Immunohistochemical localization of estrogen receptor alpha (ERα) in the oviduct of Indian buffalo during follicular and luteal phases of estrous cycle.

The localization and distribution of estrogen receptor alpha (ERα) in different segments of oviduct of buffalo during follicular and luteal phases of estrous cycle were investigated using immunohistochemistry. Tissue samples from the different segments of oviduct from 12 buffaloes (six each during follicular and luteal phases of estrous cycle) were collected from slaughter house after assessing the gross morphology of ovaries. In addition, blood samples were collected from the animals before slaughter to estimate levels of estrogen and progesterone hormones. The tissue distribution of estrogen receptor was determined by immunohistochemical technique using one-step polymer HRPO staining system. The estrogen receptor was localized in the lamina epithelialis, propria submucosa, tunica muscularis, and tunica serosa. The maximum localization was observed in the lamina epithelialis, where both ciliated and secretory cell types were positive for ERα. Percentage of positive cells varied during the follicular and luteal phases of estrous cycle. The lining epithelium of oviductal glands was also intensely positive for ERα. No immunostaining was observed in any tunic of the oviduct when the primary antibody was replaced by antibody diluent or buffer, and it served as negative control. The data showed that highest immune positive cells were observed in the ampulla region of the oviduct and these cells were lowest in the utero-tubal junction (p < 0.05). Infundibulum, ampulla, and isthmus showed a higher percentage of ERα-positive cells during follicular phase of estrous cycle as compared with those of the luteal phase of estrous cycle (p < 0.05). There was no significant difference in the percentage positive cells during the two phases of estrous cycle in the utero-tubal junction. Immunogold labeling with anti-ERα antibody confirmed the findings of immunohistochemical study at subcellular level. The higher expression during the follicular phase was directly correlated with the level of estrogen hormone.

Genotype of MTNR1A gene regulates the conception rate following melatonin treatment in water buffalo.

Buffaloes have tendency to show seasonal reproduction and remain in anestrus due to limited ovarian activity during summer. The seasonal reproductive behavior is ascribed the effect of melatonin related to photoperiod. Treating animals with melatonin could be a possible strategy to overcome the problem. The role of MTNR1A gene has not been fully explained in the buffalo. Therefore, we conducted a study on 114 buffalo heifers to detect the polymorphic site in MTNR1A gene and further treated them with melatonin implants to investigate the role of most frequent genotype following melatonin treatment on pregnancy. The present investigation is the first to investigate the association between melatonin treated different MTNR1A genotype buffalo and pregnancy. We confirmed SNP at position 72 in 812 bp fragment exon II of MTNR1A gene. RFLP of PCR products with Hpa I enzyme resulted in three genotypes: TT (812bp), CT (812, 743, 69bp) and CC (743, 69bp). Next, buffaloes of each genotype (TT, CC, CT; n = 28 for each) were treated with melatonin implants to compare the conception rate with their corresponding untreated control (n = 10 for each genotype). Melatonin concentrations were higher (P < 0.05) for the treatment groups of all genotypes compared to their respective untreated control from day 1-28. The pregnancy rate was significantly associated with the MTNR1A genotype. The conception rate was higher (P < 0.05) for TT genotype than for the other genotypes of buffaloes treated with melatonin. Furthermore, buffaloes of TT genotype treated with melatonin started exhibiting estrus activity soon from second week of melatonin treatment (14.1 ±â€¯2.1; range: 10-17 days) and were found to be 7.8 times more likely to become pregnant compared to other genotypes following melatonin treatment. In conclusion, TT genotype of MTNR1A gene is more sensitive to melatonin treatment that favours pregnancy in buffaloes during summer.

Effects of preovulatory follicle size on estradiol concentrations, corpus luteum diameter, progesterone concentrations and subsequent pregnancy rate in buffalo cows (Bubalus bubalis).

