ovary of white yak; the MT1 cDNA fragment obtained (843bp) comprised an open reading frame (827bp) encoding a protein containing 275 residues, characterized by seven transmembrane regions and an NRY motif, two distinct amino acid replacements were found. The white yak MT1 had a 83.9–98.6% protein sequence identity with that of nine other mammals. Using RT-PCR, the expression levels of MT1, MT2, and LHR in the ovary of pregnant and non-pregnant white yaks were compared, revealing higher levels of all genes in pregnant yaks: 3.83-fold increase for MT1 (P<0.05), 1.39-fold increase for MT2, and 15.32-fold increase for LHR (P<0.05). The distribution of MT1 in yak ovaries was observed using immunohistochemistry on paraffin embedded ovarian sections: MT1 was mainly present on primordial follicles (PF), granulosa cells (GCs), oocytes, and corpus luteum (CL) cells; MT1 expression showed an increasing tendency from PF to GCs to oocytes and to large CL cells. It is suggested that melatonin and MT1 are associated with the corpus luteum function of pregnancy maintenance and follicular development during oocyte maturation in the white yak.

CHARACTERIZATION AND CELULAR PROLIFERATION OF OVARIAN STRUCTURES OF EWES BY THE TECHNIQUE OF AGNOR: OVARIAN MORFOMETRY AND CORRELATIONS

The AgNOR staining technique for nucleolar organizer regions (NORs) is characterized by marking proteins related to ribossomal ribonucleic acid evaluating celular proliferation. The objective was to study the ovarian morphometry and the value of AgNOR thecnique application to evaluate the proliferation of healthy cels from ovarian structures in crossbred ewe. 102 pairs of ovaries were collected and from the largest follicle of each ovary was measured its diameter. From the largest corpus luteum (CL) was measured the diameter and width, classifying the CL in: included, protruberant, massive and cavity, followed by AgNOR staining. The significance level was 5%. It had difference (p&lt;0.05) for the diameter of the largest follicle between right (RO): 4.2±2,3mm and left (LO): 3.6±2,2mm sides. There was a correlation (p&lt;0.01) between the NORs of internal theca (IT) and external theca (ET): RO (0.46) and LO (0.61); NORs of granulosa and IT: RO (0.54) and LO (0.59); and NORs of granulosa and ET: RO (0.34) and LO (0.38). Was conclude that the NORs quantification was appropriate to study the determination of the potential for ovarian structures proliferation, which was similar for right and left sides.

The periovulatory endocrine milieu affects the uterine redox environment in beef cows.

BackgroundIn cattle, recent studies have shown positive associations between pre-ovulatory concentrations of estradiol (E2), progesterone (P4) at early diestrus and fertility. However, information on cellular and molecular mechanisms through which sex steroids regulate uterine function to support early pregnancy is lacking. Based on endometrial transcriptome data, objective was to compare function of the redox system in the bovine uterus in response to different periovulatory endocrine milieus.MethodsWe employed an animal model to control growth of the pre-ovulatory follicle and subsequent corpus luteum (CL). The large follicle-large CL group (LF-LCL, N = 42) presented greater levels of E2 on the day of GnRH treatment (D0; 2.94 vs. 1.27 pg/mL; P = 0.0007) and P4 at slaughter on D7 (3.71 vs. 2.62 ng/mL, P = 0.01), compared with the small follicle-small CL group (SF-SCL, N = 41). Endometrium and uterine washings (N = 9, per group) were collected for analyses of variables associated with the uterine redox system.ResultsThe SF-SCL group had lower endometrial catalase (0.5 vs. 0.79 U/mg protein, P < 0.001) and glutathione peroxidase (GPx; 2.0 vs. 2.43 nmol β-nicotinamide adenine dinucleotide phosphate reduced/min/mg protein, P = 0.04) activity, as well as higher lipid peroxidation (28.5 vs. 17.43 nmol malondialdehyde/mg of protein, P < 0.001) and superoxide dismutase (SOD) activity (44.77 vs. 37.76 U; P = 0.04). There were no differences in the endometrial reactive species (RS) or glutathione (GSH) concentrations between the groups. The uterine washing samples showed no differences in the concentrations of RS or GSH or in total SOD activity (P > 0.1). Additionally, catalase, GPx4, SOD1 and SOD2 gene expression was lower in the SF-SCL group than in the LF-LCL group.ConclusionsWe concluded that the intrauterine environment of cows from the LF-LCL group exhibited higher antioxidant activity than that of the cows from the SF-SCL group. We speculate that uterine receptivity and fertility are associated with an optimal redox environment, such as that present in the animals in the LF-LCL group.

Spatio-specific regulation of endocrine-responsive gene transcription by periovulatory endocrine profiles in the bovine reproductive tract.

