Plasma metabolite changes in anestrous dairy cows with negative energy balance identified using 1H NMR technology / [Alterações do metabólito plasmático em vacas leiteiras com balanço energético negativo identificado usando a tecnologia 1H NMR]

The objective of the present study was to investigate the different plasma metabolites between anestrus and estrus postpartum dairy cows and to provide a theoretical basis for prevention of anestrus in dairy farm cows. In the experiment, one hundred and sixty-seven Holstein dairy cows were selected with similar age and parity. According to the concentration of ß-hydroxybutyric acid, non-esterified fatty acids and glucose in plasma during 14 to 21 days in milk, all dairy cows were determined as having a status of energy balance. According to the results of clinical symptom, rectal and B ultrasound examination at 60 to 90 days postpartum, these cows were divided into twenty estrus and twenty-four anestrus group, other dairy cows were removed. 1H nuclear magnetic resonance technology was utilized to detect the plasma metabolites changes and screen different plasma metabolites between anestrus and estrus cows. Ten different metabolites including alanine, glutamic acid, asparagine, creatine, choline, phosphocholine, glycerophosphocholine, low-density lipoprotein, and very-low-density lipoprotein were significantly decreased in anestrous cows compared with estrous cows. Metabolic pathway analyses indicated that differential metabolites were primarily involved in amino acid and glycerophospholipid metabolism. These metabolites and their enrichment pathways indicate that reduced steroid hormone synthesis precursors result in lower levels of estradiol and progesterone and cause anestrus in negative energy balance. These data provide a better understanding of the changes that may affect estrus of postpartum dairy cows at NEB status and lay the ground for further research.(AU)

O objetivo do presente estudo foi investigar os diferentes metabolitos do plasma entre o cio e o cio pós-parto de vacas leiteiras e fornecer uma base teórica para a prevenção do cio de vacas em fazendas de leite. No experimento, foram selecionadas 127 vacas leiteiras Holstein com idade e paridade similares. De acordo com a concentração de ß- ácido hidroxibutírico, ácidos graxos não esterificados e glicose no plasma entre 14 e 21 dias no leite, todas as vacas leiteiras foram determinadas em estado de equilíbrio energético. De acordo com os resultados dos sintomas clínicos, do exame de ultra-som retal e B aos 60 a 90 dias pós-parto, estas vacas foram divididas em vinte cios e vinte e quatro grupos de cio, outras vacas leiteiras foram removidas. A tecnologia de ressonância magnética nuclear 1H foi utilizada para detectar as alterações dos metabólitos plasmáticos e para triar diferentes metabólitos plasmáticos entre as vacas do cio e do cio. Dez diferentes metabólitos incluindo alanina, ácido glutâmico, asparagina, creatina, colina, fosfocholina, glicerofosfocolina, lipoproteína de baixa densidade e lipoproteína de muito baixa densidade foram significativamente diminuídos nas vacas antróficas em comparação com as vacas estro. As análises da via metabólica indicaram que os metabólitos diferenciais estavam principalmente envolvidos no metabolismo de aminoácidos e glicerofosfolipídios. Estes metabólitos e suas vias de enriquecimento indicam que a redução dos precursores da síntese de hormônios esteróides resulta em níveis mais baixos de estradiol e progesterona e causa anestros no balanço energético negativo. Estes dados fornecem uma melhor compreensão das mudanças que podem afetar o cio das vacas leiteiras pós-parto no estado de NEB e preparam o terreno para mais pesquisas.(AU)

Effects of blood pathological changes before TAI on pregnancy of dairy cows with anestrus and estrus / [Efeitos de mudanças patológicas de plasma antes de (TAI) na gestação de vacas em anestro e estro]

The objective of this study was to investigate the influence of plasma pathological changes before timed artificial insemination (TAI) on pregnancy of cows. The contents of estrogen (E2), progesterone (P4), glucose (Glu), selenium (Se), brain-derived neurotrophic factor (BDNF), and histamine (HIS) in plasma of 48 Holstein cows were measured before TAI. According to the estrus detection, the cows were divided into estrus (E) and anestrus (A) groups. After pregnancy testing at 28 d after TAI, two groups of E and A were divided into positive pregnancy of E group (EP+), negative pregnancy of E group (EP-), positive pregnancy of A group (AP+), and negative pregnancy of A group (AP-). The contents of E2, P4, Glu, Se, BDNF and hIS significantly differed among the four groups (P<0.01). The ROC analysis was used to determine the risk of negative pregnancy test (-) after TAI was increased when plasma E2 was less than 46.45 pmol/L in cows before TAI. The changes in E2, P4,hIS, Glu, and BDNF in the blood of natural estrus and natural anestrus cows affected the pregnancy after TAI. the level of E2 in plasma may be used to assess the risk of negative pregnancy after TAI.(AU)