The present study was undertaken to investigate the effects of preovulatory follicle (POF) size on estradiol concentrations, luteal profile (CL diameter and progesterone concentration) and subsequent pregnancy rate in Murrah buffalo cows. The buffalo cows (n = 49) were synchronized for estrus by two doses of PGF2α given 11 days apart. The buffalo cows were inseminated during standing estrus and again after 24 h. Ovaries were scanned at estrus and 24 h intervals until ovulation, thereafter on days 5, 12 and 16 post-ovulation to examine the POF and CL diameter. Size of POF at estrus was divided into three categories; I: 10 to ≤12; II: >12.0 to ≤14.0; III: >14.0-16.0 mm. Blood samples were collected for estradiol (on day of estrus) and progesterone concentration (on days 5, 12 and 16). The estradiol concentrations were greater (P < 0.05) in category II than category I with the greatest (P < 0.05) concentrations estimated in category III. A positive correlation (P < 0.05) between POF and progesterone concentration, CL diameter and progesterone concentration was observed on all sampling day. Pregnant buffalo cows exhibited greater (P < 0.05) plasma progesterone as compared with their non-pregnant counterpart. Greater pregnancy rates were observed with an increased size of POF (χ2 = 2.9, P > 0.05). It was concluded that the POFs having diameters between 12 and 16 mm are mature enough to be transformed into CL of such optimum diameter and can secrete optimum progesterone concentrations that can sustain the pregnancy in Murrah buffalo cows.

Comparative efficacy of oestrus synchronization protocols in buffalo (Bubalus bubalis).

This study was conducted to test the efficacy of gonadotropic hormone (GnRH)-based synchronization protocols (Ovsynch, Heatsynch, and Ovsynch Plus) in buffaloes under field condition. Based on anamnesis and transrectal palpation twice at 10-day interval and serum progesterone (P4) concentration, 150 anoestrous buffaloes and delayed pubertal heifers were selected to induce oestrus using GnRH-based protocols. These selected animals were randomly divided into three groups: group I: Ovsynch (n = 50), group II: Heatsynch (n = 50), and group III: Ovsynch Plus (n = 50) regimen. Before treatment initiation, blood samples were collected for P4, beta-hydroxy butyric acid (ß-OHB), and mineral estimation, in addition to the monitoring of oestrus signs. In this investigation, no significant difference (P > 0.05) in oestrus signs was deduced among three groups. Oestrus induction rate (OIR) was comparable (P > 0.05) among the groups (Ovsynch 82%, Heatsynch 86%, and Ovsynch Plus 88%). Conception rate (CR) following fixed time artificial insemination (FTAI) was slightly higher with Ovsynch Plus group (28%) as compared to Ovsynch (24%) and Heatsynch (18%) groups, though non-significant. Furthermore, serum glucose, ß-OHB, macrominerals (calcium, potassium, and magnesium), and trace minerals (copper, zinc, and iron) remained comparable (P > 0.05) among the groups. In conclusion, all the protocols (Ovsynch, Heatsynch, and Ovsynch Plus) are efficient in oestrus induction in anoestrous buffaloes under field condition with Ovsynch Plus protocol resulting in higher CR as compared to other protocols.

Ovulatory and fertility response using modified Heatsynch and Ovsynch protocols in the anovular Murrah buffalo (Bubalus bubalis).

We studied the effect of modified Heatsynch and Ovsynch protocols on the ovulatory response (OR), estrus induction rate (EIR) and conception rate (CR) in the anovular postpartum Murrah buffalo (n = 35). In the modified Heatsynch protocol (Group I; n = 12), buffaloes were given two GnRH at 2 h interval on treatment day 0, PGF (PGF2α) on day 7 and estradiol (E2) 1 mg on day 8. Two FTAI were done at 20 h intervals after E2 administration. In the modified Ovsynch protocol (Group II; n = 15), GnRH was given on day 0, 7 and 16 with a PGF on day 14. Two FTAI were done; one at last GnRH and the other 20 h later. Group III served as untreated negative control (n = 8). During the treatment, ovarian changes were monitored by transrectal ultrasonography and plasma progesterone (P4) and E2. Administration of two GnRH at 2 h interval neither increased the OR nor strengthened the subsequent P4 priming. Interestingly, in group I, none of the buffalo ovulated to E2 though the EIR was 100% indicating the occurrence of behavioral, but not ovulatory estrus. Administration of GnRH 7 day prior to the commencement of Ovsynch protocol (Group II) did not improve the CR (21.4%), though the OR was 71.4%. No significant difference was found in the diameter of largest follicle between the ovulated and non-ovulated buffalo in response to GnRH suggesting that follicle of ≥9.5 mm is necessary but not sufficient to induce ovulation in the anovular buffalo. In both the protocols, the plasma P4 was higher on day 7 in those buffaloes that ovulated to GnRH. Buffaloes treated with modified Ovsynch regimens were 5.27 times more likely to become pregnant than modified Heatsynch protocol. It is concluded that modified Ovsynch protocol is superior to modified Heatsynch protocol in terms of OR and CR.

