Expression of MTNR1A, steroid (ERα, ERß, and PR) receptor gene transcripts, and the concentration of melatonin and steroid hormones in the ovarian follicles of buffalo.

High ambient temperature exhibits a retrograde effect on buffalo reproduction because of heat stress. Moreover, melatonin is known to regulate reproductive changes in seasonally reproductive animals by binding to high affinity, G protein-coupled receptors. The MTNR1A gene is a prime receptor, mediating the effect of melatonin at the neuroendocrine level to control seasonal reproduction. In sheep, the role of melatonin is well known; however, studies have not been conducted in buffalo to determine its effect during favorable and unfavorable breeding seasons. Therefore, the present study aimed to (1) determine the expression of MTNR1A, ERα, ERß, and PR gene transcripts in the ovarian follicles of buffalo during the summer and winter seasons and (2) analyze melatonin, 17ß-estradiol, and progesterone concentrations in the follicular fluid of buffalo during both seasons. Murrah buffalo ovaries were collected during both the summer (May-June) and winter (December-January) seasons. All visible ovarian follicles were allocated into one of three groups: (1) small (8-9.9 mm); (2) medium (10-11.9 mm); and (3) large (12-14 mm). Follicular fluid was aspirated from each group of follicles for hormone analyses. The granulosa cells were processed for RNA extraction. Furthermore, they were subjected to real-time quantitative PCR to analyze the expression (relative quantification) of MTNR1A, ERα, ERß, and PR in each follicular group. The expression of MTNR1A gene transcript decreased with the increasing size of the follicle and intrafollicular melatonin concentration. Expression of ERα and PR remained unaffected by the season and was similar (P > 0.05) in all groups. Expression of ERß was higher (P < 0.05) in summer than winter; nevertheless, small-sized follicles from the summer exhibited higher (P < 0.05) expressions than medium- and large-sized follicles. The overall intrafollicular melatonin concentration was positively correlated (P < 0.05) with 17ß-estradiol and progesterone concentrations. In conclusion, the decreased expression of MTNR1A and increased concentration of intrafollicular melatonin with the increasing size of the follicle indicates a probable role in folliculogenesis and ovulation in buffalo.

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.

Polymorphism of melatonin receptor (MTNR1A) gene and its association with seasonal reproduction in water buffalo (Bubalus bubalis).

The water buffalo have a seasonal pattern of reproduction with decreased sexual activity during the longer photoperiod. The present study was designed to identify a single nucleotide polymorphism (SNP) in the MTNR1 A gene and its association with seasonal reproduction and reproductive characteristics in Murrah buffalo cows. The 812 bp fragment encompassing exon II of the MTNR1 A gene was amplified from genomic DNA of 190 pluriparous Murrah buffalo cows. Amplified PCR products from 12 samples were subjected to custom sequencing of both ends (5' and 3' ends). A synonymous mutation was identified at position 72 in exon II of MTNR1 A gene. Digestion of PCR products with HpaI enzyme indicated there was one polymorphic site caused by the presence of nucleotide C at position 72 in place of T that resulted in three genotypes: T/T (812bp), C/T (812, 743 and 69 bp) and C/C (743 and 69 bp). There was an association (P < 0.05) between the MTNR1 A genotype and reproductive activity in Murrah buffalo cows such that buffalo cows with T/T genotype had less seasonal reproductive activity as compared with those with the C/C genotype. The period of greatest mating activity of buffalo cows with the C/C genotype was from November to December whereas buffalo cows with the T/T genotype mated mainly between May and July. Thus, the polymorphism of the MTNR1 A gene might be considered as a genetic marker to identify Murrah buffalo, which are able to reproduce during periods that are not a part of the typical breeding season for these buffalo.

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.

Impact of Buserelin Acetate or hCG Administration on the Day of First Artificial Insemination on Subsequent Luteal Profile and Conception Rate in Murrah Buffalo (Bubalus bubalis).

