First Day Of Estrus Research Articles

ODP428 The Effects of Asprosin Pubertal Maturation in Female Rats

Abstract Adipokines play an important role in sexual maturation and reproduction in a variety of ways. The central Asprosin administration of asprosin, a new adipokine secreted by white adipose tissue, has been found to have the potential to stimulatory effects influence on the hypothalamus-pituitary-gonadal axis (HPG)pubertal maturation in male rats. However, there is no study on the effects of asprosin on the HPG axis. Interestingly, peri-ovarian adipose tissues, which surround the ovaries and perform both autocrine and paracrine functions, suggest that asprosin may have a role in ovarian function. Asprosin has been also shown to cross the blood-brain barrier. However, the effects of asprosin, a novel adipokine, are not fully defined on the hypothalamus-pituitary-gonadal axis at the peripheral levels. The aim of the research study aims is to see how asprosin affects pubertal maturation in female rats when it is given parentally. For the experimental studies, totally a total of 24 female Sprague-Dawley rats, 21 days old and 35 ± 2 g weight, were used and randomly divided into two groups as control and asprosin groups (n=12). Asprosin (500 ng/kg) was given intraperitoneally daily in the animals in the asprosin group from postnatal day 21 for about 8 weeks. Similarly, the control group received only saline. For determining the puberty onset, vaginal opening (complete canalization of the vagina, an external index of puberty onset) was monitored daily in rats. Vaginal smears were performed daily from the date of puberty onset to determine the day of the first estrus. The ELISA method is used to analyze hormones in blood samples. Asprosin administration did not affect the onset of puberty or pubertal weight compared to the control group (p>0. 05). The first day of estrus in female rats administered with asprosin was shown to be significantly earlier than control rats (p<0. 05). Asprosin administration significantly increased the levels of FSH, oxytocin, and estradiol in the blood(p<0. 05). It significantly decreased inhibin B level (p<0. 05). There was no significant difference in LH level (p>0. 05). In conclusion, our findings suggest that asprosin may have stimulatory effects on pubertal maturation of the reproductive system in female rats. However, we believe that more research is needed to determine the mechanism by which asprosin affects the pubertal maturation of the reproductive system. This study was supported by TUBITAK (Project# 220S744). Presentation: No date and time listed

86 Ultrasonographic evaluation of preovulatory follicle and endometrial echo texture for ovulation prediction in Marwari mares

The objective of the present study was to evaluate the application of ultrasonographic changes in the preovulatory follicle and uterus for ovulation prediction in Marwari mares. Mares (n=20) were teased daily by a teaser pony and, from the day of first detected oestrus, the ovaries and uterus were examined by transrectal ultrasonography using Exago ECM ultrasound equipped with linear rectal probe (10MHz) until ovulation. Recorded data were analysed by one-way ANOVA and correlation coefficients. The duration of the oestrus period ranged from 5 to 7 days in all mares, with an average of 6.35±0.13 days, consistent with rectal findings. The last day of oestrus was marked by disappearance of the preovulatory follicle and presence of a corpus haemorrhagicum. The mean diameter of the follicles increased significantly (P<0.01) from Day 1 to Day 7, and the diameter of follicles before ovulation was 44.88±0.46 mm; follicles changed from round to oval or irregular before ovulation. In 65% (13/20) of the mares, ovulation occurred on the left ovary. The daily mean growth rate of size of ovulating follicles from the first day of oestrus until ovulation was 3.51±0.42 mm/d. The mean average growth rate of follicles was significantly different (P ≤ 0.01) on different days of oestrus. The mean endometrial echo texture score was 1.30±0.10 on Day 1, 2.10±0.16 on Day 2, 3.20±0.15 on Day 3, and highest 3.85±0.08 on Day 4, declining thereafter to reach a score of 1.36±0.11 on Day 7 of the oestrous cycle. The mean slope values of pixel intensity for the granulosa and anechoic layers of the preovulatory follicles determined by Image-Pro software (Media Cybernetics) from Days −3 to −1 was 13.8±0.7 on Day −3, 15.7±0.9 on Day −2, and 20.1±1.3 on Day −1. None of the follicles ovulated before granulosa layer echogenicity reached a score of ≥2.5 and prominence of anechoic layer reached a score of ≥2. Significant positive correlations were found between the cross-sectional diameter of the uterine horn and estrual endometrial echotexture score (P<0.01; r=0.95), estrual endometrial echo texture score and preovulatory follicle diameter (P<0.01; r=0.25), and preovulatory follicle diameter and cross-sectional diameter of uterine horn (P<0.01; r=0.36) during the oestrous cycle. We conclude that various ultrasonographic characteristics of preovulatory follicle and uterus can be used to predict ovulation in Marwari mares.

Comparative analysis of the ovarian transcriptome reveals novel insights into fertility differences in Large White sows.

