Consecutive Oestrous Cycles Research Articles

Mechanisms that clear mutations drive field cancerization in mammary tissue

Oncogenic mutations are abundant in the tissues of healthy individuals, but rarely form tumours1–3. Yet, the underlying protection mechanisms are largely unknown. To resolve these mechanisms in mouse mammary tissue, we use lineage tracing to map the fate of wild-type and Brca1−/−;Trp53−/− cells, and find that both follow a similar pattern of loss and spread within ducts. Clonal analysis reveals that ducts consist of small repetitive units of self-renewing cells that give rise to short-lived descendants. This offers a first layer of protection as any descendants, including oncogenic mutant cells, are constantly lost, thereby limiting the spread of mutations to a single stem cell-descendant unit. Local tissue remodelling during consecutive oestrous cycles leads to the cooperative and stochastic loss and replacement of self-renewing cells. This process provides a second layer of protection, leading to the elimination of most mutant clones while enabling the minority that by chance survive to expand beyond the stem cell-descendant unit. This leads to fields of mutant cells spanning large parts of the epithelial network, predisposing it for transformation. Eventually, clone expansion becomes restrained by the geometry of the ducts, providing a third layer of protection. Together, these mechanisms act to eliminate most cells that acquire somatic mutations at the expense of driving the accelerated expansion of a minority of cells, which can colonize large areas, leading to field cancerization.

Ovarian follicular dynamics, hormonal profiles and ovulation time in Mithun cows (Bos frontalis).

The study aimed to evaluate follicular dynamics and concentrations of estradiol-17β (E2), progesterone (P4), follicle stimulating hormone (FSH) and luteinizing hormone (LH) during the oestrous cycle and to determine ovulation time in Mithun cows. Ovaries of experimental cows (n=7) were examined daily by transrectal-ultrasonography for three consecutive oestrous cycles (n=21). The characteristics of follicular waves, dominant follicle, largest subordinate follicle and corpus luteum and ovulation time were evaluated. The plasma samples were analysed throughout the interovulatory interval to determine the differences in the hormonal profiles (E2, P4, FSH and LH) between different follicular wave cycles. Out of eighteen oestrous cycles analysed, three-wave follicular cycles were maximum (n=12: 66.66%) followed by two (n=4: 22.22%) and four waves (n=2: 11.11%). The two and three waves were statistically compared, and no significant (p > .05) differences were observed in day of wave emergence, number of follicles (≥3 mm) recruited, maximum diameter of the ovulatory dominant follicle, growth rates of ovulatory and anovulatory dominant follicles and maximum diameter of corpus luteum. The diameter of dominant follicles was significantly (p < .05) greater than subordinate follicles in both ovulatory and anovulatory waves. No significant differences were observed in peak concentrations of estradiol-17β and follicle stimulating hormone between ovulatory and anovulatory waves in all wave cycles. A preovulatory luteinizing hormone surge was observed a day before ovulation in all wave cycles. Progesterone concentrations were lower than 0.5ng/ml during oestrus and increased sharply to the maximum levels of ≥3.8ng/ml in all wave cycles. Ovulation time (mean ± SEM), irrespective of follicular waves was 10.5 ± 0.64 h after the end of oestrus. It was concluded that Mithun cows have a preponderance of three follicular waves with little difference between the two- and three-follicular waves and ovulation occurred 10.5h after the end of oestrus.

Ovarian follicular dynamics in Boran and Crossbred heifers in Ethiopia: Implications for assisted reproductive techniques

The study was conducted to characterize the follicular dynamics of purebred Boran (Bos indicus) and Boran * Holstein Friesian crossbred heifers during estrous cycles; for use in ovum pick up and in-vitro embryo production. Insight of reproductive physiology of cattle would help to understand and exploit the reproductive potential of elite animals for breed improvement. Follicular development, growth and atresia during estrous cycles were evaluated using a trans-rectal real-time B-mode ultrasound system for three consecutive estrus cycles. Luteal activity was evaluated by serum progesterone level. Follicular aspirations were done to investigate the potential of Boran cattle and their crosses for transvaginal oocyte production; using a vacuum pressure pump and Aloka SSD Prosound-2 ultrasound device. Boran heifers (n=15) manifested two (n = 6, 40%), three (n = 5, 33%), four (n= 3, 20%) and five (n=1, 6.7%) follicular waves. Crossbred heifers (n=14) showed one (n= 2, 14%), two (n= 6, 43%) and three (n= 6, 43%) follicular waves. Interovulatory interval was 21.1 ± 3.4 and 21.4 ± 2.7 days for Boran (n=45) and crossbred (n=42) heifers, respectively. The progesterone level in Boran and Crossbred heifers during diestrus was 9.5 ± 11.0 and 4.6 ± 8.8 ng/ml, respectively. The maximum diameter of the ovulatory follicle for crossbred heifers was higher (15.4 ± 1.6 mm) than the diameter of the Boran (14.0 ± 1.9 mm) heifers (p&lt;0.005). Differences (p&lt;0.005) were observed in the size of both right (26.6 ± 5.14 and 28.6 ± 5.1 mm) and left (21.7 ± 4.85 and 24.1 ± 5.07 mm) ovaries of Boran and Crossbred heifers, respectively. Difference (p&lt;0.005) was also observed in follicular count of the right ovaries of Boran (4.84 ± 1.96) and Crossbred (5.13 ± 2.05) heifers. Oocyte recovery rate in once weekly collection scheme for Boran and their crossbred heifers was (n=19, 42.08%) and (n=17, 42.55%), respectively. The recovery rate for twice weekly collection scheme was (n=24, 34.53%) and (n=23, 40.44%) for Boran and crossbred heifers, respectively. Follicular dynamics in Boran heifers is characterized by a higher incidence of cycles with two, three and four waves, associated with a low persistence of the dominant follicles; and smaller size of ovulatory follicles and less intense heat signs from their crossbred counterpart. Boran heifers proved to have potential for comparable number of follicular population and ease of aspiration procedures that can be tapped for advancedreproductive techniques.&#x0D; Keywords: Estrus cycle; follicular wave; ovarian follicle; ovum pickup; ultrasound.