This study was designed to investigate the impact of buserelin acetate (BA) or human chorionic gonadotropin (hCG) administration on the day of first artificial insemination (AI) on subsequent luteal profile (diameter of corpus luteum (CL) and plasma progesterone) and conception rate in Murrah buffalo. The present experiment was carried out at two locations in 117 buffalo that were oestrus-synchronized using cloprostenol (500 µg) administered (i.m.) 11 days apart followed by AI during standing oestrus. Based on treatment (i.m.) at the time of AI, buffalo were randomly categorized (n = 39 in each group) into control (isotonic saline solution, 5 ml), dAI-BA (buserelin acetate, 20 µg) and dAI-hCG (hCG, 3000 IU) group. Out of these, 14 buffalo of each group were subjected to ovarian ultrasonography on the day of oestrus to monitor the preovulatory follicle and on days 5, 12, 16 and 21 post-ovulation to monitor CL diameter. On the day of each sonography, jugular vein blood samples were collected for the estimation of progesterone concentrations. All the buffalo (n = 117) were confirmed for pregnancy on day 40 post-ovulation. The conception rate was better (p < 0.05) in dAI-BA (51.3%) and dAI-hCG (66.7%) groups as compared to their control counterparts (30.8%). Furthermore, the buffalo of dAI-hCG group had improved (p < 0.05) luteal profile, whereas the buffalo of dAI-BA group failed (p > 0.05) to exhibit stimulatory impact of treatment on luteal profile when compared to control group. In brief, buserelin acetate or hCG treatment on the day of first AI leads to an increase in conception rate; however, an appreciable impact on post-ovulation luteal profile was observed only in hCG-treated Murrah buffalo.

Estrus induction and fertility response following different treatment protocols in Murrah buffaloes under field conditions.

AIM: The aim of this study was to evaluate the efficacy of three different treatment protocols for estrus induction and conception rate in postpartum anestrus buffaloes during breeding season under field conditions. MATERIALS AND METHODS: The 47 postpartum anestrus buffaloes of the 2nd to 6th parity were divided into three groups. Group 1 (n=16): Buffaloes received cosynch treatment, that is, buserelin acetate 10 µg on day 0 and 9, cloprostenol 500 µg on day 7 followed by fixed-time artificial insemination (FTAI) at the time of second buserelin acetate and 24 h later. Group 2 (n=15): Buffaloes received norgestomet ear implant subcutaneously for 9 days, estradiol benzoate 2 mg on the day of implant insertion (day 0), pregnant mare serum gonadotropin (PMSG) 400 IU and cloprostenol 500 µg on day 9 followed by AI at 48 and 72 h after implant removal. Group 3 (Cosynch-plus, n=16): Buffaloes received Cosynch protocol as per Group 1 except an additional injection of PMSG 400 IU (i.m.) was given 3 days before the start of protocol and FTAI done at the same time of Group 1. Pregnancy diagnosis was performed after 45 days of AI. RESULTS: The estrus induction response following the treatment was 81.3%, 100%, and 93.7% in Group 1, 2, and 3, respectively. The buffaloes of Group 1, 2, and 3 expressed intense (38.4%, 60% and 46.6%, respectively) and moderate estrus (46.1%, 26.6%, and 40%, respectively). The conception rates in Group 1, 2, and 3, at FTAI and overall including subsequent estrus were 37.5% and 62.5%, 53.3%, and 66.6%, 56.3%, and 75%, respectively. CONCLUSION: All the three treatment protocols can be effectively used for induction of estrus with acceptable conception rate in postpartum anestrus buffaloes during breeding season under field conditions. However, Cosynch-plus (similar to Cosynch protocol except addition of PMSG, 400 IU 3 days before the start of first buserelin acetate administration) protocol results comparatively better pregnancy rate.

Beta-adrenergic and opioidergic modulation of cortisol secretion in response to acute stress.

The modulatory action of beta-adrenergic and opioidergic pathways on the cortisol response to acute stressors was investigated using gonadectomized male miniature pigs. Three types of stressors, nose-snare (NS, for 2 min. each on four occasions at 30 min. intervals); high intensity cracker blasts (CB, two blasts at 3 min intervals) and ACTH (1 i.u./kg BW, i.v.) were utilized 80 min after start of blood sampling. For assessment of cortisol blood samples were withdrawn every 20 min for 200 min. In addition, animals received i.v. injections of either isoproterenol (5 microg/ kg) or propranolol (0.5 mg/kg) or naloxone (1 mg/kg) 15 or 30 min before the application of stressor. Stress of repeated NS application as well as ACTH treatment, resulted in immediate secretion of cortisol (p<0.001). Blast of crackers resulted in a transient increase in cortisol (p<0.05). Isoproterenol stimulated the basal cortisol secretion for about 20 min in unstressed pigs (p <0.01) but propranolol had no effects. Isoproterenol also reinforced (p<0.05) the effect of CB, but had no effect on the cortisol response to nose-snare. In contrast, response to NS was reduced (p=0.02) by propranolol. Neither isoproterenol nor propranolol altered the cortisol response to ACTH application. Pretreatment with naloxone significantly increased the cortisol response to NS (p<0.01) and to CB (p<0.01), but had no effects on ACTH-induced cortisol release. In conclusion, the beta-adrenergic involvement is evident in the cortisol response to acute stress of nose-snare. Furthermore, the results indicate that activation of endogenous opioid system during stress mitigates adrenal response.