Impact of buserelin acetate or hCG administration on day 5 post-ovulation on subsequent luteal profile and conception rate in Murrah buffalo (Bubalus bubalis).

The present study aimed to establish the impact of buserelin acetate or hCG administration on day 5 post-ovulation on subsequent luteal profile and conception rate in buffalo. The buffalo (n=45) were subjected to an estrous synchronization protocol (synthetic analog of PGF2α administered, through intramuscular route, 11 days apart), followed by artificial insemination (AI) during mid to late estrus. On day 5 post-ovulation, buffalo were administered (i.m.) normal saline (Control, n=14), buserelin acetate (20µg, d5-BA, n=14) or human chorionic gonadotropin (3000IU, d5-hCG, n=17). Ovarian ultrasonography was conducted on the day of induced estrus and on days 0, 5, 12, 16 and 21 post-ovulation to assess preovulatory follicle or corpus luteum (CL) diameter. Also, on these days, jugular vein blood sampling was conducted for the estimation of plasma progesterone. First service conception rate was greater (χ(2)=5.18, P>0.05) in d5-BA and d5-hCG groups (71.4% and 47.1%, respectively) as compared to control (28.6%). Both treatment groups had a greater (P<0.05) CL diameter and plasma progesterone during the post-treatment period in comparison to that control treatment group. Treatment-induced accessory CL formation was observed in 92.9% and 76.5% buffalo of d5-BA and d5-hCG groups, respectively. In conclusion, buserelin acetate and hCG administration on day 5 post-ovulation leads to accessory CL formation that may have a role in enhancing conception rate.

Synchronization of follicular wave emergence following ultrasound-guided transvaginal follicle ablation or estradiol-17ß administration in water buffalo (Bubalus bubalis).

The aim of this study was to assess the synchrony in follicular wave emergence and subsequent ovulation following dominant follicle ablation or estradiol-17ß administration. Six cycling Murrah buffaloes were sequentially allotted to three groups, that is, control, follicular ablation, and estradiol-17ß groups. For the control group, buffaloes at random stages of estrous cycle were examined daily by transrectal ultrasonography for 14 days and the day of wave emergence was recorded. Following induced luteolysis and ovulation (Day 0), these buffaloes were included in the ablation group. All follicles (>5mm) were ablated on Day 3 or 5 or 7 (n=2 each day). Seven days after the ablation, these buffaloes were administered prostaglandin F2α to induce luteolysis and ovulation. Following this, buffaloes were included in the estradiol treatment group with estradiol administered on similar days as for ablation in the ablation group. Luteolysis was induced nine days after the estradiol injection. All animals of the treatment groups were subjected to transrectal ultrasound and blood samplings daily from treatment to induced ovulation. The follicular waves emerged significantly earlier (P=0.001) in both the ablation (2.1±0.79 days) and estradiol (4.0±0.25 days) treatment groups than the control group (8.3±0.88 days). The deviation from mean day of ovulation was greater (P=0.02) for the control group buffaloes (1.66±0.3 day) than those of the treatment groups (ablation, 0.76±0.2 and estradiol, 0.58±0.2 day). In conclusion, both ablation and estradiol resulted in synchronous emergence of a new follicular wave irrespective of stage at which the treatment was given, with greater synchrony of ovulations in water buffalo.

Detection of estrous behavior in buffalo heifers by radiotelemetry following PGF2α administration during the early or late luteal phase.