Ovarian follicular dynamics and uterine changes during the ovulatory wave predicts imminent ovulation in Mares

Equine reproduction is unique by having long behavioral estrus and variations in ovarial follicular dynamics that made difficulty to standardize breeding time in mares. There are limited data on equine reproduction and breeding in Ethiopia. An experimental study was conducted at Balderas sport horses and recreational center, Addis Ababa, Ethiopia to determine ovarian follicular dynamics in local and exotic cross breed mares. A daily transrectal ultrasonography was carried out to evaluate uterine changes and ovarian structures for 2 consecutive estrus cycles, as well as teasing scores estimated during estrus. The mean (±SEM) of cross sectional uterine diameter were 44.4±0.5mm and 45±0.5mm for local and cross breed mares respectively; whereas scores of endometrial fold was 3.1±0.1 in local and 2.8±0.1 in cross breed mares with significant difference at P &lt; 0.05. The mean (±SEM) diameter of preovulatory follicle for local and cross breed mares were 49.1±1.0mm and 50.1±0.8mm, respectively.Length of estrus was 7.0±0.9 days for local and 6.1±0.6 days for cross breed mares. Fast growth of dominant follicles in cross breed shorten interovulatory interval than local breed mares.A positive correlation of teasing scores, uterine diameter and endometrial fold with diameter of preovulatory follicles (r=0.5) in the present study has been used to estimate imminent ovulation.Ovulation occurred within 6-7days of mean scores of 2.8-3.1endometrial fold, 44.4-45mm of uterine diameter and 2-4 teasing scores for both mares.In conclusion, imminent ovulation can be predicted by measuring uterine diameter, scoring endometrial fold and considering teasing scores in relation to diameter of preovulatory follicles when breeding mares.

Investigation of Ovarian Follicular Waves and Major Hormonal Profile in Red Chittagong Cattle

Red Chittagong Cattle (RCC) is one of the renowned varieties of cattle which is indigenous and most potential type of domestic animal genetic resource in Bangladesh with distinct phenotypic characteristics. Reproductive management might be a process for multiplying this cattle variety. Therefore, the goals of this study were to characterize the follicular waves of RCC and to determine the Progesterone (P4), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH) profile during three consecutive estrus cycle. Follicular wave emergence (Day 0) was identified using trans-vaginal ultrasound technique and follicular growth was then monitored from Day 0 to Day 23 during three consecutive estrus cycle and blood samples were collected. After measuring the growth and diameter of the follicles, hormonal assays of collected blood samples were performed using Enzyme Linked Immunosorbent Assay (ELISA). We found that, both RCC cows and heifers showed two waves inter-ovulatory intervals. The length of inter-ovulatory intervals and the diameter of the ovulatory follicle of RCC cows and heifers were slightly different from each other. The identification of the dominant follicle was confirmed from the start of its deviation from other subordinate follicles till it reached to the maximum diameter and identified as dominant follicle which increases linearly for approximately six days (growing phase). This dominant follicle is remained approximately same size for five days (static phase), and then begins to regress (regressing phase) decreasing the antral diameter. The follicular waves are first detectable as 4 - 5 mm follicles approximately at day 0 and day 10 both for RCC cows and heifers which showed two waves inter-ovulatory intervals. The circulating varied concentrations of LH, FSH and P4 were determined using ELISA in case of both cows and heifers and the concentration curves followed the similar pattern of change during the estrus cycle. Hence, the knowledge about ovarian follicular waves will create a base line for reproductive management of RCC as well as to allow reproductive biotechnologies to improve and multiply this cattle variety.