Effect of transport on pulsatile and surge secretion of LH in ewes in the breeding season.

The aim of this study was to elucidate the mechanism(s) involved in stress-induced subfertility by examining the effect of 4 h transport on surge and pulsatile LH secretion in intact ewes and ovariectomized ewes treated with steroids to induce an artificial follicular phase (model ewes). Transport caused a greater delay in the onset of the LH surge in nine intact ewes than it did in ten ovariectomized ewes (intact: 41.0 +/- 0.9 h versus 48.3 +/- 0.8 h, P < 0.02; ovariectomized model: 40.8 +/- 0.6 h versus 42.6 +/- 0.5 h, P < 0.02). Disruption of the hypothalamus-pituitary endocrine balance in intact ewes may have reduced gonadotrophin stimulation of follicular oestradiol production which had an additional effect on the LH surge mechanism. In the ovariectomized model ewes, this effect was masked by the exogenous supply of oestradiol. However, in these model ewes, there was a greater suppression of maximum LH surge concentrations (intact controls: 29 +/- 4 ng ml-1 versus intact transported 22 +/- 5 ng ml-1, P < 0.02; ovariectomized model controls: 35 +/- 7 ng ml-1 versus model transported 15 +/- 2 ng ml-1, P < 0.02). Subsequent exposure to progesterone for 12 days resulted in the resumption of a normal LH profile in the next follicular phase, indicating that acute stress leads to a temporary endocrine lesion. In four intact ewes transported in the mid-follicular phase, there was a suppression of LH pulse amplitude (0.9 +/- 0.3 versus 0.3 +/- 0.02 ng ml-1, P < 0.05) but a statistically significant effect on pulse frequency was not observed (2.0 +/- 0.4 versus 1.7 +/- 0.6 pulses per 2 h). In conclusion, activation of the hypothalamus-pituitary-adrenal axis by transport in the follicular phase of intact ewes interrupts surge secretion of LH, possibly by interference with LH pulsatility and, hence, follicular oestradiol production. This disruption of gonadotrophin secretion will have a major impact on fertility.

Effect of transport on pituitary responsiveness to exogenous pulsatile GnRH and oestradiol-induced LH release in intact ewes.

This study examined the effect of transport on GnRH self-priming in vivo as well as the consequential effects on the oestradiol-induced LH surge. The follicular phases of ewes (eight per group) were synchronized with progestin sponges, and 50 micrograms oestradiol benzoate was injected 24 h (time zero) after sponge removal to improve precision in the timing of the LH surge. Beginning 8 h after oestradiol, saline or GnRH (500 ng, i.v.) was given at 2 h intervals with or without 8 h transport beginning 0.5 h before the first GnRH injection (late transport) and effects were compared with those observed during early transport, that is, starting 2.5 h before the first GnRH injection. In all ewes, GnRH alone induced a maximum LH response of 1.9 +/- 0.4 ng ml-1 after the first challenge. The response was enhanced (P < 0.01) after the second and third GnRH injections (7.4 +/- 1.4 ng ml-1 and 7.6 +/- 1.7 ng ml-1, respectively). This self-priming effect after the second GnRH was reduced by late transport (7.4 +/- 1.4 versus 4.2 +/- 0.8 ng ml-1; P < 0.05) but not early transport, that is, transport initiated closer to the time of GnRH administration had greater suppressive effects on LH secretion. Throughout transport, spontaneous LH pulse frequency was lower in treated than it was in control ewes (2.38 +/- 0.53 versus 4.50 +/- 0.53 pulses per 8 h; P < 0.01), with marked effects in the first 4 h of transport (1.0 +/- 0.19 versus 2.63 +/- 0.38 pulses per 4 h; P < 0.02). Spontaneous pulse amplitude also tended to decrease during transport (0.13 +/- 0.02 versus 0.20 +/- 0.03 ng ml-1; P = 0.07). When LH turnover was stimulated by exogenous GnRH, the onset of the LH surge was delayed (controls: 20.5 +/- 2.0 h versus GnRH alone: 25.3 +/- 1.5 h; P < 0.05) and the duration was reduced (8.5 +/- 0.9 versus 6.5 +/- 0.4 h; P < 0.05). Transport tended to delay the LH surge in saline-treated ewes (20.5 +/- 2.0 versus 22.9 +/- 1.9 h; P = 0.08), with a further delay imposed by late transport plus GnRH (27.5 +/- 1.6 h; P < 0.05) but not by early transport plus GnRH (27.8 +/- 2.5 versus 26.4 +/- 2.4 h; P > 0.05), that is, effects mediated by increasing LH turnover were only manifest if transport occurred near the LH surge, when there was insufficient time to replenish stores of releasable LH. In all transported ewes, plasma cortisol increased from 4.5 +/- 1.0 ng ml-1 to 29.2 +/- 5.5 ng ml-1 (P < 0.001) within 15 min of the start of transport and was significantly lower (P < 0.01) by 6.5 h. Plasma progesterone also increased from 0.30 +/- 0.04 to 0.38 +/- 0.04 ng ml-1 (P < 0.05). In conclusion, transport affected the oestradiol-induced LH surge by causing a 50% reduction in the self-priming effect of exogenous GnRH, but hypothalamic effects were also revealed by a two-fold decrease in spontaneous LH pulse frequency in saline-treated ewes.