Fertility is the most important economic trait in sows, as it is critical for profitability. Considerable phenotypic variation in litter size exists in Large White sows. However, relatively little is known about the underlying molecular and genetic bases. An experiment was conducted to screen key genes that affect the fecundity of pigs during the luteal (L) and follicular phases (F) of the estrous cycle. Eight sows (n = 4 for high fertility sows and n = 4 for low fertility sows) were sacrificed on day 14 (day 1 = first day of estrus) after estrus in the L phase. Another eight sows were slaughtered on day 20 of the estrous cycle in the F phase. Sixteen ovarian tissue samples were collected at the different sacrifice time points. Total RNA extracted was used to construct the library and then sequence on an Illumina HiSeq X10 system. Differentially expressed genes (DEGs) between high and low fertility in Large White sows were identified, and their potential biological functions were analyzed using bioinformatics analysis. In total, 457 DEGs (161 up-regulated and 296 down-regulated genes) were detected in the ovarian tissues of the high and low fertility groups in the L phase of the estrous cycle. Furthermore, 475 DEGs (253 up-regulated and 222 down-regulated genes) were identified in the F phase. Twenty-nine DEGs were common to both comparisons. Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the DEGs were mainly associated with steroid biosynthesis, the Hippo signaling pathway, and lysosomes. Others, such as MSMO1, CYP27B1, and CTSB, were related to reproduction. These results will contribute to a better understanding of the individual differences in fertility at the transcriptome level, which may provide useful information to explore new ways to improve fertility in pigs.

In Utero and Lactational Exposure to Flame Retardants Disrupts Rat Ovarian Follicular Development and Advances Puberty.

Brominated flame retardants (BFRs), including polybrominated diphenyl ethers and hexabromocyclododecane, leach out from consumer products into the environment. Exposure to BFRs has been associated with effects on endocrine homeostasis. To test the hypothesis that in utero and lactational exposure to BFRs may affect the reproductive system of female offspring, adult female Sprague Dawley rats were fed diets formulated to deliver nominal doses (0, 0.06, 20, or 60 mg/kg/day) of a BFR dietary mixture mimicking the relative congener levels in house dust from prior to mating until weaning. Vaginal opening and the day of first estrus occurred at a significantly earlier age among offspring from the 20 mg/kg/day BFR group, indicating that the onset of puberty was advanced. Histological analysis of ovaries from postnatal day 46 offspring revealed an increase in the incidence of abnormal follicles. A toxicogenomic analysis of ovarian gene expression identified upstream regulators, including HIF1A, CREB1, EGF, the β-estradiol, and PPARA pathways, predicted to be downregulated in the 20 or 60 mg/kg/day group and to contribute to the gene expression patterns observed. Thus, perinatal exposure to BFRs dysregulated ovarian folliculogenesis and signaling pathways that are fundamental for ovarian function in the adult.

Dietary supplementation of n-3 polyunsaturated fatty acid alters endometrial expression of genes involved in prostaglandin biosynthetic pathway in breeding sows (Sus scrofa)

The present investigation was designed to study the effect of dietary supplementation of omega-3 (n-3) PUFA on endometrial expression of fertility-related genes in breeding sows. Sixteen crossbred sows were randomized to receive diets containing 4% (wt/wt) flaxseed oil as n-3 PUFA source (TRT group) or iso-nitrogenous, iso-caloric standard control diet (CON group), starting from the first day of estrus up to 40 days and were artificially bred on the second estrus. Endometrial samples were collected during days 10–11 and 15–16 post-mating for studying relative expression profile of candidate genes viz. Prostaglandin F Synthase (PGFS), microsomal Prostaglandin E Synthase-1 (mPGES-1) and Carbonyl Reductase-1 (CBR-1) using quantitative Real-Time PCR. Expression level of mPGES-1 gene transcript was 2.1-fold higher (P < 0.05) during 10–11 days of pregnancy and 1.4-fold higher (P > 0.05) during 15–16 days of pregnancy in TRT group as compared to CON group. Relative expression of PGFS gene transcript was significantly lower (P < 0.05) during 10–11 days of pregnancy in TRT group while there was no significant effect (P > 0.05) of dietary supplementation during 15–16 days of pregnancy. Endometrial mRNA level of CBR1 was significantly lower (P < 0.05) with 3.93-fold decrease in TRT group during 10–11 days of pregnancy whereas 2.82-fold reduction in expression (P > 0.05) was observed subsequently during 15–16 days of pregnancy as compared to CON group. Collectively, these results indicate that dietary n-3 PUFA supplementation can modulate gene expression of key enzymes in prostaglandin biosynthetic pathway during early gestation, which in turn might have beneficial impact on overall reproductive response in breeding sows. These findings partly support strategic dietary supplementation of plant-based source of n-3 PUFA with an aim to improve overall reproductive performance in sows.

