Plasma extracellular vesicle miRNAs as potential biomarkers of superstimulatory response in cattle.

The ability to predict superstimulatory response would be a beneficial tool in assisted reproduction. Using small RNAseq technology, we profiled extracellular vesicle microRNA (EV-miRNA) abundance in the blood plasma of heifers exhibiting variable responses to superstimulation. Estrous synchronized crossbred beef heifers (n = 25) were superstimulated and blood samples were collected from each heifer on Day 7 of consecutive unstimulated (U) and superstimulated (S) cycles. A subset of high (H) and low (L) responders was selected depending on their response to superstimulation and EV-miRNA profiles were analysed at both time-points in each heifer. Approximately 200 known miRNAs were detected in each sample with 144 commonly detected in all samples. A total of 12 and 14 miRNAs were dysregulated in UH vs. UL and in SH vs. SL heifers, respectively. Interestingly, miR-206 and miR-6517 exhibited the same differential expression pattern in H compared to L heifers both before and after superstimulation. Pathway analysis indicated that circadian rhythm and signaling pathways were among the top pathways enriched with genes targeted by dysregulated miRNAs in H vs. L responding heifers. In conclusion, heifers with divergent ovarian responses exhibited differential expression of plasma EV-miRNAs which may be used as a potential biomarker to predict superstimulation response.

Onset of oestrus and periovulatory events in sheep exposed to 5 and 14 days of CIDR treatment with and without eCG.

The present study supports that 5-day short-term CIDR treatments without administration of eCG are equally effective for inducing oestrus behaviour, preovulatory LH discharge and ovulation in sheep than classical protocols based on 14-day treatments plus eCG at CIDR withdrawal. However, the implementation of a 5-day protocol without eCG for fixed-time artificial insemination would be adapted to a later timing of ovulation (p < .05).

RNA sequencing and differential expression reveals the effects of serial oestrus synchronisation on ovarian genes in dairy goats.

A total of 24 female Xinong Saanen dairy goats were used to examine differentially expressed genes (DEGs) in the ovaries of goats treated once or three times for oestrus synchronisation (ES). The goats were randomly divided into two groups: one group received three ES treatments at fortnightly intervals (repeated or triple ES group), whereas the other was only treated once on the same day as the third ES treatment for the triple group (control group) during the breeding season. Ovaries of three goats in oestrus from each group were collected for morphological examination and transcriptome sequencing, while the rest of the goats were artificially inseminated twice. Litter size and fecundity rate tended (P=0.06) to be lower in the triple ES group. A total of 319 DEGs were identified, including carbohydrate sulphotransferase 8 (CHST8), corticosteroid-binding globulin (CBG), oestradiol 17-ß-dehydrogenase 1 (DHB1), oestrogen receptor 1 (ESR1), progestin and adipoQ receptor family member 4 (PAQR4), PAQR9, prostacyclin synthase (PTGIS), contactin-associated protein (CNTNAP4), matrix metalloproteinase-2 (MMP-2), regulator of G-protein signalling 9-2 (RGS9-2) and sperm surface protein Sp17 (Sp17); these were the most promising novel candidate genes for reproductive performances in goats. Our study indicates that triple ES could cause DNA damage and alter gene expression in goat ovaries, potentially affecting ovary function, neural regulation and hormone secretion.

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles.

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17ß followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.

The expression of ERα, OTR, cPLA(2), COX-2, and PPARγ in the cervix of the ewe during the estrous cycle.

The ovine cervix relaxes at estrus allowing easier entry of spermatozoa into the uterus. The mechanism responsible for this relaxation is not fully elucidated and we hypothesized that cervical relaxation at estrus is induced by ovarian and pituitary hormones stimulating the local production of prostaglandin E(2) via a biosynthetic pathway involving a number of mediators including oxytocin, phospholipase A(2) (cPLA(2)), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor gamma (PPARγ). The aim of this study was to investigate the cervical expression of estradiol receptor alpha (ERα), oxytocin receptor (OTR), cPLA(2), COX-2, and PPARγ at three stages of the estrous cycle (the luteal phase and two times during the follicular phase, just before and just after the LH surge). An experiment was conducted during the breeding season, in 25 ewes to test this hypothesis. Samples of cervical tissue were collected from groups of ewes at three stages of the estrous cycle: the luteal (N = 8), "pre-LH surge" (N = 8), and "post-LH surge" (N = 9) stages. Cervical tissue from uterine, mid, and vaginal regions of the cervix were analyzed by Western immunoblot analysis for ERα, OTR, cPLA(2,) COX-2, and PPARγ. The results showed that the levels of all five proteins were lowest during the luteal phase of the estrous cycle in all regions of the cervix. The levels of all except cPLA(2), increased significantly during the "pre-LH surge" stage. The levels of cPLA(2) and ERα increased in the "post-LH surge" stage and those for OTR and PPARγ were unchanged and those for COX-2 were lower. These data show that the cervical levels of all five of the intermediates in the synthesis of prostaglandin E(2) that were examined in this study were higher in the "pre-" and "post-LH surge" stages compared with the luteal phase of the estrous cycle and these findings are consistent with our hypothesis.