124 Ovarian follicular development and steroid secretion during oestrous cycle of Lohi sheep

Pakistan has 30.9 million heads of sheep; however, little information is available on their reproductive aspects. The objective of this study was to document ovarian physiology and endocrinology of Lohi ewes during the oestrous cycle. Nine Lohi ewes, synchronized by administering single prostaglandin F2α (PGF2a; Cyclomate, Star Laboratories), were monitored for ovarian follicular dynamics using transrectal ultrasonography (7.5MHz, HS-1500, Honda) for two consecutive oestrous cycles during the breeding season (September to November 2018). Changes in plasma progesterone and oestradiol-17β concentrations of ewes (n=9) were also determined during the oestrous cycle using radioimmunoassay. The interovulatory interval of Lohi ewes averaged 17.0±0.1 days, and the duration of follicular and luteal phases was 4.6±0.2 and 11.3±0.2 days, respectively. Follicles emerged in either 3- or 4-wave patterns, but the frequency of the 3-wave pattern was higher than that of the 4-wave (87 vs. 13%, respectively; P=0.05). Following ovulation (Day 0), follicles (=3mm) in 3-wave cycles (n=14) emerged on Days 0.7, 5.2, and 10.5, whereas in 4-wave cycles (n=2) follicles emerged on Days 0.1, 4, 8.5, and 11.5. The maximum diameter of preovulatory follicles and corpora lutea (CL) were 5.4±0.3 and 10.4±0.3mm, respectively. Regardless of the wave pattern, single ovulation occurred in each cycle. The CL was first detectable on Day 4±0.1, it reached maximum diameter on Day 9±0.1, and luteolysis began on Day 12.2±0.2 of the cycle. The peak plasma oestradiol-17β concentration (42.5±2.6 pgmL−1) was observed 48h before ovulation and correlated with the diameter of the preovulatory follicle during the follicular phase (r=0.84; P&amp;lt;0.05). The peak plasma progesterone concentration (11.8±1.7ngmL−1) was observed on Day 9±0.1 and coincided with the diameter of CL throughout the oestrous cycle (r=0.93; P&amp;lt;0.05). In conclusion, the majority of oestrous cycles in Lohi ewes had a 3-wave pattern and were mono-ovulatory in nature.

The role of exogenous melatonin and photoperiod on productive and reproductive performance of Ossimi sheep.

To determine effect of the administration of exogenous melatonin and photoperiod on initiating the lambing season and improving reproductive efficiency of sheep, a total of thirty-four multiparous, non-pregnant Ossimi ewes aged 3-5 years with average body weight 55.94±1.80 kg were used. After weaning and the starting of the breeding season, the ewes were divided into four groups; the 1st group (7 ewes) was exposed to natural daylight (NL) and severed as a control group, the 2nd group (9 ewes) was exposed to 16-hour artificial lighting (AL), the 3rd group (10 ewes) was exposed to natural daylight+18-mg/h melatonin (NL+MEL), while the 4th group (8 ewes) was exposed to 16-hour artificial lighting+18-mg/h melatonin (AL+MEL). The treatment of the ewes continued throughout the mating season and for three consecutive estrus cycles (60 days). Ewes were weighed before mating and after parturition. Reproductive traits were recorded after parturition. Results revealed that exogenous melatonin (induced by either natural daylight or artificial photoperiod) exhibited significantly (P≤0.05) higher body weights (6.20±1.711 and 4.14±1.91kg for NL+MAL and AL+MEL groups more than the control group). Both of the melatonin treatment and the use of artificial light for 16 h insignificantly influenced reproductive parameters (percentages of fertility, conception rate, scanning, lambing rate, and fecundity rate of Ossimi ewes. Fertility and conception rates tended to be higher in (NL+MEL) and (AL+MEL) groups, while SP percentage, lambing and fecundity rates were higher in treated groups than control. The percentage of the number of services per conception (NSC) and abortion rate were insignificantly reduced in treated ewes as compared with the control. Both weaning rate and the survival rate was elevated (P≥0.05) in treated ewes than untreated ones. Lambing interval and days open were significantly lower (P≤0.05) in AL+MEL group than AL and NL+MEL groups. The lambs’ weight at birth, weaning, net gain, and average daily gain tended to be higher in (NL+MET) and (AL+MEL) groups than other groups with insignificant differences. It can be concluded that, stimulation of melatonin (induced by natural daylight and artificial photoperiod manipulation) might be a useful tool in improving reproductive performance and lamb production in Ossimi sheep.

Research Article Effect of You-Gui-Wan on endometrial angiogenesis in controlled ovarian hyperstimulation mice model

Purpose: To explore the mechanism of You-Gui-Wan (YGW) in improving endometrial receptivity in mouse model of controlled ovarian hyperstimulation (COH). Methods: Sixty female mice with two consecutive oestrous cycles were randomly divided into COH model group (n = 50) and normal control group (n = 10). The COH model was created by using GnRH-a + PMSG + HCG and further randomly divided into 5 subgroups of simple model, YGW high-dose, YGW medium-dose, YGW low-dose and aspirin control, 10 in each subgroup. YGW or aspirin or saline was administrated to the mice by gavage once a day for 11 days. Afterwards, blood and uterine samples were collected for examination of serum levels of estradiol (E2) and progesterone (P4), endometrial thickness and number of blood vessels as well as expressions of angiogenic factors of VEGF, Ang-1, Ang-2 and Tie-2 at transcription and translation levels by using H& E staining, ELISA, real-time qRT-PCR, and immunohistochemistry, respectively. Results: COH per se increased the serum concentrations of E2 and P4i¼Œbut decreased the number of endometrial blood vessels and the protein expressions of the angiogenic factors when compared to the normal control group (P< 0.05 or 0.01). YGW increased the mRNA expressions of all angiogenic factors and restored the effect of COH on number of endometrial blood vessels (P< 0.01), and the protein expressions of the angiogenic factors when compared to the COH simple model group (P< 0.05 or 0.01). In comparison, aspirin could only restore the number of endometrial blood vessels and the protein expressions of Ang-2 and Tie-2 (P< 0.05 or 0.01). Conclusions: YGW can restore the side effect of COH on number of endometrial blood vessels and the expressions of angiogenic factors, thus improve endometrial receptivity in COH mice

The effect of select seminal plasma proteins on endometrial mRNA cytokine expression in mares susceptible to persistent mating-induced endometritis.