This study examined the usefulness of radiotelemetry for estrous detection in buffalo heifers and the impact of prostaglandin F2α (PGF2α) administration during the early or late luteal phase on estrous behavior and ovulatory follicle variables. Induction of estrus with PGF2α at a random stage of the estrous cycle was followed by the arbitrary division of heifers into groups receiving a second dose of PGF2α during either the early (n=33) or late (n=17) luteal phase (6-9 or 11-14 days after estrus, respectively) for the induction of synchronized estrus. The electronic detection of synchronized estrus by radiotelemetry was confirmed using ultrasonography every 6h until ovulation. Radiotelemetry was 90% efficient and 100% accurate for estrous detection. Intervals between the PGF2α dose and the beginning of synchronized estrus (40.7 ± 10.9 vs. 56.7 ± 12.8h) or ovulation (70.0 ± 11.3 vs. 85.6 ± 12.5h) were shorter (P<0.05) for heifers receiving PGF2α during the early luteal phase. PGF2α administration during the early or late luteal phase produced similar (P>0.05) results for the duration of estrus, the intervals from the beginning or end of estrus to ovulation, the number and duration of mounts per estrus, the duration of mounts, the diameter of the ovulatory follicle and the luteal profile on day 5 after estrus. In conclusion, radiotelemetry was a suitable tool for the efficient and accurate detection of estrus in buffalo heifers. Treatment with PGF2α during the early luteal phase had a shorter interval to synchronized estrus and ovulation; however, estrous behavior, ovulatory follicle dynamics and subsequent luteal activity were similar following PGF2α administration during the early or late luteal phase.

Correlation of blood flow of the preovulatory follicle to its diameter and endocrine profile in dairy buffalo.

Blood flow of the preovulatory follicle (POF) wall can be used as a predictor of the quality of POF. Our aim was to determine the correlation of blood flow of POF with the POF diameter, and intra-follicular and plasma concentrations of Insulin-like Growth Factor-I (IGF-1) and oestradiol in dairy buffalo. Nine Murrah buffalo subjected to an ovulation synchronization protocol (Ovsynch) were assessed on day 10 of the protocol for diameter and blood flow of POF, followed by the aspiration of follicle fluid. Prior to follicular aspiration, blood samples were obtained from jugular vein for estimation of IGF-1 and oestradiol. The vascularity of POF was determined (Range: 250-967 pixel(2) ) along with intra-follicular and plasma concentration of IGF-1 (Range: 9.3-31.8 ng/ml and 14.7-29.7 ng/ml respectively) and oestradiol (Range: 124.2-447.9 ng/ml and 0.25-1.05 ng/ml respectively). Diameter of the POF was weakly correlated (r = 0.21, p < 0.01) with blood flow to it. As compared to POF diameter, the blood flow of POF had greater positive correlation with intra-follicular and plasma concentrations of hormones (IGF-1 and oestradiol). A strong positive correlation was recorded between intra-follicular IGF-1 and oestradiol. Also, plasma concentrations of oestradiol and progesterone were negatively correlated In brief, assessment of the blood flow of the POF is a non-invasive and reliable indicator of its functional competence as compared to the POF diameter.

Impact of buserelin acetate or hCG administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffalo (Bubalus bubalis).

The present study investigated the impact of gonadotropic hormone administration on day 12 post-ovulation on subsequent luteal profile and conception rate in buffaloes. All the buffaloes (n=48) were estrus synchronized by a synthetic analogue of prostaglandin F(2α) (PGF(2α)), administered 11 days apart, followed by insemination during mid to late estrus. To examine the effect of mid-luteal phase hormonal treatment, buffaloes were randomly divided into control (normal saline, n=14), d12-BA (buserelin acetate, 20µg, n=17) and d12-hCG (hCG, 3000IU, n=17) groups. Ovaries were scanned on the day of induced estrus to measure the preovulatory follicle (POF) diameter and on days 5, 12, 16 and 21 post-ovulation to examine the alterations in corpus luteum (CL) diameter. On the day of each sonography, blood samples were collected for the estimation of plasma progesterone. In treatment groups, luteal profile (CL diameter and plasma progesterone) on day 16-21 post-ovulation was better (P<0.05) as well as first service conception rate was higher (52.9% in each treatment group vs. 28.6%, P>0.05) compared to controls. All the pregnant buffaloes exhibited higher (P<0.05) plasma progesterone on various post-ovulation days than their respective non-pregnant counterparts. Treatment-induced accessory corpus luteum (ACL) formation was observed in 58.8 per cent and 70.6 per cent buffaloes of d12-BA and d12-hCG group, respectively, that also had higher (P<0.05) plasma progesterone compared to controls. Compared to the spontaneous CL, the diameter of ACL was less (P<0.05) in the treatment groups. In conclusion, buserelin acetate and hCG administration on day 12 post-ovulation leads to accessory CL formation, improves luteal profile and consequently increases conception rate in buffaloes.