Placental development during early pregnancy in sheep: effects of embryo origin on fetal and placental growth and global methylation.

The origin of embryos including those created through assisted reproductive technologies might have profound effects on placental and fetal development, possibly leading to compromised pregnancies associated with poor placental development. To determine the effects of embryo origin on fetal size, and maternal and fetal placental cellular proliferation and global methylation, pregnancies were achieved through natural mating (NAT), or transfer of embryos generated through in vivo (NAT-ET), IVF, or in vitro activation (IVA). On Day 22 of pregnancy, fetuses were measured and placental tissues were collected to immunologically detect Ki67 (a marker of proliferating cells) and 5-methyl cytosine followed by image analysis, and determine mRNA expression for three DNA methyltransferases. Fetal length and labeling index (proportion of proliferating cells) in maternal caruncles (maternal placenta) and fetal membranes (fetal placenta) were less (P < 0.001) in NAT-ET, IVF, and IVA than in NAT. In fetal membranes, expression of 5-methyl cytosine was greater (P < 0.02) in IVF and IVA than in NAT. In maternal caruncles, mRNA expression for DNMT1 was greater (P < 0.01) in IVA compared with the other groups, but DNMT3A expression was less (P < 0.04) in NAT-ET and IVA than in NAT. In fetal membranes, expression of mRNA for DNMT3A was greater (P < 0.01) in IVA compared with the other groups, and was similar in NAT, NAT-ET, and IVF groups. Thus, embryo origin might have specific effects on growth and function of ovine uteroplacental and fetal tissues through regulation of tissue growth, DNA methylation, and likely other mechanisms. These data provide a foundation for determining expression of specific factors regulating placental and fetal tissue growth and function in normal and compromised pregnancies, including those achieved with assisted reproductive technologies.

Differences in uterine immunoexpression of PR, ERα and OTR when comparing prostaglandin- to progestagen-based protocols for ovine estrus synchronization.

The aim of this work was to compare PR, ERα and OTR uterine expression between days 9 and 21 of pregnancy in ewes whose estrus had been synchronized with two different protocols. Sixty-four adult Manchega ewes were synchronized with either conventional progestagens (P) or prostaglandin analogues (PG), and mated. Uterine samples were obtained from pregnant animals (group P, n=24; group PG, n=25) on days 9 post coitus (pc), 13pc, 15pc, 17pc and 21pc. Immunohistochemical detection of progesterone receptor (PR), estrogen receptor-α (ERα) and oxytocin receptor (OTR) was assessed in different uterine cell compartments including luminal and glandular epithelium, stroma and myometrium. Interaction day × treatment was obtained when assessing PR expression in the caruncular stroma (P=0.027) and myometrium (P=0.000), as well as for ERα in the superficial stroma (P=0.05). Significant "day post coitus" effect was found regarding to PR (P<0.01, with the exception of the superficial stroma, deep stroma and myometrium), ERα (P<0.01), and OTR (P<0.05, except in the deep compartments). No significant "treatment" effect was found for PR, ERα or OTR protein immunoexpression. This study supports the implication of PR, ERα and OTR within days 9-21 of the ovine pregnancy. Moreover, different expression pattern of PR and ERα proteins has been found between treatments in various compartments studied. Collectively, these results indicate that PR, ERα and OTR expression during early pregnancy is similar between ewes treated with either progestagens or prostaglandin analogues-based protocols for estrus synchronization.

Melanin concentrating hormone (MCH) in the cerebrospinal fluid of ewes during spontaneous oestrous cycles and ram effect induced follicular phases.