In the horse, breeding induces a transient endometrial inflammation. A subset of mares are unable to resolve this inflammation, and they are considered susceptible to persistent mating-induced endometritis PMIE Select seminal plasma proteins cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin have been shown to affect the innate immune response to sperm in vitro. The objective of this study was to determine whether the addition of CRISP-3 and lactoferrin at the time of insemination had an effect on the mRNA expression of endometrial cytokines in susceptible mares after breeding. Six mares classified as susceptible to PMIE were inseminated during four consecutive oestrous cycles with treatments in randomized order of: 1mg/ml CRISP-3, 150μg/ml lactoferrin, seminal plasma (positive control) or lactated Ringer's solution (LRS; negative control) to a total volume of 10ml combined with 1 × 109 spermatozoa pooled from two stallions. Six hours after treatment, an endometrial biopsy was obtained for qPCR analysis of selected genes associated with inflammation (pro-inflammatory cytokines interleukin (IL)-1β, IL-8, tumour necrosis factor (TNF)-α, interferon (INF)-γ, anti-inflammatory cytokines IL-1RN and IL-10, and inflammatory-modulating cytokine IL-6). Seminal plasma treatment increased the mRNA expression of IL-1β (p=.019) and IL-8 (p=.0068), while suppressing the mRNA expression of TNF (p=.0013). Lactoferrin also suppressed the mRNA expression of TNF (p=.0013). In conclusion, exogenous lactoferrin may be considered as one modulator of the complex series of events resulting in the poorly regulated pro-inflammatory response seen in susceptible mares.

High interindividual and intraindividual variation of oxytocin secretion in estrous mares exposed to stallions, but no significant link to mate preferences

Oxytocin is a hormone that may not only influence reproductive mechanisms in mammals but also their social behavior, including pair bonding. We therefore tested if the concentrations of oxytocin and other hormones reveal mate preferences of 13 mares in estrus. Each mare was first exposed to two stallions (haphazardly selected out of seven) and her behavior recorded. The mare was then returned to her box (i.e., no contact to stallions during that time). Approximately 4.5 hours later, venous blood samples were collected every minute during 30 minutes preceding exposure to one of the two previously used stallions, 6 minutes during exposure, and 30 minutes after exposure back in the mare's box. The procedure was repeated in the consecutive estrus cycle, with the difference that the mare was each exposed to the other of the two stallions during oxytocin measurements. In 20 of the 26 trials, oxytocin concentrations were significantly elevated during exposure to the stallion, without significant associations to cortisol and estradiol concentrations. We found no significant association between oxytocin secretion and preferences in the previous choice situation. While estradiol concentration showed a high repeatability over the two cycles, we found considerable intraindividual differences in oxytocin and cortisol plasma concentration among the two cycles. Partially, the variation in oxytocin concentrations could be linked to the time of ovulation, with lower oxytocin plasma concentrations in mares which ovulated later than expected. In conclusion, when teasing under experimental conditions, we found high interindividual and intraindividual variation among mares in the increase of oxytocin plasma concentrations, depending on the timing of ovulation. However, oxytocin levels seemed to be no predictor of mare preference.

Effectiveness of a recombinant human follicle stimulating hormone on the ovarian follicles, peripheral progesterone, estradiol-17β, and pregnancy rate of dairy cows.

Aims:This study aimed at elucidating the effects of recombinant human follicle stimulating hormone (r-hFSH) on the ovarian follicular dynamics, progesterone, estradiol-17β profiles, and pregnancy of dairy cows.Materials and Methods:Three groups (G, n=5 cows) of multiparous dairy cows were used. G1 (C) control cows were given controlled internal drug release (CIDR) and prostaglandin F2α; G2 (L) cows were given low dose (525 IU and G3 (H) cows were given high dose (1800 IU) of r-hFSH on twice daily basis at the last 3 days before CIDR removal. All cows were ultrasonically scanned for follicular growth and dynamics, and blood samples were collected every other day for two consecutive estrus cycles for the determination of estradiol-17β and progesterone.Results:Estrus was observed in all C and L but not in H cows. Dominant follicle was bigger in L compared to C and H cows. Dominant follicle in C (16.00±2.5 mm) and L cows (17.40±2.3 mm) disappeared at 72 h after CIDR removal. However, in H cows, no ovulation has occurred during 7 days post-CIDR removal. Progesterone was not different (p>0.10) among groups, whereas estradiol-17β revealed significant (p<0.01) reduction in H (15.96±2.5 pg/ml) cows compared to C (112.26±26.1 pg/ml) and L (97.49±15.9 pg/ml) cows. Pregnancy rate was higher in L cows (60%) compared with C cows (20%). However, H cows were not artificially inseminated due to non-ovulation. Only a cow of C group has calved one calf, however, 2 of the L cows gave birth of twins and a cow gave single calf.Conclusion:Administration of a low dose (525 IU) of r-hFSH resulted in an optimal size of dominant follicle, normal values of progesterone and estradiol-17β, and 40% twinning rate, howeverusing 1800 IU of r-hFSH, have adverse effects on ovarian follicular dynamics and hormonal profiles with non-pregnancy of dairy cows raised under hot climate.