Improvement of conception rate in postpartum flaxseed supplemented buffalo with Ovsynch+CIDR protocol.

The present study was conducted on lactating Murrah buffalo to assess the effect of crushed flaxseed (a source of omega-3 fatty acids) supplementation (300g/100kg bwt/day for 60 days), over and above the routine feed, on luteolytic signal (PGF2α), luteal function (progesterone) and conception rate. In first experiment, on day 50 post-calving, six non-supplemented buffalo were treated to synchronize time of ovulation using an Ovsynch+Controlled Internal Drug Release (CIDR) protocol followed by intravenous oxytocin treatment (OT; 100IU) on day 15 post-ovulation. Blood samples were collected at 15min interval, 1h before to 4h after OT challenge. Thereafter, the same buffalo were supplemented with flaxseed, treated to synchronize time of ovulation starting on day 35 post-supplementation using the same protocol and subjected to OT treatment and blood sampling on day 15 post-ovulation. The PGF2α response was measured as the venous concentration of 13,14-dihydro-15-keto PGF2α (PGFM). The mean hourly concentration of PGFM subsequent to flaxseed supplemented was less (P<0.05) than in the pre-supplementation period at all the occasions. Flaxseed supplementation did not affect plasma fatty acids and other plasma metabolites except for an increase (P<0.05) in plasma cholesterol and plasma alanine transaminase. In the second experiment, 31 buffalo were randomly assigned to a control (n=16) and flaxseed supplemented (n=15) group. The latter group was supplemented with flaxseed starting from day 15 post-calving. On day 50-post-calving, buffalo of both groups were treated to synchronize time of ovulation among animals as described for the first experiment followed by artificial insemination (AI). Post-AI luteal phase plasma progesterone was greater (P<0.05) in the supplemented group compared to controls. Conception rate on day 63 post-AI was 66.7% in supplemented and 31.2% in controls (P<0.05). The present study indicated the beneficial impact of dietary supplementation of crushed flaxseed on conception rate through attenuation of luteolytic signal and improvement in post-breeding luteal profile.

Attenuation of luteolytic response following fish meal supplementation in dairy buffaloes (Bubalus bubalis).

Luteolysis of corpus luteum, due to un-inhibited PGF(2α) secretion, has been reported to be a cause of early embryonic mortality in dairy animals. The objective of this study was to determine the effects of fish meal (FM) supplementation on the uterine secretion of PGF(2α) and hence establish its supplementation as an antiluteolytic strategy in dairy buffaloes. Five cycling Murrah buffaloes were supplemented with 250g FM daily for 55 days in addition to their routine feed and seven buffaloes were kept as non-supplemented control. After 30 days of FM supplementation, the oestrus was synchronized in all the buffaloes using Ovsynch protocol. On day 15 of synchronized cycle, animals were challenged with oxytocin (OT; 100IU) intravenously and blood samples were collected at 15min interval, 1h before to 4h after OT challenge. The PGF(2α) response was measured as the venous concentration of 13,14-dihydro-15-keto PGF(2α) (PGFM). The mean hourly concentration of PGFM in FM supplemented buffaloes was lower than in the control buffaloes at all the occasions. During peak response (1h post-OT challenge), PGFM concentration was significantly lower (P<0.05) in FM supplemented buffaloes than in the control (197.4±41.7pg/ml versus 326.3±33.5pg/ml, respectively). Also the percent rise in PGFM after OT-challenge in FM supplemented buffaloes was less than the control (11.73% versus 22.47%). The dietary supplementation did not affect the size of corpus luteum (CL) and plasma progesterone concentration. Plasma glucose and total protein concentrations remained within the normal physiological limits during FM supplementation. The present study indicated that supplementing FM decreased the concentrations of PGF(2α) without alterations in the size of CL and plasma progesterone concentrations in dairy buffaloes.

Impact of pre-ovulatory follicle diameter on plasma estradiol, subsequent luteal profiles and conception rate in buffalo (Bubalus bubalis).