The melanin-concentrating hormone (MCH) is a neuropeptide synthesized by neurons of the lateral hypothalamus and incerto-hypothalamic area that project throughout the central nervous system. The aims of the present report were: (1) to determine if MCH levels in cerebrospinal fluid (CSF) of ewes vary between the mid-luteal and the oestrous phase of spontaneous oestrous cycles; and (2) to study if MCH levels in CSF of ewes vary acutely during the follicular phase induced with the ram effect in anoestrous ewes. In the first experiment, CSF was collected from 8 adult ewes during spontaneous oestrous and during the mid-luteal phase (8-10 days after natural oestrus). In the second experiment, performed during the mid non-breeding season, a follicular phase was induced with the ram effect. After isolating a group of 16 ewes from rams, CSF was obtained from 5 of such ewes (control group). Three rams were joined with the ewes, and samples were obtained 12h (n=6) and 24h (n=5) later. In Experiment 1, there were no differences in MCH concentrations in CSF measured during the mid-luteal phase and spontaneous oestrus (0.14 ± 0.04 vs. 0.16 ± 0.05 ng/mL respectively). In Experiment 2, MCH concentrations tended to increase 12h after rams introduction (0.15 ± 0.08 vs. 0.35 ± 0.21 ng/mL, P=0.08), and increased significantly 24h after rams introduction (0.37 ± 0.15 ng/mL, P=0.02). We concluded that MCH concentration measured in the CSF from ewes increased markedly during the response to the ram effect but not during the natural oestrous cycle of ewes.

Characterisation of endometrial gene expression and metabolic parameters in beef heifers yielding viable or non-viable embryos on Day 7 after insemination.

The aim of the present study was to compare the hormonal and metabolic characteristics and endometrial gene expression profiles in beef heifers yielding either a viable or degenerate embryo on Day 7 after insemination as a means to explain differences in embryo survival. Oestrus was synchronised in cross-bred beef heifers (n = 145) using a controlled internal drug release (CIDR)-prostaglandin protocol. Heifers (n = 102) detected in standing oestrus (within 24-48 h after CIDR removal) were inseminated 12-18 h after detection of oestrus (Day 0) with frozen-thawed semen from a single ejaculate of a bull with proven fertility. Blood samples were collected from Day 4 to Day 7 after oestrus to measure progesterone (on Days 4, 5 and 7), insulin and insulin-like growth factor (IGF)-I (on Days 4 and 6) and urea (on Day 7) concentrations. All animals were killed on Day 7. Uterine pH was determined at the time of death. Animals from which an embryo was recovered were classified as either having a viable embryo (morula/blastocyst stage; n = 32) or a retarded embryo (arrested at the two- to 16-cell stage; n = 19). In addition, 14 single-celled unfertilised oocytes were recovered, giving an overall recovery rate of 64%. There was no significant difference in the blood parameters determined or uterine pH at the time of death between heifers with either a viable or retarded embryo. The relative abundance of nine transcripts (i.e. MOGAT1, PFKB2, LYZ2, SVS8, UHRF1, PTGES, AGPAT4, DGKA and HGPD) of 53 tested in the endometrial tissue differed between heifers with a viable or retarded embryo. Both LYZ2 and UHRF1 are associated with regulation of the immune system; PFKFB2 is a mediator in glycolysis; MOGAT, AGPAT4 and DGKA belong to the triglyceride synthesis pathway; and PTGES and HGPD belong to the prostaglandin pathway. Both these metabolic pathways are important for early embryonic development. In conclusion, retarded embryo development in the present study was not related to serum progesterone, IGF-I, insulin or urea concentrations, nor to uterine pH at the time of death. However, altered expression of genes involved in the prostaglandin and triglyceride pathways, as well as two genes that are closely associated with the regulation of immunity, in the endometrium may indicate a uterine component in the retardation of embryo development in these beef heifers.

In vivo effect of growth hormone on the expression of connexin-43 in bovine ovarian follicles.