Serial transvaginal ultrasound-guided biopsy of the porcine corpus luteum in vivo.

The aims of the present study was to develop and describe a transvaginal ultrasound-guided biopsy method for luteal tissue in the porcine and to evaluate the effects of the method on the reproductive tract, ovarian status and pregnancy status. Biopsies were performed in four multiparous sows on Days 9 and 15 of three consecutive oestrous cycles; the size and histological composition of the samples obtained were evaluated and the reproductive tract of the sows was monitored. Furthermore, biopsies were performed in 26 multiparous sows on Days 10 and 13 after insemination, and the pregnancy rate, gestation length and subsequent litter size were evaluated. RNA was extracted from the samples obtained and the quality and quantity were determined. Altogether, 76 biopsies were performed and 38 samples were obtained. Compared with sows from which no samples were obtained (n=6), sows from which one or more samples were obtained (n=24) were older (parity 5.0±2.8 vs 2.2±0.4, mean±s.d.), heavier (290±26 vs 244±27kg) and had higher back fat (11.4±2.7 vs 6.4±2.5mm; P<0.05 for all). No effect of the biopsies (P>0.05) was observed on the cyclicity and reproductive organs of the sows, or on corpus luteum diameter on Day 13 (8.9±1.0 vs 9.2±1.1mm), pregnancy rate (95% vs 96%), gestation length (115±1 vs 115±1 days) and subsequent litter size (12.7±2.5 vs 13.3±2.8) between sows from which samples were obtained and those from which no samples were obtained. The samples obtained had a diameter of 1mm and contained heterogeneous tissue with various cell types. The RNA quantity was 520±160µg per sample and the RNA integrity number was 8.5±1.0. In conclusion, an ultrasound-guided biopsy method for ovarian tissue, which can be used for gene expression studies, was established in the porcine. No effect on corpus luteum function was found.

Ovarian function and pregnancy outcome in pony mares following immunocontraception with native and recombinant porcine zona pellucida vaccines.

Few studies have investigated ovarian function in the mare undergoing porcine zona pellucida (pZP) immunocontraception despite reported ovarian dysfunction in other species. This study aimed to describe ovarian function and oestrous cyclicity in pony mares following treatment with either the conventional pZP vaccine or a novel recombinant form of the vaccine derived from porcine ZP3 and ZP4 (reZP). In addition, the contraceptive efficacy of pZP vs. reZP was assessed. Blinded, randomised, prospective clinical trial. Mares (n = 21) were randomised into 3 groups of 7: Group I received the pZP vaccine, with a booster 5 weeks later; Group II received the reZP vaccine, with a booster 5 weeks later; and Group III (controls) received 2 treatments, 5 weeks apart, of saline and adjuvant alone. Mares underwent weekly monitoring via transrectal palpation and ultrasound examination of the reproductive tract, with daily monitoring during oestrus. Data were collected over a 24 week period coinciding with the physiological breeding season; treatments commenced in Week 4. Serum samples were obtained for antibody titres and ovarian steroid level analyses at 7 day intervals. Cycling mares were bred via fresh semen artificial inseminations over a maximum of 2 consecutive oestrous cycles, commencing 5 weeks post booster vaccination. Control mares cycled throughout the trial. After treatment, 6 of 7 pZP mares (86%) and one reZP mare (14%) had an extended anoestrus that correlated with basal serum oestradiol and progesterone levels. All mares resumed cyclicity by 10 months post treatment. Pregnancies were diagnosed in all controls, 4 reZP- (57%) and none of the pZP-immunised mares. The current study demonstrates the reversible suppression of ovarian function in pony mares following treatment with pZP. The effect of the reZP vaccine on pregnancy outcome requires further investigation.

A novel method of extraction of bamboo seed oil (Bambusa bambos Druce) and its promising effect on metabolic symptoms of experimentally induced polycystic ovarian disease.