Endometrial blood perfusion as assessed using a novel laser Doppler technique in Angus cows

Previous studies have characterized ovarian steroid synthesis which directly affects uterine environment and blood flow. Clearance of steroids occurs primarily in hepatic tissues, however, it was discovered that there is an abundant activity of the phase II steroid metabolizing enzyme UDP-glucuronosyltransferase (UGT) in uterine biopsies. No minimally invasive techniques for collecting endometrial perfusion, which is affected by steroids and indicative of reproductive health, have been developed for livestock. The objective of the present study was to characterize UGT activity and endometrial blood perfusion during a normal estrous cycle of cattle. It was hypothesized that there would be increased steroid metabolism during the luteal phase of the estrous cycle and in the uterine horn ipsilateral to the corpus luteum (CL). During the first synchronized estrous cycle, progesterone and UGT activity increased on Day 6 compared with 0 and 3, with the first day of estrus being considered Day 0 of the study. Endometrial perfusion was greater ipsilateral to the CL compared with contralateral on Day 12, and was less ipsilateral to the CL compared with contralateral on Day 18. Similar to perfusion results, nitric oxide metabolites (nitrites) were greatest in the endometrium ipsilateral as compared with contralateral to the CL. Moreover, there was a positive correlation (r = 0.28; P = .04) between endometrial perfusion and nitrite concentration. It is concluded that activity of UGT within the endometrium is affected by the contralateral or ipsilateral location of the CL, and collection of endometrial perfusion data using a laser Doppler probe could be a viable measurement technique as indicated by associated nitrite concentrations in the present study.

14 ENHANCEMENT OF THE EARLY RECOVERY OF OVARY AND UTERUS FUNCTIONS BY TREATMENT OF eCG AND POVIDONE-IODINE IN POSTPARTUM JAPANESE BLACK CATTLE

The aim of this study was to evaluate the effect of administration of eCG and intrauterine infusion of povidone-iodine to promote the early recovery of ovary and uterus functions in early stage after delivery on postpartum beef cows. Thirty-three postpartum Japanese Black cows without retention of placenta were used for this investigation. After delivery, cows nursed colostrums to calves for several hours in a pen, and then were separated from calves and returned into a barn managed in a group. In experimental group (n = 14), on Day 10 (Day 0 = day of parturition), an intramuscular administration of 500 IU of eCG and infusion of 30 mL of 2% povidone-iodine into the uterus body were conducted. The untreated cows delivered in the same period were compared as a control group (n = 19). Cows were inseminated artificially using commercial Japanese Black frozen–thawed semen when standing oestrus was detected until Day 90. Pregnancy diagnosis was performed 30 days after insemination by ultrasonography. For all of the experimental group and 8 cows in the control group, the diameter of follicles &gt;8 mm and the diameter of cross-section between endometrium in uterus of the implanted side at the point of ~2 cm from bifurcation of the uterus were measured using an ultrasound scanning machine connected to a 7.5-MHz convex probe. This monitoring was carried out every 10 days from Day 10 until the day of first oestrus (= insemination) or until Day 80 for non-returned oestrus cows. Data were analysed using Fisher’s exact test and ANOVA. The ratio of returned oestrus by Day 90 was 100% (14/14) in experimental group and 89.5% (17/19) in control group. The first oestrus day postpartum in the experimental group and the control group was the almost same: 52.9 ± 12.1 (24–88) days and 57.2 ± 19.9 (29–78) days, respectively. The conception rate until Day 90 tended to be higher (P = 0.07) in the experimental group (78.6%, 11/14) than in the control group (47.4%, 9/19). The days inseminated to cows that were conceived, was almost same between the experimental group (57.5 ± 18.6, 24–87 d) and the control group (55.4 ± 12.9, 36–78 d). The ratio of cows observed follicles more than 8 mm, was higher in the experimental group than control group on Day 50 (90.9% v. 50.0%; P = 0.09) and Day 60 (66.7% v. 0%; P &lt; 0.05), respectively. The diameter of uterus tended to be lesser in the experimental group than in the control group on Day 30 (16.9 ± 2.8 mm v. 19.2 ± 2.8 mm; P = 0.09), and that regressed linearly to Day 30 in the experimental group however that was prolonged until Day 40 (16.4 ± 1.7 mm) in the control group. These results indicate that administration of eCG and uterus infusion of povidone-iodine at an early stage postpartum may promote the early recovery of ovary and uterus functions in beef cows. This research was supported by a grant from the Research Program of MAFF, (REP1004) Development of Innovative Technology for Animal Reproduction.