This study assessed the in vivo effects of recombinant growth hormone (rGH) administration on the expression of connexin-43 (Cx43) in bovine ovarian follicles. Two independent experiments were carried out using either estrous unsynchronized or synchronized multiparous Aberdeen Angus cows. rGH-treated animals were inoculated with a single dose of hormone (500 mg, intramuscular) while control animals were inoculated with hormone diluent. Five and 14 days after treatment (Experiments 1 and 2, respectively), ovarian Cx43 and apoptosis expression were assessed using immunohistochemistry. In both experiments primary, secondary, and tertiary follicles from rGH-treated and control groups distinctly expressed Cx43 protein. Primordial and atretic follicles were Cx43-negative. Interestingly, the number of Cx43 dots per granulosa cell did not show significant variation at different folliculogenesis stages neither in the rGH-treated nor in the control group. In unsynchronized animals, Cx43-positive follicles per total number of follicles ratio showed an interaction between stage of folliculogenesis and treatment due to significant differences between treatment groups in the early secondary follicle stage. In synchronized animals, there were significant differences between treatment groups and folliculogenesis stage. In both experiments, atretic follicles showed apoptosis-related DNA-fragmentation as determined by terminal uridin nick end labeling (TUNEL) assay. Tertiary follicles presented moderate TUNEL staining. Our results show significant increment in the number of ovarian follicles expressing the gap junction subunit Cx43 after in vivo rGH treatment. Therefore, we conclude that growth hormone can modulate in vivo gap junction assembly at early stages of folliculogenesis.

Proteinase-activated receptors in ovine cervical function.

In sheep, inflammation not only functions in cervical dilation at parturition, but also plays an important part in the non-pregnant ewe cervix, as demonstrated by the high level of expression of interleukin (IL)-8 at oestrus. Ewes artificially induced to ovulate have significantly lower levels of IL-8 gene expression at oestrus compared with natural oestrus, indicating an inhibition of inflammation and function, offering an explanation for the low rates of conception in vaginally inseminated synchronised ewes. To identify potential pro-inflammatory agents to combat the anti-inflammatory effects of hormonal synchronisation of oestrus, we have investigated the role of proteinase-activated receptor (PAR)-1 and PAR-2. To localise and measure the level of expression of these receptors, ovine-specific probes were derived for PAR-1 and PAR-2 and used for quantitative in situ hybridisation in the ovine cervix. Both PAR-1 and PAR-2 were expressed in the luminal epithelium of the cervix throughout the oestrous cycle, with expression being highest at oestrus. The gene expression of PAR-2 at oestrus was approximately 30% higher than that of PAR-1. Artificial synchronisation of oestrus by either an intravaginal progesterone sponge or prostaglandin F2+/- injections did not inhibit PAR-1 or PAR-2 expression at oestrus; rather, in the case of PAR-2, progesterone synchronisation increased it. Both synchronising procedures increased the expression of PAR-1 and PAR-2 during the luteal phase of the cycle. Therefore, agonists of PAR-1 and PAR-2 may be potentially useful pro-inflammatory agents countering the inhibition of inflammation by hormonal synchronisation.

Luteal stage dependence of pituitary response to gonadotrophin-releasing hormone in cyclic dairy ewes subjected to synchronisation of ovulation.

Possible hormonal aberrations precluding conception or maintenance of pregnancy in dairy ewes subjected to ovulation synchronisation were investigated in this study. The pituitary response to exogenous gonadotrophin-releasing hormone (GnRH) was tested at different luteal stages in 36 ewes. Oestruses were synchronised by using progestagen-impregnated sponges and the animals were randomly allotted into one of three treatment groups (A, B and C; n = 12 for each). Treatments commenced on Days 4, 9 and 14 of the new cycle (oestrus was defined as Day 0). Ewes were given two GnRH injections, 5 days before and 36 h after a prostaglandin F2+/- (PGF2+/-) injection, and the animals were inseminated 12-14 h after the second GnRH injection (modified OVSYNCH). For luteinising hormone (LH) determination blood samples were withdrawn from six ewes of each group at the time of GnRH administration, and 30, 90, 180, 270 and 360 min later. Progesterone was assayed in samples taken every other day starting from oestrus and for 17 days after the second GnRH injection, and in an additional sample collected on the day of insemination. After the first GnRH injection, the LH concentration was higher in Group C than in Groups B and A (mean +/- s.d.: 64.8 +/- 10.0 ng mL(-1), 41.3 +/- 3.7 ng mL(-1) and 24.6 +/- 9.0 ng mL(-1), respectively; P < 0.05), whereas after the second GnRH injection a uniform LH release was found in all groups. PGF2+/- caused a significant decrease in progesterone (P4) concentration in all groups; however, at artificial insemination ewes that conceived had significantly lower P4 concentration in comparison with those that failed to conceive. As early as Day 5, pregnant animals had higher P4 concentrations than non-pregnant animals. Overall, 21 animals conceived (seven, nine and five ewes from Groups A, B and C, respectively). These results indicate that the proposed protocol is equally effective in inducing a preovulatory LH surge at any stage of the luteal phase, and that elevated P4 concentration along with a delayed P4 increase should be considered as a causative factor for inability to conceive.