Objective:To evaluate the potential effect of bamboo seed oil in decreasing the major metabolic symptoms associated with letrozole-induced polycystic ovarian disease using female rat model.Materials and Methods:A new method of microwave-assisted extraction was developed. Female rats were grouped into four with six animals each. All rats were daily administered with letrozole (1 mg/kg b.wt.) for 21 days except control, and during this period, changes in estrous cycle were observed. After letrozole treatment, Group 2 was considered negative control, Groups 3 and 4 were treated orally with bamboo oil, 0.5 ml/kg b.wt. and 1 ml/kg b.wt., respectively, for 3 weeks (five consecutive estrus cycles). Various parameters such as estrus cycle, blood sugar level, lipid profile, and weights of reproductive system were determined. The characteristics of cystic ovaries were evaluated by histopathological studies.Results:The isolated bamboo oil restored estrus cyclicity showed hypoglycemic and hypolipidemic effects. 1 ml/kg b.wt. of bamboo oil showed a marked glucose reduction from 254.04 ± 2.08 to 92.6 ± 1.63, and levels of total cholesterol, very low-density lipoprotein, triglyceride were reduced from 186.45 ± 2.28, 30.07 ± 2.36, 100.36 ± 2.35 to 152.14 ± 2.63, 25.94 ± 1.66, 93.32 ± 1.09, respectively. Histopathological results showed the presence of ovulation and recovery from cystic ovaries.Conclusion:A novel and promising drug was isolated in the treatment and maintenance of various metabolic symptoms associated with polycystic ovary disease.

Does Clinical Treatment with Phenylbutazone and Meloxicam in the Pre-ovulatory Period Influence the Ovulation Rate in Mares?

The presence of anovulatory haemorrhagic follicles during the oestrous cycle of mares causes financial impacts, slowing conception and increasing the number of services per pregnancy. Non-steroidal anti-inflammatory drugs (NSAIDs) such as meloxicam and phenylbutazone are used in the treatment of several disorders in mares, and these drugs can impair the formation of prostaglandins (PGs) and consequently interfere with reproductive activity. This study aimed to evaluate the effects of treatment with NSAIDs on the development of pre-ovulatory follicles in mares. In total, 11 mares were studied over three consecutive oestrous cycles, and gynaecological and ultrasound examinations were performed every 12 h. When 32-mm-diameter follicles were detected, 1 mg of deslorelin was administered to induce ovulation. The first cycle was used as a control, and the mares received only a dose of deslorelin. In the subsequent cycles, in addition to receiving the same dose of deslorelin, each mare was treated with NSAIDs. In the second cycle, 4.4 mg/kg of phenylbutazone was administered, and in the third cycle, 0.6 mg/kg of meloxicam was administered once a day until ovulation or the beginning of follicular haemorrhage. All of the mares ovulated between 36 and 48 h after the induction in the control cycle. In the meloxicam cycle, 10 mares (92%) did not ovulate, while in the phenylbutazone cycle, nine mares (83%) did not ovulate. In both treatments, intrafollicular hyperechoic spots indicative of haemorrhagic follicles were observed on ultrasound. Thus, our results suggested that treatment with meloxicam and phenylbutazone at therapeutic doses induced intrafollicular haemorrhage and luteinization of anovulatory follicles.

The effect of deslorelin acetate on the oestrous cycle of female guinea pigs

Deslorelin acetate, a GnRH agonist, is commonly used to prevent folliculogenesis in several species. However, little is known of the effect of deslorelin acetate implants on the oestrous cycle of female guinea pigs. Fifteen intact healthy female guinea pigs were investigated in this study. Signs of sexual behaviour, the presence of a vaginal membrane along with plasma concentrations of oestradiol (E 2 ) and progesterone (P 4 ), were monitored during two consecutive oestrous cycles. At the beginning of the third oestrous cycle each guinea pig was administered an implant of the GnRH analogue, deslorelin acetate, (4.7 mg). When compared to the untreated state, deslorelin implantation was associated with altered signs of oestrus. The average time to opening of the vaginal membrane was delayed. After opening, the vaginas were found to be variably opened and closed. A significant reduction in P 4 (to less than 1.0 ng/ml) and cessation of P 4 cyclical variation was observed. Plasma E 2 remained high during the whole experimental period. This study shows that cessation of the oestrous cycle by the deslorelin implant might be useful in preventing pregnancy in guinea pigs.