In cattle, pro-oestrous oestradiol and dioestrous progesterone concentrations modulate endometrial gene expression and fertility. The aim was to compare the effects of different periovulatory endocrine profiles on the expression of progesterone receptor (PGR), oestrogen receptor 2 (ESR2), oxytocin receptor (OXTR), member C4 of aldo-keto reductase family 1 (AKR1C4), lipoprotein lipase (LPL), solute carrier family 2, member 1 (SLC2A1) and serpin peptidase inhibitor, clade A member 14 (SERPINA14): (1) between uterine horns ipsi- and contralateral to the corpus luteum (CL), (2) between regions of the ipsilateral horn and (3) in the vagina. Endometrium and vagina tissue samples were collected from cows that ovulated a larger (large follicle-large CL, LF-LCL; n=6) or smaller follicle (small follicle-small CL, SF-SCL; n=6) 7 days after oestrus. Cows in the LF-LCL group had a greater abundance of transcripts encoding ESR2, AKR1C4, LPL, SLC2A1 and SERPINA14, but a reduced expression of PGR and OXTR in the endometrium versus the SF-SCL group (PPGR and OXTR was greater in the contralateral compared with the ipsilateral horn (PPGR, ESR2, LPL, SLC2A1 and SERPINA14 (P<0.05). Different periovulatory endocrine profiles, i.e. LF-LCL or SF-SCL, did not influence gene expression in the vagina and had no interaction with inter- or intra-uterine horn gene expression. In conclusion, inter- and intra-uterine horn variations in gene expression indicate that the expression of specific genes in the bovine reproductive tract is location dependent. However, spatial distribution of transcripts was not influenced by distinct periovulatory sex-steroid environments.

Molecular characterization and hormonal regulation of tissue inhibitor of metalloproteinase 1 in goat ovarian granulosa cells

Tissue inhibitor of metalloproteinase 1 (TIMP1) belongs to a group of endogenous inhibitors that control the activity of matrix metalloproteinases and other metalloproteinases. TIMP1 is ubiquitously expressed and implicated in many physiological and pathologic processes. In this study, the full-length complementary DNA of goat (Capra hircus) Timp1 was cloned from adult goat ovary for the first time to better understand the regulatory role of TIMP1. The putative TIMP1 protein shared a high amino acid sequence identity with other species. Real-time polymerase chain reaction results showed that Timp1 was widely expressed in adult goat tissues, and messenger RNA expression was higher in the ovary than in other tissues; meanwhile, increasing expression of Timp1 was also discovered during the process of follicle growth and corpus luteum. We then investigated Timp1 expression patterns in different types of ovarian follicular cells from goats. In small or large antral follicles, Timp1 expression was higher (P < 0.05) in theca cells than in granulosa cells, cumulus cells, and oocytes. Increasing expression of Timp1 in theca and granulosa cells was observed as the variation of the follicle size. Immunohistochemical analyses further revealed the presence of the TIMP1 proteins in follicles at all antral stages of development. The most intense staining for TIMP1 was observed in the theca cells and granulosa cells of large antral follicles and corpus luteum. Timp1 was highly (P < 0.05) induced in granulosa cells in vitro after treatment with the luteinizing hormone agonist, human chorionic gonadotropin. Treatments with forskolin, phorbol 12-myristate 13-acetate, or phorbol 12-myristate 13-acetate + forskolin could also stimulate Timp1 messenger RNA expression. The effects of human chorionic gonadotropin were reduced (P < 0.05) by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase, indicating that Timp1 expression could be adjusted by luteinizing hormone–initiated activation of these signaling mediators. Our results suggested that TIMP1 may be involved in regulating ovarian follicle development and ovulation.

144 PREGNANCY RATES FOLLOWING THE TRANSFER OF IN VITRO FERTILIZED EMBRYOS TO RECIPIENTS ON DAY 6, 7, OR 8 AFTER OESTRUS

The aim of this experiment was to investigate the effect of day of synchrony on the pregnancy rate of recipients following the transfer of Day 7 IVF embryos. In addition, the effect of IVF embryo grade and corpus luteum (CL) grade of recipients was determined. A total of 317 cumulus-oocyte complexes collected from 24 dry Brahman cows by TVR were matured, fertilized, and cultured under standard in vitro production procedures (Fry et al. 2003 Theriogenology 59, 446). A total of 89 (44 Grade 1, 43 Grade 2, and 2 Grade 3, IETS classification) in vitro-produced embryos were transferred to parous 4- to 9-year-old dry Brahman cross recipient cattle 7 days after IVF. Two groups of recipient cows were synchronised one day apart with an 8-day CIDR/pg protocol so that oestrous would be concentrated over 3 days with the middle day aligning with the day of IVF (Day 0). Donors that produced a large number of IVF embryos had these divided and transferred into recipients either on Day –1 or Day +1 of synchrony, and those producing less than 4 IVF embryos were transferred into recipients on Day 0. At embryo transfer the ovaries of the recipient were palpated and then scanned by rectal ultrasound and the grade of CL noted (Grade 1 = large distinct CL by palpation, Grade 2 = small distinct CL by palpation, Grade 3 = CL not distinguishable by palpation). Pregnancy was diagnosed by ultrasound scanning on Day 92. Although recipient numbers were low, differences in pregnancy rate between groups were analysed by Chi-squared. Data from the 2 Grade 3 embryos transferred were not included in the analysis (0/2 pregnant). Similar (P &gt; 0.05) pregnancy rates were found when Day 7 IVF embryos were transferred to either Day 6 (17/32 = 53%), Day 7 (9/24 = 38%), or Day 8 (14/31 = 45%) recipients. Furthermore, neither the grade of the embryo (Grade 1: 20/44 = 45%, Grade 2: 20/43 = 47%) nor the grade of recipient CL (Grade 1: 17/45 = 38%, Grade 2: 17/29 = 59%, Grade 3: 6/13 = 46%) effected pregnancy rate (P &gt; 0.05). This experiment demonstrates the flexibility of the IVF embryo to achieve an acceptable pregnancy rate over a range of recipient stages thereby allowing a high usage rate of good-quality recipients in an IVF embryo transfer program.