Inhibition of serotonin reuptake in the prepubertal rat ovary by fluoxetine and effects on ovarian functions.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor is an antidepressant in the treatment of mood disorders. Its impact on reproductive processes is incompletely known. The present study analyzed the reproductive effects of FLX in prepubertal female rats. Two experiments were conducted. First (acute administration), 30-day-old female rats were injected intraperitoneally with 5mg/kg of fluoxetine-hydrochloride, and were terminated 24, 48 or 72h after the treatment. Second (subchronic administration), FLX was injected on days 30-33 of age, and the animals were terminated the day of first estrus. In acute treatment estradiol concentration increased to 72h. In subchronic treatment increased serotonin concentration in ovaries and decreased the number of ova shed. An increase in number of atretic follicles and oocyte fragmentation was observed in these animals. The results suggest that FLX acts on the ovary or hypothalamus-pituitary axis resulting in modifications of the follicular development and ovulation.

Fecal endocrine monitoring of reproduction in female snow leopards (Uncia uncia)

Although the snow leopard (Uncia uncia) is a common endangered felid species in zoos, little is known about the complex endocrine interactions controlling ovarian function and conception in this species. The goal of this work was to characterize ovarian activity throughout the estrous cycle, nonpregnant luteal phase (pseudopregnancy), and gestation in female snow leopards. This goal was accomplished using an enzyme immunoassay to measure fecal concentrations of estrogen metabolites (E) and progesterone metabolites (P). Fecal samples were collected from 12 female snow leopards (ages 18 months to 18 years) during one to three breeding seasons. In each breeding season, the majority of females (78%, 88%, and 100%, respectively) began to exhibit ovarian activity in December or January. The estrous cycle, defined by the first day of estrus (E ≥ 2 × basal concentration) to the first day of the subsequent estrus, was 12.7 ± 0.6 days (n = 145 cycles). Estrus lasted 4.3 ± 0.4 days with mean concentrations of fecal E during the follicular phase (1661 ± 139 ng/g feces) increasing 3.2-fold above basal concentrations (515 ± 32 ng/g feces). No spontaneous ovulations were observed in any of the cycling females. Nonpregnant luteal phases were observed in eight females that bred but did not become pregnant. The length of the nonpregnant luteal phase ranged from 11 to 72 days (45.7 ± 5.7 days; n = 10) with mean concentrations of fecal P during the luteal phase (12.46 ± 1.7 μg/g feces) increasing 6.2-fold above basal concentrations of P (2.01 ± 0.2 μg/g feces). Three of the females in the study became pregnant and gave birth after a gestation of 93 (n = 2) and 95 (n = 1) days. Fecal P concentrations during pregnancy increased to 11.64 ± 1.3 μg/g feces, or 5.8-fold above basal concentrations. The results of this study provide a comprehensive characterization of reproductive endocrinology in snow leopards, and confirm that fecal hormone monitoring is an effective way to monitor female snow leopards throughout the breeding season.

Uncovering Novel Reproductive Defects in Neurokinin B Receptor Null Mice: Closing the Gap Between Mice and Men

Patients bearing mutations in TAC3 and TACR3 (which encode neurokinin B and its receptor, respectively) have sexual infantilism and infertility due to GnRH deficiency. In contrast, Tacr3(-/-) mice have previously been reported to be fertile. Because of this apparent phenotypic discordance between mice and men bearing disabling mutations in Tacr3/TACR3, Tacr3 null mice were phenotyped with close attention to pubertal development, estrous cyclicity, and fertility. Tacr3(-/-) mice demonstrated normal timing of preputial separation and day of first estrus, markers of sexual maturation. However, at postnatal d 60, Tacr3(-/-) males had significantly smaller testes and lower FSH levels than their wild-type littermates. Tacr3(-/-) females had lower uterine weights and abnormal estrous cyclicity. Approximately half of Tacr3(-/-) females had no detectable corpora lutea on ovarian histology at postnatal d 60. Despite this apparent ovulatory defect, all Tacr3(-/-) females achieved fertility when mated. However, Tacr3(-/-) females were subfertile, having both reduced numbers of litters and pups per litter. The subfertility of these animals was not due to a primary ovarian defect, because they demonstrated a robust response to exogenous gonadotropins. Thus, although capable of fertility, Tacr3-deficient mice have central reproductive defects. The remarkable ability of acyclic female Tacr3 null mice to achieve fertility is reminiscent of the reversal of hypogonadotropic hypogonadism seen in a high proportion of human patients bearing mutations in TACR3. Tacr3 mice are a useful model to examine the mechanisms by which neurokinin B signaling modulates GnRH release.