Induction of estrus in non-lactating dairy goats with different estrous synchrony protocols.

The objective of this study was to evaluate two protocols of estrous synchronization in non-lactating Toggenburg goats. Nineteen goats were allocated, according to body condition score and weight, into two groups (A and B) and evaluated utilizing two treatments (T1 and T2). Animals in the T1 and T2 groups received an intravaginal sponge (day 0) containing 60 mg medroxyprogesterone acetate for 6 and 9 days, respectively, plus 200 IU equine chorionic gonadotrophin (eCG) and 22.5 microg cloprostenol 24 h before sponge removal. Females were bred only at the second estrus and received 22.5 microg cloprostenol 7 days later to prevent pregnancy. Percentages of animals in estrus did not differ (P > 0.05) between T1 (89.5%) and T2 (84.2%). From 33 females in estrus (T1 + T2), 28 (84.8%), 2 (6.1%), and 3 (9.1%) were identified in estrus at 06:00, 12:00 and 18:00 h, respectively. Additionally, 6 (18.2%), 0 (0.0%) and 27 (81.8%) were no longer detected to be on estrus at 06:00, 12:00 and 18:00 h, respectively. Interval from sponge removal and the onset of estrus (IE) did not differ (P > 0.05) between T1 (46.1 +/- 15.0 h) and T2 (53.6 +/- 16.1 h). Duration of estrus did not differ (P > 0.05) between T1 (30.0 +/- 12.0 h) and T2 (27.2 +/- 11.2 h). Both protocols were effective in inducing estrus in non-lactating goats. The onset and end of the estrus relative to hour of the day should be considered in estrous detection, natural breeding, and artificial insemination in goats.

Regulation of gonadotropin releasing hormone release by neuropeptide Y at the median eminence during the preovulatory period in ewes.

The median eminence (ME) of the hypothalamus is known to be an important brain site where hypophysiotropic release might be regulated by excitatory and inhibitory signals impinging on their neuronal terminals. Since a role for neuropeptide Y (NPY) on preovulatory luteinizing hormone (LH) release has been suggested, we hypothesized that NPY might act at the ME to control preovulatory gonadotropin-releasing hormone (GnRH) release and thus the onset of the preovulatory surge of LH. To examine this possibility, we used the ewe as an animal model to determine: (a) immunocytochemical distribution of GnRH and NPY in the ewe ME; (b) changes in in vivo release of NPY and GnRH using ME push-pull cannula (PPC) perfusate samples, as well as in plasma LH, during the luteal, follicular and preovulatory phases of a synchronized estrous cycle, and (c) effects of ME perfusion of NPY or a Y1-NPY antagonist, or an NPY antiserum on in vivo release of ME-GnRH and plasma LH during a synchronized follicular phase. Immunolocalization reveals a dense plexus of beaded GnRH-containing neurites in the arcuate nucleus and in its vicinity, the pituitary stalk and the palisade. In contrast, a dense plexus of NPY-containing neurites occurs in the internal layer, with occasional fibers found in the intermediate and lateral external zone of the ME. In the area between the lateral internal and lateral external layers, both NPY and GnRH-containing processes were found, thus providing opportunities for synaptic and/or paracrine interactions between NPY- and GnRH-containing neurons. Hormonal analysis indicated that a synchronized preovulatory surge of LH is elicited within a 2-hour window by the sequential implantation and removal of silastic-encased estradiol (E2) or progesterone (P4) implants. In this paradigm, there was a parallel increase in ME release of both NPY and GnRH preceding the synchronized LH surge. The onset of this synchronized LH surge was advanced by ME perfusion of exogenous NPY and was both delayed and blunted by ME perfusion with the NPY antagonist (both were perfused through the PPC probe for 2 h, starting 2-3 h before the expected onset of the LH surge). In addition, NPY perfusion in the ME increases, while perfusion of the Y1-NPY antagonist or of the NPY antiserum decreases ME-PPC GnRH content and plasma levels of LH in early follicular ewes. Finally, perfusion of NPY antiserum during an ongoing LH surge disrupted LH release. These results suggest that interactions between NPY and GnRH neurons are important in controlling the timing, magnitude and maintenance of the preovulatory LH surge.