112 OVARIAN FOLLICLE RESERVE IN NELLORE AND ANGUS HEIFERS WITH LOW AND HIGH FOLLICLE COUNT

Cattle from Bos indicus breeds recruit and develop more follicles per oestrous cycle compared with cattle from Bos taurus breeds. The number of antral follicles in the ovaries is a decisive factor for the success of reproductive technologies such as ovum pick-up and embryo transfer. The reasons why indicine cattle recruit and develop more follicles are not known. Our objectives were to evaluate the ovarian reserve of Nellore and Angus heifers and determine at which stage of follicle development Nellor ovarian reserve differs from Angus ovarian reserve. To determine if animals were in the low or high follicle count group (LFC or HFC), antral follicles (≥3 mm) were scanned with an ultrasound device (US; Mindray Vet DPS 2200, Sao Paulo, Brazil) equipped with a 7.5-MHz probe Day 1 after ovulation (Day 0) for 3 consecutive oestrous cycles. Eighteen Nellore heifers (11 LFC and 7 HFC) and 25 Angus heifers (12 LFC and 13 HFC) were used in the experiment. Oestrous cycles were synchronized with 2 doses of PGF2α ± 11 days apart. Animals were slaughtered ~24 h after ovulation of the third oestrous cycle. Ovulation was determined by oestrous observation, absence of the dominant follicle on the ultrasound, and presence of the corpus luteum after slaughter. One ovary from each animal was used for histology. The ovary was sliced longitudinally in 6 parts, fixed in 10% paraformaldehyde for 24 h, and stored in 70% ethanol until processing. Two longitudinal slices were blocked in paraffin. One section (8 μm thick) was stained after every 50 sections, totaling 10 sections per longitudinal slice of ovarian tissue. Sections were stained using eosin and hematoxylin. Follicles were counted and classified under a stereo microscope with a 40× objective; only follicles with a visible nucleus were counted. Follicles were classified as primordial, primary, and secondary. The area of each section was measured using the hand tool on the ImageJ software. Area measurements were expressed as pixels squared. The density of each follicle classification was calculated as follicle/log10 [pixel2]. Data were analysed using PROC GLM procedure of SAS 9.2 considering the effects of breed and group (LFC or HFC). Angus heifers had more primordial follicles (P &lt; 0.02) when compared with Nellore heifers, but there was no difference within groups. There was no difference in the number of primary follicles within breeds or groups. Secondary follicle numbers tended to be higher (P = 0.09) in Nellore heifers when compared with Angus heifers and was higher within groups (P &lt; 0.02), with HFC Angus and Nellore heifers having more follicles than LFC heifers. It is possible that HFC heifers have a mechanism that is developing more follicles beginning at the secondary stage, especially Nellore heifers. The development of more secondary follicles may contribute to the depletion of primordial follicles observed in Nellore heifers. This research is funded by Sao Paulo Research Foundation (FAPESP) grant #2011/50964-0. Scholarships for Favoureto, Queiroz, Loureiro, Ereno, and Pupulin are also from FAPESP.

176 EXPRESSION OF INSULIN-LIKE GROWTH FACTOR FAMILY MEMBERS AND GROWTH DIFFERENTIATION FACTOR 9 ON NELORE AND ANGUS HEIFERS WITH A LOW AND HIGH OVARIAN FOLLICLE COUNT

Members of the IGF family are key intra-ovarian regulators of follicle growth, selection, and atresia. Growth differentiation factor 9 (GDF9) induces follicular somatic cells to undergo mitosis and differentiation during follicular development. Cattle from Bos indicus breeds are slower to reach sexual maturity and have longer calving intervals when compared with Bos taurus breeds (Luna-Nevarez et al. 2011). On the other hand, indicine cattle have greater number of ovarian follicles recruited per oestrous cycle when compared with taurine breeds (Alvarez et al. 2000). Our objective was to evaluate the expression of IGF1, IGFR1, IGF2, and GDF9 genes in follicles dissected from Nelore and Angus heifers with high (HFC) and low (LFC) follicle counts. Eighteen Nelore heifers and 22 Angus heifers (≈24 months old) were kept on Brachiaria brizantha grass and fed with a mix of grains with minerals and water ad libitum. Oestrous cycle was synchronized with 2 doses of PGF2α 11 days apart. Heifers were scanned with an ultrasound device (US; Mindray Vet DPS 2200, São Paulo, Brazil) with a 7.5-MHz probe 1 day after ovulation for 3 consecutive oestrous cycles. Animals were slaughtered ≈24 h after ovulation of the third cycle; 3 follicles from 2 to 4 mm in diameter were dissected from the ovary contralateral to the CL. Total RNA was extracted using the RNeasy Microarray Tissue Mini Kit (Qiagen, Valencia, CA, USA). Gene expression was evaluated using oligo-dT reverse transcription, Sybr Green Master Mix and Step One Plus (AB, Foster City, CA, USA) Real-Time PCR Detection System. Samples were analysed in duplicates and CT values were normalized to the housekeeping gene PPIA using the ΔCT method. Results were analysed using the PROC MIXED procedure of SAS with follicle as the repeated measure and cow as the subject. Individual differences were analysed using contrast (SAS 9.2). The effects of group (LFC × HFC), breed, and follicle diameter on mNRA abundance were tested. Follicle LSmean was higher (P &lt; 0.05) in Nelore heifers (32 ± 3.1; LFC = 18; HFC = 52) when compared with Angus heifers (20 ± 2.6; LFC = 10; HFC = 27). Follicle diameter did not differ between breeds or groups. Expression of IGF1 and GDF9 was not different between follicles from Nelore and Angus heifers or between groups. Expression of IGFR1 was higher in follicles from Angus heifers (fold change 1.74; P &lt; 0.04) when compared with follicles derived from Nelore heifers but not different between HFC and LFC groups within each breed. Expression of IGF2 mRNA was also higher (fold change 1.70; P &lt; 0.04) in Angus heifers but not different between HFC and LFC groups within each breed. In conclusion, the higher expression of IGFR1 and IGF2 in Angus early antral follicles might be involved in important reproductive traits commonly observed in Bos taurus breeds. However, expression of IGF1, IGFR1, IGF2, and GDF9 in the follicle are not involved in the mechanisms determining different number of follicle recruited through the oestrous cycle in the same breed. This research and scholarship for Loureiro, Ereno, Favoureto, and Pupulim was from FAPESP.