Transcriptional Profiling by Deep Sequencing Identifies Differences in mRNA Transcript Abundance in In Vivo-Derived Versus In Vitro-Cultured Porcine Blastocyst Stage Embryos1

In vitro embryo culture systems promote development at rates lower than in vivo systems. The goal of this project was to discover transcripts that may be responsible for a decrease of embryo competency in blastocyst-stage embryos cultured in vitro. Gilts were artificially inseminated on the first day of estrus, and on Day 2, one oviduct and the tip of a uterine horn were flushed and the recovered embryos were cultured in porcine zygote medium 3 for 4 days. On Day 6, the gilts were euthanized and the contralateral horn was flushed to obtain in vivo derived embryos. Total RNA was extracted from three pools of 10 blastocysts from each treatment. First and second strand cDNA was synthesized and sequenced using Illumina sequencing. The reads generated were aligned to a custom-built database designed to represent the known porcine transcriptome. A total of 1170 database members were different between the two groups (P < 0.05), and 588 of those had at least a 2-fold difference. Eleven transcripts were subjected to real-time PCR that validated the sequencing. There was an overall decrease in inner cell mass (ICM) and trophectodermal (TE) cell numbers in embryos cultured in vitro; however, no difference in the ICM:TE ratio was found. Interestingly, the transcript SLC7A1 was higher in the in vitro cultured group. This difference disappeared after addition of arginine to the 4-day culture. Illumina sequencing and alignment to a custom transcriptome identified a large number of genes that yield clues on ways to manipulate the culture media to mimic the in vivo environment.

The effects of simazine, a chlorotriazine herbicide, on pubertal development in the female Wistar rat

Chlorotriazine herbicides, such as atrazine and its metabolites, have been shown to target the neuroendocrine regulation of male and female reproductive development. However, no studies have evaluated the effects of the chlorotriazine simazine on pubertal development in the female rat. Here we report the effects of a 21- and 41-day exposure to simazine on pubertal development and estrous cyclicity in the female rat using the U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program, Pubertal Development and Thyroid Function in Intact/Juvenile Peripubertal Female Rats (Tier 1) protocol. In the first study, Wistar rats were exposed orally to 0, 12.5, 25, 50, or 100 mg/kg of simazine from postnatal day 22 to 42. In the second study, rats were exposed from PND 22 until the first day of estrus after PND 62 to 0, 12.5, 25, 50, 100 or 200 mg/kg of simazine. In the 21-day exposure, vaginal opening (VO) was delayed, the number of normal cycles was significantly decreased, and the day of first estrus was delayed compared to controls. In the 41-day exposure, VO and the day of first estrus was delayed, but the number of normal estrous cycles was not different than controls. In addition, both studies showed a significant decrease in serum prolactin (PRL) following simazine exposure. This data clearly demonstrates that simazine delays the onset of puberty in the female rat and decreases serum PRL similar to other chlorotriazines. The extended dosing period after VO provides a sufficient time period to monitor the effects of a toxicant on estrous cyclicity, an important measure for reproductive competence.

Differential Gene Expression in Oviducts Collected During Proestrus from Ewes Fasted During the Luteal Phase of the Estrous Cycle.

Short-term fasting during the luteal phase of the estrous cycle perturbs circulating concentrations of progesterone and estradiol, causing a delay in the onset of the surge release of LH. Although ovulation rate is not affected by fasting in this model, number of lambs born is decreased. Fasting may affect fertility by decreasing ova quality and/or altering the oviduct/uterine environment. The objective of this study was to elucidate differences in gene expression in the oviduct near the time of expected ovulation. Estrous cycles were synchronized with PGF2α. Control ewes were given ad libitum access to good-quality grass hay throughout the experiment. Ewes in the fasted group were withheld from feed on d 7 through d 11 of the estrous cycle (d 0 = first day of estrus). On d 12 all ewes were treated with 10 mg PGF2α, and fasted ewes were returned to ad libitum feed. Oviduct tissue was collected from fasted and fed ewes during the expected peri-ovulatory period (72 hr following PGF2α and realimentation). At 72 hr, ewes were euthanized and mid sections of oviduct were dissected and snap-frozen for RNA analysis. Purified RNA was screened using the Affymetrix bovine gene chip containing ~24,000 genes at the Montana State University Functional Genomics facility. Although the RNA was ovine in origin, there is at least an 80% presence/identity when screening bovine chips with ovine RNA. Genes differentially expressed on the microarray chip were analyzed using DAVID (NIAID, NIH) for functional annotation and identification of expression patterns, and genes of interest were confirmed using semi-quantitative real time RT-PCR. Differential gene expression was observed in oviducts of the fasted ewes compared to control ewes. Affected intracellular processes include ADP ribosylation, transcription, transport, phosphorylation, ubiquitinylation, apoptosis, proteasomal degradation, GTP binding, signal transduction, carbohydrate transport and metabolism, regulation of transcription, and cell adhesion. Some genes found to be up-regulated in the oviducts of fasted ewes by microarray analysis include ARF-1, ARF-3, RhoGDP inhibitor, ataxin, aquaporin, ubiquitin, proteasomal subunit 8, HSD17B1, and a wide array of zinc finger proteins (P< 0.05). Genes down regulated by microarray analysis include, but are not limited to, TNFα, MX1, MDA5, and IRF 7 (P< 0.05). The mRNA for ARF-3 was one of several transcripts found to be up regulated by real time PCR (P< 0.01) while oxytocin receptor was found to be down- regulated (P< 0.05). These results imply a role for nutrition in molecular mechanisms regulating the environment of the oocyte at the site of fertilization which may impact overall fertility. Future studies will involve similar genetic analyses of the endometrium as well as the follicle and the study of genes/gene products which may alter the outcome of reproductive efficiency in farm animal species.