The present study was designed to investigate the impact of pre-ovulatory follicle (POF) diameter on the day of estrus on plasma estradiol concentration, subsequent luteal profile (corpus luteum, CL, diameter and plasma progesterone concentration) and conception rate in buffaloes. Twenty-eight buffaloes were synchronized with synthetic analogue of prostaglandin F(2α) (PGF(2α)) administered 11 days apart. Transrectal ultrasonography and jugular vein blood sampling was carried out on the day of estrus and on days 0 (day of ovulation), 5, 12, 16 and 21 post-ovulation. Out of 28 buffaloes, 11 (39.3%) were diagnosed pregnant on day 40 post-ovulation. Retrospective analysis revealed that the buffaloes getting pregnant had larger (p<0.05) POF diameter. In fact, all the buffaloes (n=5/5) having POF diameter between >14 and 16 mm conceived, whereas, conception rate in buffaloes with POF diameter between >12 and ≤14 mm (n=6/17) or <12 mm (n=0/6) was 35.3% and 0.0%, respectively. A positive correlation (r=0.57, p<0.05) was observed between POF diameter and plasma estradiol concentration at estrus. On day 5 post-ovulation, luteal profile was positively correlated (CL: r=0.34, p<0.05; plasma progesterone concentration: r=0.27, p>0.05) with POF diameter. Further, on the same day, plasma progesterone concentration was positively correlated (r=0.47, p<0.05) with CL diameter, however, this correlation was absent (r=0.05, p>0.05) during the subsequent luteal phase. Nevertheless, the post-ovulation luteal profile of pregnant buffaloes was higher (p<0.05) compared to non-pregnant counterparts. In conclusion, the diameter of POF in buffaloes has positive impact on plasma estradiol concentration at estrus, post-ovulation luteal profile and conception rate. The diameter of CL can be used as an indicator of luteal function at early but not at mid or late luteal phase of estrus cycle in buffaloes.

Neuronal responses in the brainstem and hypothalamic nuclei following insulin treatment during the late follicular phase in the ewe.

The aim of this study was to determine the neuronal responses following insulin administration during the late follicular phase. Intact ewes were given either saline or insulin (5 IU/kg, i.v.) at 35 h after progesterone withdrawal and killed 3 h later. There was a marked increase in the number of Fos-positive noradrenergic neurones in the caudal brainstem of insulin-treated ewes. In the hypothalamic paraventricular nucleus, insulin treatment increased the presence of Fos-positive corticotrophin-releasing hormone neurones (from 2% to 98%) and Fos-positive arginine vasopressin parvocellular neurones (from 2% to 46%). Interestingly, after insulin treatment, despite a general increase in Fos-positive neurones in the arcuate nucleus (ARC), there was a marked reduction (from 47% to 1%) in Fos-positive ß-endorphin neurones. Similarly, colocalized Fos and oestradiol receptor (ER) α-positive neurones decreased in the ARC after insulin (from 7% to 3%). Conversely, in the ventromedial nucleus, ERα-positive neurones with Fos increased (from 7% to 22%) alongside a general increase in Fos-positive neurones. Overall, a complex system of neurones in brainstem and hypothalamus is activated following insulin administration during the late follicular phase.

Integration between different hypothalamic nuclei involved in stress and GnRH secretion in the ewe.

This study investigated possible integrated links in the neuroanatomical pathways through which the activity of neurones in the paraventricular nucleus and arcuate nucleus may modulate suppression of gonadotrophin-releasing hormone (GnRH) secretion during stressful situations. Double-label immunofluorescence and laser scanning confocal microscopy were used to examine the hypothalamic sections from the follicular phase ewes. Noradrenergic terminals were in close contact with 65.7 ± 6.1% corticotrophin-releasing hormone (CRH) and 84.6 ± 3.2% arginine vasopressin (AVP) cell bodies in the paraventricular nucleus but not with ß-endorphin cell bodies in the arcuate nucleus. Furthermore, γ-amino butyric acid (GABA) terminals were close to 80.9 ± 3.5% CRH but no AVP cell bodies in the paraventricular nucleus, as well as 60.8 ± 4.1%ß-endorphin cell bodies in the arcuate nucleus. Although CRH, AVP and ß-endorphin cell terminals were identified in the medial pre-optic area, no direct contacts with GnRH cell bodies were observed. Within the median eminence, abundant CRH but not AVP terminals were close to GnRH cell terminals in the external zone; whereas, ß-endorphin cells and terminals were in the internal zone. In conclusion, neuroanatomical evidence is provided for the ewe supporting the hypothesis that brainstem noradrenergic and hypothalamic GABA neurones are important in modulating the activity of CRH and AVP neurones in the paraventricular nucleus, as well as ß-endorphin neurones in the arcuate nucleus. These paraventricular and arcuate neurones may also involve interneurones to influence GnRH cell bodies in medial pre-optic area, whereas the median eminence may provide a major site for direct modulation of GnRH release by CRH terminals.