171 EXPRESSION OF ANDROGEN-PRODUCING ENZYMES IN NELORE AND ANGUS HEIFERS WITH LOW AND HIGH NUMBERS OF FOLLICLES IN THE OVARY

During follicle development, androgens are synthesised by thecal cells under LH influence through the enzymatic conversion of the androgen precursors. In cattle, androgen concentration is positively correlated with the number of antral follicles. This experiment was designed to assess mRNA levels of androgen-producing enzymes (CYP11A1, CYP17A1, HSD3B, HSD17B) in Nelore and Angus heifers with high (HFC) and low follicle counts (LFC). Eighteen Nelore and 22 Angus heifers (≈24 months old) were kept on Brachiaria brizantha grass supplemented with a mix of grains. To determine the number of follicles, heifers were scanned with an ultrasound device (Mindray Vet DPS 2200, São Paulo, Brazil) with a 7.5-MHz probe 1 day after ovulation for 3 consecutive oestrous cycles. The first and third (before slaughter) oestrous cycles were synchronized with 2 doses of PGF2α 11 days apart. Animals were slaughtered ≈24 h after ovulation of the third cycle; 3 follicles from 2 to 4 mm in diameter were dissected from the ovary contralateral to the CL. Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Gene expression was evaluated using oligo-dT reverse transcription, Sybr Green Master Mix, and Step One Plus (AB, Foster City, CA, USA) Real-Time PCR System. Samples were analysed in duplicates, and CT values were normalized to the housekeeping gene PPIA using the ΔCT method. The ΔCT values were compared using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC, USA) with follicle as the repeated measure and cow as the subject. We tested the effects of breed, follicle number, and millimeters on gene expression. Individual differences between groups (HFC or LFC) within breed were analysed using the Diff function (SAS 9.2). Results were significant when P &lt; 0.05. Follicle count was higher (P &lt; 0.04) in Nelore 32 ± 3.1 (LFC = 16; HFC = 52) when compared with Angus 20 ± 2.6 (LFC = 10; HFC = 27) heifers. Follicle diameter did not differ between breeds or groups. Expression of CYP11A1 mRNA was higher in Angus heifers (P &lt; 0.01). However, there was no difference in CYP11A1 mRNA expression within groups in Angus heifers, whereas HFC Nelore heifers expressed higher levels of CYP11A1 mRNA than LFC Nelore heifers (P &lt; 0.04). Expression of CYP17A1 was not different between breeds or groups within each breed. Expression of HSD3B mRNA was higher (P &lt; 0.04) in Nelore heifers (P &lt; 0.004) when compared with follicles derived from Angus heifers but not different between HFC and LFC groups within each breed. Expression of HSD17B was higher in Nelore heifers (P &lt; 0.001) but not different between HFC and LFC groups within each breed. In conclusion, Nelore heifers had a higher expression of hydroxysteroid dehydrogenases (HSD3B and HSD17B). As androgens have been demonstrated to stimulate early follicle development (Yang and Fortune 2006 Biol. Reprod. 75, 924–932), these data are indicative that androgens may be involved in the mechanisms determining different follicle counts across cattle subspecies but not between groups within each breed. Funding and scholarship for Loureiro, Ereno, Favoureto, and Pupulim was from FAPESP.

Effect of immunomodulatory therapy on the endometrial inflammatory response to induced infectious endometritis in susceptible mares

The objective of the present study was to evaluate the effect of immunomodulatory therapy (glucocorticoids (GC) and mycobacterium cell wall extract (MCWE)) on the endometrial gene expression of inflammatory cytokines in susceptible mares with induced infectious endometritis. Endometrial gene expression of pro- and anti-inflammatory cytokines; interleukin (IL)-1β, IL-6, IL-8, IL-10, tumor necrosis factor (TNF)-α, IL-1 receptor antagonist (ra), acute phase protein (APP) serum amyloid A (SAA) and clinical parameters were evaluated. Five mares were classified as susceptible to persistent endometritis based on their endometrial histopathology and ability to clear an induced uterine inflammation. To investigate the effect of immunomodulatory therapy, the mares were inoculated with 105 colony forming units (CFU) Escherichia coli in three consecutive estrus cycles in a modified cross-over study design. Thus, each mare served as its own control and the treatment type was performed in randomized order. The effect of treatment with MCWE (1.5 mg Settle IV), dexamethasone (0.1 mg per kg IV) or no treatment was investigated. All mares were free from uterine inflammation before each E. coli inoculation. Endometrial biopsies were recovered 3, 24 and 72 h post inoculation. Relative gene-expression analyses were performed by quantitative reverse transcriptase PCR (qRT-PCR). Endometrial gene expression of inflammatory cytokines was modulated by administration of GC. Expression of proinflammatory cytokines (IL-1β, IL-6, IL-8) and SAA was significantly lower in the GC treated group late in the study period (72 h) compared to “no treatment” and MCWE treatment. Increased expression of the anti-inflammatory cytokine IL-10 was observed 3 and 24 h after E. coli infusion and GC treatment. A significant decrease of SAA expression was observed after MCWE treatment compared to “no treatment”. MCWE and GC treatment had a significant effect on the clearance of uterine pathogens and number of mares retaining fluid after E. coli infusion. The results of the current investigation suggest that GC is capable of effectively modulating the innate immune response to induced infectious endometritis in susceptible mares.