Estradiol interactions with dopamine antagonists in mares: Prolactin secretion and reproductive traits

Two experiments studied the effects of pretreatment with estradiol benzoate before treatment with a dopamine antagonist on prolactin secretion and reproductive traits in mares during (1) the seasonal anovulatory period and (2) the normal breeding season. Experiment 1 was performed in winter with 17 mares selected for low follicular activity. Nine mares received estradiol benzoate injections every other day for a total of 10 injections; 8 mares received similar injections of vehicle. Ten days after onset of injections, all mares were placed on daily injections of sulpiride (250 mg) for 35 days or until ovulation. Plasma prolactin concentrations were higher ( P < .001) in mares receiving estradiol than in controls for all assessments from days 12 through 36. Plasma luteinizing hormone (LH) concentrations were also increased ( P < .05) by estradiol treatment from days 14 to 23. Mean day of first ovulation was 73.6 for control mares and 29.0 for estradiol-treated mares ( P = .016). Estradiol treatment greatly enhanced prolactin secretion in response to sulpiride and increased LH secretion in seasonally anovulatory mares, which together hastened the date of first ovulation by an average of 45 days. Experiment 2 was designed to assess the efficacy of a long-acting, single-injection microparticle preparation of another dopamine antagonist, domperidone, for increasing prolactin secretion in cyclic mares in the summer. The experimental design and procedures used in experiment 1 were repeated, except that a single 3-g domperidone-microparticle injection was administered on day 11 rather than 45 days of sulpiride injections. Day 0 was the first day of estrus for each mare. Prolactin concentrations were higher ( P < .05) in mares receiving estradiol than in control mares from days 12 through 25 and after a thyrotropin-releasing hormone injection on d 21. Estrous cycle traits (time to ovulation and time of luteal regression) were not affected ( P > .1) by treatment. Estradiol enhanced the prolactin response to a single injection of 3 g domperidone in cyclic mares in the summer in a manner similar to the estradiol enhancement of prolactin secretion in response to daily sulpiride injections in anovulatory mares in winter. Thus, the single injection of domperidone could possibly replace the daily sulpiride injections used in experiment 1 to induce ovulation in seasonally anovulatory mares; this needs to be tested in future experiments.

Mouse Major Urinary Proteins Trigger Ovulation via the Vomeronasal Organ

The major urinary proteins are a species-specific complex of proteins excreted by male mice that influence the reproductive behavior and the neuroendocrine condition of female mice through the olfactory system. The aim of this work is to determine their influence on ovulation. The major urinary proteins isolated from the urine of adult male mice were voided of bound odorants, dissolved at a physiological concentration in urine of prepubertal mice, and put on the nostril of reproductively cycling female mice housed in groups, the first day of estrus at 1100. The eggs shed in the oviducts were counted under dissection the morning of the second day of estrus. The results showed that 1) a single stimulus of the major urinary proteins increased ovulation nearly as much as the whole urine of male mice, 2) the effect was not elicited by male rat urine which contains different proteins, 3) a peptide with four residues of the amino-terminal sequence of the major urinary proteins stimulated ovulation, and 4) mice that had been isolated or had the vomeronasal organ (VNO) removed did not respond to the major urinary proteins and had a high spontaneous ovulation. The results suggest that the major urinary proteins activate the neuroendocrine system through the VNO and trigger ovulation.