Induction of ovulation of ovulatory size non-ovulatory follicles and initiation of ovarian cyclicity in summer anoestrous buffalo heifers (Bubalus bubalis) using melatonin implants.

This study was conducted on summer anoestrous buffalo heifers to monitor the efficacy of melatonin for induction of ovulation and ovarian cyclicity. During pre-treatment period of 24 days, the ovarian dynamics of five cycling and 10 summer anoestrous heifers was monitored on each alternate day using a transrectal ultrasound scanner. Thereafter, during treatment period, these 10 anoestrous heifers along with additional seven anoestrous heifers were randomly allocated into non-implanted (n = 5) and implanted (n = 12, one melatonin implant/50 kg, 18 mg melatonin/implant) group. Non-implanted heifers were monitored on each alternate day till the confirmation of second-ovulation in implanted heifers. Pre-treatment period revealed the presence of dominant follicles in anoestrous heifers which attained the diameter comparable with ovulatory follicles of cycling heifers but failed to ovulate and regressed. Between 6 and 36 days (15.3 +/- 2.9 days) post-treatment, all the implanted heifers (p < 0.05) exhibited ovulation of dominant follicles; however none of the non-implanted heifers ovulated during the corresponding period. The first-interovulatory period in implanted heifers ranged between 8 and 28 days (18.0 +/- 1.8 days). The implanted heifers with short (

Follicle and oocyte morphology in ewes after treatment with insulin in the late follicular phase.

Stress reduces fertility in ruminants. Various experimental models, such as insulin-induced hypoglycaemia, have been used to investigate the mechanisms involved, and have revealed abnormal LH profiles (both pulse and surge secretion). This disruption affects follicular function and it is proposed there may be negative consequences on subsequent oocyte morphology. Insulin (5iu/kg), administered to ewes in the late follicular phase, induced hypoglycemia for 10h, decreased estradiol concentrations for 8-12h and delayed the LH surge by 15h. Although the diameters of dominant follicles just before ovulation were not affected, granulosa cells had fewer pycnotic nuclei, less apoptosis and increased proliferation 16-17h after the LH surge. Nevertheless, we did not observe gross ultra-structural differences in nuclear, cytoplasmic or cumulus maturity between oocytes from insulin-treated and control animals. This suggests that reduced LH pulsatility and a delay in the LH surge may only produce very subtle changes in gross oocyte morphology, imperceptible by electron microscopy.

Noradrenergic control of GnRH release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study investigates the influence of alpha(1)-adrenoreceptors in GnRH release in vitro and determines whether oestradiol modulates alpha(1)-adrenoreceptor-GnRH interaction. Within 10 min after ewe sacrifice, saggital midline hypothalamic slices were dissected, placed in oxygenated Minimum Essential Media-alpha (MEM-alpha) at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without oestradiol (24 pg/ml). After 4-h equilibration, 10-min fractions were collected for 4 h interposed with a 10-min exposure at 60 min to specific alpha(1)-adrenoreceptor agonist (methoxamine) or antagonist (thymoxamine) at various doses (0.1-10 mm). The alpha(1)-adrenoreceptor agonist (10 mm) increased (p < 0.05) GnRH release at 90 min both in presence and absence of oestradiol. However, in presence of oestradiol, alpha(1)-adrenoreceptor agonist (10 mm)-induced GnRH release remained elevated (p < 0.05) for at least 60 min. The bioactivity of the released GnRH was studied using a hypothalamus-pituitary sequential double-chamber perifusion. Only after exposure of hypothalamic slices to alpha(1)-adrenoreceptor agonist (10 mm), did the hypothalamic eluate stimulate LH release from pituitary fragments (n = 9, 7.8 +/- 12.3-36.2 +/- 21.6 ng/ml) confirming that the alpha(1)-adrenoreceptor agonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is under stimulatory noradrenergic control and this is potentiated in the presence of oestradiol.