Effect of adrenocorticotrophic hormone (ACTH1-24) on ovine pituitary gland responsiveness to exogenous pulsatile GnRH and oestradiol-induced LH release in vivo.

The present experiment was designed to determine if and how exogenous ACTH replicates the effects of stressors to delay the preovulatory LH surge in sheep. Twenty-four hours after oestrous synchronisation with prostaglandin in the breeding season, groups of 8-9 intact ewes were injected with 50 microg oestradiol benzoate (0 h) followed 8 h later by 3 injections of saline or GnRH (500 ng each, i.v.) at 2 h intervals (controls). Two further groups received an additional 'late' injection of ACTH (0.8 mg i.m.) 7.5 h after oestradiol, i.e., 0.5 h before the first saline or GnRH challenge. To examine if the duration of prior exposure to ACTH was important, another group of ewes was given ACTH 'early', i.e. 2.5 h before the first GnRH injection. The first GnRH injection produced a maximum LH response of 1.9+/-0.4 ng/ml which was significantly (p < 0.01) enhanced after the second and third GnRH challenge (7.1+/-1.5 ng/ml and 7.0+/-1.7 ng/ml, respectively; 'self-priming'). Late ACTH did not affect the LH response after the first GnRH challenge (1.9+/-0.4 vs. 1.8+/-0.3 ng/ml; p > 0.05) but decreased maximum LH concentrations after the second GnRH to 35% (7.1+/-1.5 vs. 4.6+/-1.1 ng/ml; p = 0.07) and to 40% after the third GnRH (7.0+/-1.7 vs. 4.0+/-0.8 ng/ml; p = 0.05). When ACTH was given early, 4.5 h before the second GnRH, there was no effect on this LH response suggesting that the effect decreases with time after ACTH administration. Concerning the oestradiol-induced LH surge, exogenous GnRH alone delayed the onset time (20.5+/-2.0 vs. 27.8+/-2.1 h; p > 0.05) and reduced the duration of the surge (8.5+/-0.9 vs. 6.7+/-0.6 h; p > 0.05). The onset of the LH surge was observed within 40 h after oestradiol on 29 out of 34 occasions in the saline +/- GnRH treated ewes compared to 11 out of 34 occasions (p < 0.05) when ACTH was also given, either late or early. In those ewes that did not have an LH surge by the end of sampling, plasma progesterone concentrations during the following oestrous cycle increased 2 days later suggesting a delay, not a complete blockade of the LH surge. In conclusion, we have revealed for the first time that ACTH reduces the GnRH self-priming effect in vivo and delays the LH surge, at least partially by direct effects at the pituitary gland.

Effect of adrenocorticotrophic hormone on gonadotrophin releasing hormone-induced luteinizing hormone secretion in vitro.