263 USE OF PORCINE PARTHENOTES AND GENE EXPRESSION PROFILING USING MICROARRAYS FOR IDENTIFICATION OF IMPRINTED GENES

Genomic imprinting arises from differential epigenetic markings including DNA methylation and histone modifications and results in one allele being expressed in a parent-of-origin specific manner. For further insight into the porcine epigenome, gene expression profiles of parthenogenetic (PRT; two maternally derived chromosome sets) and biparental embryos (BP; one maternal and one paternal set of chromosomes) were compared using microarrays. Comparison of the expression profiles of the two tissue types permits identification of both maternally and paternally imprinted genes and thus the degree of conservation of imprinted genes between swine and other mammalian species. Diploid porcine parthenogenetic fetuses were generated using follicular oocytes (BOMED, Madison, WI, USA). Oocytes with a visible polar body were activated using a single square pulse of direct current of 50 V/mm for 100 �s and diploidized by culture in 10 �g/mL cycloheximide for 6 h to limit extrusion of the second polar body. Following culture, BP embryos obtained by natural matings, and PRT embryos, were surgically transferred to oviducts on the first day of estrus. Fetuses recovered at 28-30 days of gestation were dissected to separate viscera including brain, liver, and placenta; the visceral tissues were then flash-frozen in liquid nitrogen. Porcine fibroblast tissue was obtained from the remaining carcass by mincing, trypsinization, and plating cells in �-MEM. Total RNA was extracted from frozen tissue or cell culture using RNA Aqueous kit (Ambion, Austin, TX, USA) according to the manufacturer's protocol. Gene expression differences between BP and PRT tissues were determined using the GeneChip� Porcine Genome Array (Affymetrix, Santa Clara, CA) containing 23 256 transcripts from Sus scrofa and representing 42 genes known to be imprinted in human and/or mice. Triplicate arrays were utilized for each tissue type, and for PRT versus BP combination. Significant differential gene expression was identified by a linear mixed model analysis using SAS 5.0 (SAS Institute, Cary, NC, USA). Storey's q-value method was used to correct for multiple testing at q d 0.05. The following genes were classified as imprinted on the basis of their expression profiles: In fibroblasts, ARHI, HTR2A, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, SGCE, SNRPN, and UBE3A; in liver, IGF2, PEG3, PLAGL1, PEG10, and SNRPN; in placenta, HTR2A, IGF2, MEST, NDN, NNAT, PEG3, PLAGL1, PEG10, and SNRPN; and in brain, none. Additionally, several genes not known to be imprinted in humans/mice were highly differentially expressed between the two tissue types. Overall, utilizing the PRT models and gene expression profiles, we have identified thirteen genes where imprinting is conserved between swine and humans/mice, and several candidate genes that represent potentially imprinted genes. Presently, our efforts are focused in the identification of single nucleotide polymorphisms (SNPs) to more carefully evaluate the behavior of these genes in normal and abnormal gestations and to test whether the candidate genes are indeed imprinted. This research was supported by USDA-CSREES grant 524383 to J. P. and B. F.

Effect of housing system and boar exposure on estrus expression in weaned sows.

Reproductive efficiency depends on detection of estrus, which may be influenced by housing and boar exposure. This experiment investigated the effects of housing system and boar contact on measures of estrus in weaned sows. Mixed-parity sows were randomly assigned to be weaned into gestation crates away from boars (AWC, n = 45), into pens away from boars (AWP, n = 42), or into pens adjacent to a mature boar (ADJ, n = 46). Estrus detection was initiated at approximately 0700 (0 h) and again at 0.25-, 0.5-, 1-, 2-, 4-, and 8-h intervals beginning on d 4 and continuing through d 7 following weaning. Estrus detection involved observation of the standing response after application of nose-to-nose boar exposure, backpressure, and side rubbing. For the AWC sows, a mature boar was moved to the front of the crates for a 10-min period and then removed. Sows housed in AWP were moved approximately 15 m to an empty pen adjacent to a mature boar for a 10-min period, and then returned to their pen. Sows housed ADJ were not moved and estrus detection was performed in their home pen for a 10-min period. The proportion of sows expressing estrus within 7 d from weaning was lowest for ADJ (80%, 37/46) compared with AWP (98%, 41/42) and AWC (96%, 43/45; P < 0.05). There was an effect of interval from weaning to estrus on the percentage of sows expressing estrus, but there was no interaction with treatment. Sows in AWC and AWP (4.7 d) had decreased (P = 0.01) intervals from weaning to estrus compared with ADJ (5.2 d). The duration of estrus was also shorter (P < 0.001) for ADJ (45 h) compared with AWC (58 h) or AWP (62 h). There was a treatment x interval x day of estrus effect for the percentage of sows expressing estrus. After detection of the first standing response on the first day of estrus, only 62 to 82% of sows were detected standing over the next 2 h for all treatments. However, at 4 to 8 h, this increased to 85 to 98% for the AWC and AWP sows, but <73% of the ADJ sows were detected during this period. On the second day of estrus, estrus expression was not influenced by interval for the AWC and AWP sows and was between 90 to 100% during the 8-h period, whereas ADJ sow detection rates were between 68 to 88%. These data suggest that housing sows adjacent to boars negatively affects estrus expression and detection. In addition, refractory behavior occurs in approximately 30 to 40% of sows and is influenced by housing relative to the boar, day of estrus, and interval from last boar exposure.