GABA control of GnRH release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study examines the involvement of GABA(A or B) receptors in gonadotrophin-releasing hormone (GnRH) release in vitro and determines whether oestradiol modulates gamma-aminobutyric acid (GABA)-GnRH interaction. Within 10 min after ewe killing, hypothalamic slices were dissected and placed in oxygenated Minimum Essential Media (MEM)-alpha at 4 degrees C; within 2 h, slices were singly perifused at 37 degrees C with oxygenated MEM-alpha (0.15 ml/min), with or without oestradiol (24 pg/ml). After 4 h equilibration, fractions were collected for 4 h interposed with a 10 min exposure to specific GABA(A or B) receptor ligands (0.1-10 mM). The GABA(A or B) agonists (muscimol or baclofen) did not greatly influence GnRH release. However, GnRH increased (p < 0.05) after exposure to 10 mM GABA(A or B) antagonists (bicuculline or CGP52432, respectively). The GABA(A) antagonist stimulated greater sustained GnRH release (p < 0.05) in the absence of oestradiol than in its presence. The bioactivity of the released GnRH was studied using a hypothalamus-pituitary sequential double-chamber perifusion. Only after exposure of hypothalamic slices to the GABA(A) antagonist, did the hypothalamic eluate stimulate luteinizing hormone release from pituitary fragments (p < 0.05) confirming that the GABA(A) antagonist stimulated release of biologically active GnRH. In summary, GnRH release from the hypothalamus is predominantly under GABA(A) receptor inhibitory control and this is attenuated in the presence of oestradiol.

Noradrenergic control of arginine vasopressin release from the ewe hypothalamus in vitro: sensitivity to oestradiol.

The present study aims at ascertaining the influence of alpha(1)-adrenoreceptors on arginine vasopressin (AVP) release in vitro and determine whether E(2) modulates the alpha(1)-adrenoreceptor and AVP interaction. Ten minutes after ewe killing, sagittal midline hypothalamic slices (from the anterior preoptic area to the mediobasal hypothalamus with the median eminence, 2 mm thick, 2 per sheep) were dissected, placed in oxygenated minimum essential media-alpha (MEM-alpha) at 4 degrees C and within 2 h were singly perifused at 37 degrees C with oxygenated MEM-alpha (pH 7.4; flow rate 0.15 ml/min), either with or without E(2) (24 pg/ml). After 4 h equilibration, 10 min fractions were collected for 4 h interposed with 10 min exposure at 60 min to a specific alpha(1)-adrenoreceptor agonist or antagonist at various doses (0.1-10 mm). At the end of all perifusions, slices responded to KCl (100 mm) with AVP efflux (p < 0.05). Release of AVP was enhanced (p < 0.05) by the alpha(1)-adrenoreceptor agonist (methoxamine 10 mm; no E(2), n = 7 perifusion chambers: from 14.3 +/- 2.7 to 20.9 +/- 3.9, with E(2), n = 10: from 10.7 +/- 1.2 to 18.4 +/- 3.4 pg/ml) or the antagonist (thymoxamine 10 mm; no E(2), n = 5: from 9.5 +/- 3.1 to 30.4 +/- 6.0, with E(2), n = 10: from 10.8 +/- 0.9 to 39.1 +/- 6.3 pg/ml). With the agonist, the response occurred only at 80 min (p < 0.05) both in the presence and absence of E(2). Whereas, after the antagonist, values were higher (p < 0.05) throughout the post-treatment period (80-170 min) without E(2), but declined by 150 min in the presence of E(2). Furthermore, the response to the alpha(1)-adrenoreceptor antagonist was greater (p < 0.05; 90-140 min) than the agonist only in the presence of E(2). In conclusion, these results reveal direct alpha(1)-adrenoreceptor-mediated control of the hypothalamic AVP neuronal system which is modulated by E(2).