Modulation of periovulatory endocrine profiles in beef cows: consequences for endometrial glucose transporters and uterine fluid glucose levels

In beef cattle, proestrus estradiol and subsequent progesterone (P4) concentrations can regulate the endometrial characteristics and thereby determine maternal receptivity toward the embryo. However, the underlying mechanisms linking periovulatory endocrine profiles to receptivity, which is crucial to obtain pregnancy, need to be elucidated. We hypothesized that the size of the preovulatory follicle (POF) and subsequent circulating P4 concentrations, during early diestrus, modulate endometrial levels of glucose transporter transcripts and proteins, and subsequently affect the luminal glucose availability in the uterus. Therefore, follicle growth of Nelore cows was manipulated, and cows were assigned to 2 experimental groups: (1) large follicle and large corpus luteum (LF-LCL) group with a large POF and corpus luteum (CL); and (2) small follicle and small corpus luteum (SF-SCL) group with a small POF and CL. At day 7 post gonadotropin-releasing hormone induced ovulation (gonadotropin-releasing hormone treatment = day 0), animals were slaughtered (n = 18 per group), and uterine tissues and washings were collected for characterization of glucose transporters and glucose levels, respectively. The diameter of POF was larger (P < 0.05) in the LF-LCL cows compared with their SF-SCL counterparts (12.8 ± 0.4 vs 11.1 ± 0.4 mm). Furthermore, CL size (17.49 ± 0.88 vs 14.48 ± 0.52 mm) and circulating P4 concentrations at day 7 (4.5 ± 1.0 vs 3.3 ± 1.1 ng/mL, P < 0.05) were significantly higher in the LF-LCL cows compared with the SF-SCL cows. No differences (P > 0.05) were detected in gene expression patterns of SLC2A1, SLC2A3, SLC2A4, SLC2A5, SLC5A1, ATP1A2, ATP1B2, and SLC37A4. However, the protein abundance of endometrial SLC2A1was increased in the LF-LCL group compared with the SF-SCL group (P < 0.05). SLC2A1 and SLC2A4 protein products were mainly identified at the endometrial luminal and glandular epithelium membranes as well as in the endometrial stroma. Glucose concentrations in uterine washings were similar between groups. In conclusion, we provided information on the potential link between endocrine profiles and glucose transport pathways in the bovine endometrium. More specifically, our data reveal that the size of the POF, and subsequent P4 concentrations, do not functionally affect the main endometrial glucose transporter pathways or uterine fluid glucose concentrations during diestrus.

A field study to unravel factors that are significantly associated with the secretory activity of the corpus luteum during the first three postpartum cycles in high yielding dairy cows, based on the amount of steroidogenic and endothelial cells present in the luteal tissue.

Fourteen multi- and eight primiparous high-yielding dairy cows were followed from the first till the fourth ovulation postpartum. Cows were randomly divided into two groups and supplemented with soybean (group I; n = 11) or rapeseed meal (group II; n = 11). Both groups were subjected to a biopsy sampling of the corpus luteum (CL) at cycle day 9. The luteal capillary network (visualized by Bandeiraea simplicifolia) was denser in cycles 2 and 3 (p = 0.0005). The same was seen for the surface occupied by steroidogenic cells (visualized by 3β-hydroxysteroiddehydrogenase) (p = 0.0001). The peripheral blood progesterone concentration showed an increasing trend with increasing cycle number and was higher in primiparous cows (p = 0.013), which had also larger glands on cycle day 9. The area occupied by endothelial cells was positively correlated with the area occupied by steroidogenic cells (r = 0.59; p < 0.0001). Both the areas occupied by endothelial and by steroidogenic cells were negatively correlated with the blood concentration of nonesterified fatty acids (NEFAs) (respectively, r = -0.377; p = 0.004 and r = -0.355; p = 0.007). We can conclude that primiparous cows generally have higher peripheral progesterone levels during the first three cycles after calving which is associated with a larger CL. In comparison with those of the first post-partum cycle, corpora lutea of cycles 2 and 3 have a denser capillary network and a larger area of steroidogenic cells, while these are only associated with a trend of higher peripheral progesterone concentrations.