Produção in vitro de embriões bovinos com soro de égua ou de vaca em estro com ou sem a adição de LH/FSH

Mil duzentos e setenta e um oócitos foram divididos em 4 tratamentos com a finalidade de se avaliar a influência da adição de LH e FSH na produção in vitro de embriões bovinos com soro de vaca em estro (SVE) ou soro de éguas obtido no 1º dia do estro (SE). Independente do tratamento os oócitos foram maturados com TCM199 + 5,95mg/ml de Hepes, 0,025mg/ml de piruvato de sódio e 2,2mg/ml de bicarbonato de sódio, sendo adicionado 10% de soro de égua (ES), 10% de SVE (VS), 10% de soro de égua + 0,5mg/ml de hormônio luteinizante bovino (LHb) + 0,01UI/ml de hormônio folículo estimulante recombinante humano-rFSHh (EH) e 10% SVE + LHb + rFSHh (VH). Os oócitos assim tratados, foram maturados em estufa com 5% de CO2 a 39ºC sob umidade saturada por 22-24h. Depois, foram fecundados em TALP-FERT por 18-20h e cultivados por 8 dias em meio SOF + 5% de SE (ES e EH) ou SVE (VS e VH). As taxas de clivagem de 72% obtidas no grupo VH (229/316) e 61% no grupo VS (193/315) foram significativamente menores (p&lt;0,05) que as dos grupos ES (80% - 254/317) e EH (80% - 257/323). A produção de embriões (blastocistos iniciais, blastocistos, blastocistos expandidos e eclodidos) no D7 após a inseminação para os grupos ES (32%), EH (28%) e VH (27%), foi significativamente superior aos 20% obtidos no VS (p&lt;0,05). No D9, verificou-se uma diferença significativa (p&lt;0,05) entre os grupos ES (31%) e EH (29%) quando comparados com o grupo VS (22%), mas não houve diferença quando comparamos com o VH (24%). A taxa de eclosão em D9 foi de 11% (ES), 11% (EH), 10% (VS) e 5% (VH). Não houve diferença entre as médias do número de células dos embriões (Blastocistos expandidos e eclodidos) obtidos no D9. Conclui-se, com estes resultados que, para a obtenção de taxas regulares de blastocistos, não seja necessária a adição de hormônios quando se utilize o soro de égua, e que os hormônios devam ser adicionados quando se utilize soro de vaca.

Mating System in Mouse Lemurs: Theories and Facts, Using Analysis of Paternity

Among nocturnal Malagasy prosimians, the grey mouse lemur (Microcebus murinus) is considered a solitary species which has a promiscuous mating system. Indirect indicators, such as the lack of sexual dimorphism, the overlapping of male and female home ranges with each other, the synchronism of seasonal oestrus and the high relative testes size of males, support the presence of sperm competition. In captivity, an intense sexual precopulatory competition develops among males, leading to the emergence of a dominant male who fathers the majority of the litters. Although multiple mating did occur, the dominant male achieved the majority of the matings on the first day of oestrus. A ‘mate-guarding’ behaviour, exhibited by the dominant male, was observed in 11 groups out of 15, on only the first day of the vaginal opening and was significantly more often directed towards younger females. Females also played an important role in sexual competition among males, since their presence enhances the aggressive interactions between males. Difference in aggressive behaviours of females, in response to male sexual solicitations, suggests female mate choice. Compared with data from wild animals, it may be hypothesised that alternative mating strategies can be used by male grey mouse lemurs to enhance their reproductive success, depending on the availability and distribution of receptive females.

Androgen receptor and follicle-stimulating hormone receptor in the pig ovary during the follicular phase of the estrous cycle.

Follicle-stimulating hormone (FSH) is an important regulator of follicular development. Some effects of FSH on ovarian follicles might be enhanced by androgens. The main objectives of the present study were to examine expression of the androgen receptor (AR) and FSH receptor (FSHR) in late developing follicles in pigs. Ovaries were collected from gilts on days 13, 15, 17, and 19 of the estrous cycle (day 0 = first day of estrus, n = 4 gilts/day), a period coincident with the follicular phase. One ovary was processed for immunohistochemistry (IHC) of AR. Samples of surface wall from the largest follicles (4-5 per gilt) were dissected from the other ovary, pooled and processed for determination of AR and FSHR mRNAs using reverse transcription-polymerase chain reaction (RT-PCR). Intense AR immunostaining was present in nuclei of granulosa cells of preantral and antral follicles. AR immunoreactivity was also present in the nuclei of oocytes. Weak staining for AR was observed in cells of the theca interna, ovarian surface epithelium, and in most cells of the ovarian stroma. Relative amounts of immunoreactive AR in granulosa cells of late developing follicles, or small antral follicles (< 2 mm), did not differ between days 13, 15, 17, and 19. However, amounts of AR in granulosa cells of small antral follicles was greater (P < 0.05) than in the largest follicles present in the same ovary. The relative amounts of AR mRNA in tissue from the largest follicles on days 13, 15, 17, and 19 did not differ; however, amounts of FSHR mRNA in the same follicles were not different between days 13, 15, and 17, but decreased (P < 0.05) by day 19. Results indicate that during the follicular phase in gilts, the AR protein is mainly present in granulosa cells. Relative amounts of AR protein in granulosa cells and mRNA in walls of late developing follicles did not significantly change from day 13 to 19; however, amounts of FSHR mRNA decreased in preovulatory follicles by day 19 of the estrous cycle.