An in vitro perifusion study investigated the effect of different forms of adrenocorticotrophic hormone (ACTH) on gonadotrophin releasing hormone (GnRH)-induced luteinizing hormone (LH) secretion, particularly GnRH self-priming, and oestradiol sensitisation of the ovine pituitary. Fragments of pituitaries were obtained from mixed-breed adult nonpregnant female sheep (without corpora lutea, unless otherwise stated). The amount of LH released by different doses of GnRH (2.5 x 10(-10) M (n = 9 chambers), 1 x 10(-10) M (n = 9), or 5 x 10(-11) M (n = 6)) was evaluated by giving two GnRH pulses (5 min each) 2 h apart. In a duplicate set of chambers, ACTH1-24 (5 x 10(-7) M) was included in the perifusate 0.5 h before the first GnRH challenge. Potassium chloride (KCl; 100 mM) was administered 2 h after the second GnRH challenge to assess the viability of the tissue and the size of the releasable LH pool. Results were expressed as percentage of LH secretion. The influence of ACTH1-24 on oestradiol sensitisation was also examined using pituitaries obtained during the luteal phase. Pituitary tissues were perifused throughout with 1 x 10(-9) M or 6 x 10(-11) M oestradiol in the medium. The LH response to the second GnRH challenge (GnRH 2) was significantly greater (p < 0.01) than after the first (GnRH 1) at the highest dose of GnRH (2.5 x 10(-10) M; 2547 +/- 804 vs. 4547 +/- 1013%), but at the lower doses (1 x 10(-10) M or 5 x 10(-11) M), the self-priming effect of GnRH was not evident (3016 +/- 550 vs. 2932 +/- 490% and 841 +/- 205 vs. 711 +/- 87%). Treatment with ACTH1-24 (5 x 10(-7) M) did not affect tonic LH secretion nor the LH response to the first or second GnRH challenge at any of the GnRH doses tested. The LH released in response to KCl was also similar from control and ACTH1-24-treated tissue at all GnRH doses. Both lower doses of GnRH (1 x 10(-10) M or 5 x 10(-11) M) produced the self-priming effect when the pituitary tissue was sensitised with the higher dose of oestradiol (1 x 10(-9) M; 1711 +/- 239 vs. 5085 +/- 1307%, and 1502 +/- 376 vs. 2619 +/- 629%). In the presence of lower concentrations of oestradiol (6 x 10(-11) M), self-priming was observed only after the higher dose of GnRH (1 x 10(-10) M; 1293 +/- 214 vs. 2865 +/- 436%), not the lower dose (5 x 10(-11) M; 985 +/- 203 vs. 1271 +/- 436%). In spite of these differences, ACTH1-24 treatment did not affect LH secretion (neither basal nor potassium-induced). The effect of ACTH1-39 (1 x 10(-8) M or 5 x 10(-7) M; n = 6 chambers per combination) on GnRH-induced LH secretion was examined using the higher (2.5 x 10(-10) M) or lower dose of GnRH (1 x 10(-10) M), with or without oestradiol sensitisation (1 x 10(-9) M). At the lower dose (1 x 10(-8) M), ACTH1-39 influenced neither tonic nor GnRH-induced LH secretion. The LH released by KCl was also similar to the control and ACTH-treated tissue. In contrast, the higher dose of ACTH1-39 (5 x 10(-7) M) increased tonic LH secretion immediately after inclusion in the medium (104 +/- 3 vs. 161 +/- 20%), but suppressed the GnRH self-priming effect after 2.5 x 10(-10) M, i.e., the LH responses to GnRH 1 and 2 were similar (1786 +/- 294 vs. 1553 +/- 373%). However, the LH response to KCl was not significantly different (p > 0.05) between the control and ACTH-treated tissues (2333 +/- 286 vs. 2638 +/- 431%). When the effect of this higher dose of ACTH1-39 on oestradiol-priming was investigated, ACTH increased tonic LH secretion but suppressed the self-priming effect of GnRH (1 x 10(-10) M GnRH; 945 +/- 274 vs. 922 +/- 323%; p > 0.05), and decreased (p < 0.05) the LH released in response to KCl compared to the controls (1803 +/- 409 vs. 4302 +/- 1017%). In summary, in vitro, ACTH1-24 did not affect either tonic LH secretion, the GnRH self-priming effect, or oestradiol sensitisation. The entire ACTH1-39 increased tonic LH secretion, but reduced GnRH self-priming and oestradiol sensitisation. (ABSTRACT TRUNCATED)