Hormonal characterization of the reproductive cycle and pregnancy in the female Mohor gazelle (Gazella dama mhorr).

The oestrous cycles of seven captive Mohor Gazelles (Gazella dama mhorr) were investigated. Hormone profiles obtained from faecal samples collected each day from cyclic females indicated that the mean duration of the oestrous cycle was 18.62 +/- 0.26 days (range 16-22 days; n = 37 oestrous cycles). No inter-individual differences in the concentration of faecal progestagen metabolites excreted were observed, but mean faecal oestrogen excretion during both the luteal and inter-luteal phases of the oestrous cycle varied among females (P < 0.001 and P = 0.070, respectively). Oestrous cycles were synchronized using controlled internal drug release (CIDR) devices, before natural mating with an intact male. Concentrations of faecal progestagen metabolites remained approximately constant for the first 10 weeks of gestation (mean +/- SEM = 4048 +/- 407 ng g(-1) faeces), before increasing to a mean of 23 556 +/- 1176 ng g(-1) faeces. Two of seven female gazelles conceived immediately after removal of the CIDR device, a similar proportion to that conceived at the postpartum oestrus under natural conditions. Life history data for these individuals indicated that the mean time to conception in female gazelles is positively correlated with peak values in the ratio of excreted oestrogen : progestagen during the inter-luteal period of their oestrous cycles (R(2) = 0.58; P < 0.05). This finding indicates that interactions between steroid production and metabolism may influence the likelihood of conception occurring in this species.

Regulation of follicle waves to maximize fertility in cattle.

Cattle have recurrent follicular waves every 7-10 days in most physiological situations; an FSH increase is associated with emergence of the wave and LH pulse frequency determines the fate of the dominant follicle. To control oestrus with hormones it is necessary to ensure that either induced corpus luteum regression or the termination of a progestogen treatment coincides with the selection of the dominant follicle during the wave, to give a precise onset of oestrus and high fertility. The exogenous administration of progesterone or progestagen blocks the normal turnover of the dominant follicle once the corpus luteum regresses. Thus, the effects of duration of dominance of the preovulatory follicle on onset of oestrus and fertility were examined. The variation in onset of oestrus was reduced but occurred 5-9 h later after 4 versus 8 days of dominance; pregnancy rate was also affected with dominance periods of 2-4, 4-8 and > 10 days resulting in 0, 10-15% or 20-50% reduction in pregnancy rates, respectively. The necessity for short duration of dominance of the preovulatory follicle means that to ensure high fertility the follicular wave needs to be regulated when using hormones to control oestrus. Two approaches were examined, namely the use of GnRH or oestradiol at time of progesterone intravaginal releasing device insertion. The effect of 250 micrograms of synthetic GnRH on the fate of an existing follicle wave was to ovulate the dominant follicle (20/20 cows) and a new wave emerged 1.6 +/- 0.3 days later; however, there was no effect of GnRH on the wave if administered before dominant follicle selection. The effect of oestradiol concentrations on suppression of FSH in ovariectomized heifers showed that increasing oestradiol to 10-15 pg ml-1 caused a 37 +/- 6.9% decrease in FSH for 24 h, with a subsequent increase to pretreatment values by 57 +/- 13 h. In cyclic heifers, increasing oestradiol to > 10 pg ml-1 in conjunction with progesterone treatment at emergence of the first wave of the cycle affected the current follicle wave by either preventing dominant follicle selection or decreasing diameter of the dominant follicle, without consistently affecting the interval to new wave emergence. Increase of oestradiol after dominance, however, delayed new wave emergence by 2-5 days. A better understanding of the hormonal control of follicle waves will lead to development of improved hormonal regimens to control oestrus sufficiently to give high pregnancy rates to a single AI without recourse to detection of oestrus.

Gonadotrophin storage patterns in the ewe during the oestrous cycle or after long-term treatment with a GnRH agonist.

The storage pattern of gonadotrophins in the ewe pituitary was investigated during the oestrous cycle and after desensitization to GnRH using long-term treatment with a GnRH agonist, buserelin. Oestrous cycles in ewes were synchronized with progestagen sponges. Animals were allocated to two experiments. In the first, ewes were killed 36 h (before the preovulatory surge, n = 4), 48 h (end of the preovulatory surge, n = 5), 72 h (post-ovulation, n = 4) and 240 h (luteal phase, n = 3) after sponge removal. In the second experiment, another progestagen sponge was inserted in ewes 84 h after removal of the first sponge. Four ewes were infused continuously with buserelin (50 micrograms/day) for 15 days before killing. A further four ewes received no buserelin (controls). Pituitaries were collected and processed for immunocytochemistry to detect monohormonal (LH or FSH) and multihormonal (LH/FSH) cells. The percentages of LH or FSH immunoreactive cells in the pituitary were lower at the end of the preovulatory surge (7.4 +/- 0.3% and 1.2 +/- 0.3% respectively) compared with the other stages (11.4 +/- 0.5% and 5.4 +/- 0.7% respectively). Analysis of dual immunostaining showed the existence of monohormonal cells for LH and multihormonal cells (LH/FSH). No monohormonal cell for FSH was detected except at the end of the preovulatory surge when a few monohormonal FSH cells appeared (0.1 +/- 0.01% of pituitary cells). The percentage of monohormonal LH cells in the pituitary gland was similar in all studied stages of the oestrous cycle, whereas the percentage of multihormonal cells was lower at the end of the surge. In agonist-treated ewes, the percentages of LH or FSH immunoreactive cells (5.3 +/- 0.5% and 1.5 +/- 0.8% respectively) were decreased compared with controls (9.4 +/- 1% and 7.5 +/- 1.1% respectively). Analysis of the double immunostaining revealed a few monohormonal FSH cells (0.2 +/- 0.01% of pituitary cells) in agonist-treated ewes but not in controls. The percentage of monohormonal LH cells in the pituitary gland increased from 1.9 +/- 0.2% in controls to 3.8 +/- 0.3% in agonist-treated ewes, whereas multihormonal cells dropped from 7.5 +/- 1.1% to 1.3 +/- 0.7%. Our data suggest, therefore, that multihormonal cells contribute to gonadotrophin secretion, either during the preovulatory surge of the oestrous cycle or during the 'flare-up' effect initially induced by a GnRH agonist. Moreover, the appearance of monohormonal FSH cells in some conditions reflects a differential regulation of LH and FSH.

Natural and induced ovarian synchrony in golden lion tamarins (Leontopithecus rosalia).

Ovarian cycle synchrony was assessed in spontaneously cycling female golden lion tamarins by monitoring longitudinal (16 mo) urinary steroid metabolite (estrone conjugates; pregnanediol-3 alpha-glucuronide, PdG) excretion in four pairs (n = 8) of females isolated from males. The overall mean ovarian cycle duration was 18.5 +/- 0.3 days (n = 136 cycles; mean range, 15.7-21.0 days), and there was no evidence of reproductive seasonality. Laparoscopic ovarian examinations confirmed that cyclic fluctuations in urinary steroid metabolite excretion were temporally associated with the formation and demise of corpora lutea. Evaluation of ovarian synchronization tested the null hypothesis that urinary hormone cycles were expressed randomly relative to those of cagemates or other females housed in separate cages but within close proximity. Natural ovarian synchrony (expressed as the mean difference in ovarian cycle onset) between cagemates (4.1 +/- 0.4 days) and among noncagemates (4.2 +/- 0.2 days) did not differ (p > 0.05) from a random ovarian cycle distribution. Two trials also were conducted to evaluate the efficacy of the prostaglandin (PG) F2 alpha analogue, cloprostenol, for artificially synchronizing ovarian cycles. Induced ovarian synchrony was not achieved with a single 0.8-microgram i.m. injection of cloprostenol. However, doubling the cloprostenol dose (1.6 micrograms) caused a rapid decrease in mean urinary PdG (p < 0.05) within 2 days, and synchronous ovulation was demonstrated by an increase (p < 0.01) in mean urinary PdG 10 days after cloprostenol administration. In summary, females housed in pairs, in the absence of males, exhibit spontaneous, year-round ovarian cycles with no evidence of among-female ovarian synchrony. Results also suggest that this New World primate has a reduced sensitivity to cloprostenol (compared to common marmosets) but that a single, midcycle cloprostenol injection of 1.6 micrograms effectively induces luteolysis and synchronous ovulation.