O objetivo do presente estudo foi investigar a influência de mudanças patológicas de plasma antes de inseminação artificial (TAI) na gestação de vacas. O conteúdo de estrogênio (E2), progesterona (P4), glucose (Glu), selênio (Se), fator neurotrófico derivado do cérebro (BDNF), e histamina (HIS) no plasma de 48 vacas Holstein foi medido antes de TAI. De acordo com a detecção de estro, as vacas foram divididas em dois grupos: estro (E) e anestro (A). Após teste de gestação 28 d após TAI, dois grupos de E e A foram formados em gestação positiva do grupo E (EP+), gestação negativa do grupo E (EP-), gestação positiva do grupo A (AP+), e gestação negativa do grupo A (AP-). Os valores de E2, P4, Glu, Se, BDNF e hIS foram significativamente diferentes entre os quatro grupos (P<0,01). A análise ROC foi utilizada para determinar o risco de teste de gestação negativo (-) após aumento de TAI quando plasma E2 estava abaixo de 46,45 pmol/L em vacas antes de TAI. Alterações em E2, P4,hIS, Glu e BDNF no sangue de estro natural e anestro natural em vacas afetou a gestação após TAI. O nível de E2 no plasma pode ser usado para avaliar o risco de gestação negativa após TAI.(AU)

Association of CYP19A1 gene polymorphisms with anoestrus in water buffaloes.

Cytochrome P450 aromatase (encoded by the CYP19A1 gene) regulates oestrogen biosynthesis and so plays an essential role in female fertility. We investigated the genetic association of CYP19A1 with the risk of anoestrus in Egyptian water buffaloes. A total of 651 animals (326 anoestrous and 325 cycling) were used in this case-control study. Using single-strand conformation polymorphisms and sequencing, four single nucleotide polymorphisms (SNPs) were detected; c.-135T>C SNP in the 5'UTR and three non-synonymous SNPs: c.559G>A (p. V187M) in Exon 5, c.1285C>T (p. P429S) and c.1394A>G (p. D465G) in Exon 10. Individual SNP-anoestrus association analyses revealed that genotypes (CC, AA and GG) and alleles (C, A and G) of the -135T>C, c.559G>A and c.1394A>G SNPs respectively were high risk for anoestrus. A further analysis confirmed that these three SNPs were in linkage disequilibrium. Additionally, haplotypes with two (TAG/122 and CAA/221) or three (CAG/222) risk alleles were significantly associated with susceptibility to anoestrus, lower blood levels of both oestradiol and antioxidant enzymes (superoxide dismutase, glutathione peroxidase (GPX) and catalase) and downregulated expression levels of CYP19A1, oestrogen receptor α and Gpx3 in the ovary, as well as increased serum level of malondialdehyde. This suggests the occurrence of a high incidence of oxidative ovarian damage and subsequently ovarian inactivity in buffaloes carrying risk alleles. Therefore, with this study we suggest the selection of buffaloes with protective alleles at these SNPs to improve the reproductive efficiency of the herd.

The Two Populations of Kisspeptin Neurons Are Involved in the Ram-Induced LH Pulsatile Secretion and LH Surge in Anestrous Ewes.

Exposure to a ram during spring stimulates luteinizing hormone (LH) secretion and can induce ovulation in sexually quiescent ewes ("ram effect"). Kisspeptin (Kiss) present in the arcuate nucleus (ARC) and the preoptic area (POA) is a potent stimulators of LH secretion. Our aim was to investigate whether Kiss neurons mediate the increase in LH secretion during the ram effect. With double immunofluorescent detection, we identified Kiss neurons (Kiss IR) activated (Fos IR) by exposure to a ram for 2 hours (M2) or 12 hours (M12) or to ewes for 2 hours (C). The density of cells Kiss + Fos IR and the proportion of Kiss IR cells that were also Fos IR cells were higher in M2 and M12 than in C in ARC (P < 0.002) and POA (P < 0.02). In ARC, these parameters were also higher in M12 than in M2 (P < 0.02 and P < 0.05). Kiss antagonist (P234 10-6M) administered by retrodialysis in POA for 3 hours at the time of introduction of the ram reduced the amplitude of the male-induced increase in LH concentration compared with solvent (P < 0.02). In ARC, P234 had a more limited effect (P < 0.038 1 hour after P234) but pulse frequency increased less than after solvent (P = 0.07). In contrast, Kiss antagonist (P271 10-4M) infused in ARC but not POA 6 to 18 hours after introduction of the ram prevented the LH surge in the ewe (0/6 vs 4/5 and 4/6 in C). These results suggest that both populations of Kiss neurons are involved in the ram-induced pulsatile LH secretion and in the LH surge.

Biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in hypothalamic-pituitary unit of anoestrous and cyclic ewes.

This study was performed to explain how the molecular processes governing the biosynthesis of gonadotropin-releasing hormone (GnRH) and GnRH receptor (GnRHR) in the hypothalamic-pituitary unit are reflected by luteinizing hormone (LH) secretion in sheep during anoestrous period and during luteal and follicular phases of the oestrous cycle. Using an enzyme-linked immunosorbent assay (ELISA), we analyzed the levels of GnRH and GnRHR in preoptic area (POA), anterior (AH) and ventromedial hypothalamus (VM), stalk-median eminence (SME), and GnRHR in the anterior pituitary gland (AP). Radioimmunoassay has also been used to define changes in plasma LH concentrations. The study provides evidence that the levels of GnRH in the whole hypothalamus of anoestrous ewes were lower than that in sheep during the follicular phase of the oestrous cycle (POA: p < 0.001, AH: p < 0.001, VM: p < 0.01, SME: p < 0.001) and not always than in luteal phase animals (POA: p < 0.05, SME: p < 0.05). It has also been demonstrated that the GnRHR amount in the hypothalamus-anterior pituitary unit, as well as LH level, in the blood in anoestrous ewes were significantly lower than those detected in animals of both cyclic groups. Our data suggest that decrease in LH secretion during the long photoperiod in sheep may be due to low translational activity of genes encoding both GnRH and GnRHR.

Anti-Müllerian Hormone in the Domestic Dog during the Anestrus to Oestrous Transition.

The reproductive cycle of the domestic dog features a long period of relative ovarian inactivity or anestrus. The mechanism of anestrous termination/oestrous resumption is not yet fully understood, which presents a challenge to the development of oestrous induction protocols. In this study, we assess the possibility that anti-Müllerian hormone (AMH) might play a role in this transition by characterizing its patterns of expression in the circulation during the transition from anestrus to oestrous and in all stages of ovarian follicular growth. Serum samples from five beagles (2.0-4.5 years) were collected three times per week at least 30 days prior to the onset of oestrous and assessed for AMH concentrations. Serum AMH concentration increased significantly during the transition from anestrus to proestrus and then declined back to the anestrous baseline beginning on day -4 before the luteinizing hormone surge, which was determined by changes in serum progesterone concentrations. Cortical sections of ovaries from females undergoing routine ovariohysterectomy (aged 8 months-5 years, n = 4) were evaluated for AMH by immunohistochemistry. Pre-antral and small antral follicles were most strongly immunoreactive for AMH. These data suggest that the increase in the number of antral follicles is associated with the rise in serum AMH as the anestrous period comes to an end. The rise in AMH might be useful in predicting the onset of oestrus and therefore assist with the optimization of oestrous induction protocols and possibly other assisted reproductive technologies.

Effects of senktide, a neurokinin 3 receptor agonist, on luteinizing hormone secretion and follicular development in anestrous Shiba goats: a pilot study.

BACKGROUND: Recent studies suggest that neurokinin B and its receptor, neurokinin 3 receptor, have an essential role in the regulation of gonadotropin-releasing hormone and luteinizing hormone (LH) release in several mammalian species. As the first trial, this pilot study reports the effect of intravenous treatment with senktide, a selective agonist of neurokinin 3 receptor, on LH secretion, follicular development in female goats that were clinically diagnosed with anestrus. FINDINGS: Anestrous goats were intravenously administered 200 nmol senktide at 4-h intervals for 24 h. Most of them examined (5/6 cases) showed a pulsatile increase in LH secretion after each injection of senktide, whereas the remaining one case showed a surge-like increase of LH secretion. Ovulation was confirmed in 5/6 cases at the range of 48-96 h after the beginning of treatment. CONCLUSIONS: This pilot study demonstrated that intravenous treatment with senktide has therapeutic action in goats with anestrus by inducing LH release, which could promote follicular development and ovulation.

The pattern of LH secretion and the ovarian response to the 'ram effect' in the anoestrous ewe is influenced by body condition but not by short-term nutritional supplementation.

In sheep, the 'ram effect' induces out-of-season fertility and good nutrition increases prolificacy. This experiment determined if fatness or short-term nutritional supplementation modified the response to the 'ram effect'. A group of 48 Île-de-France ewes were fed diets that produced groups with body-condition scores (BCS) of >3.0 and <2.0. Within each BCS group animals were supplemented daily with 500g of lupins from Day -5 to Day 0 (ram introduction) resulting in four groups: low BCS, supplemented (n=7) and non-supplemented (n=8) and high BCS, supplemented (n=12) and non-supplemented (n=11). The blood concentrations of glucose and insulin and the LH response to gonadotrophin-releasing hormone (GnRH) were determined. After the 'ram effect' the pattern of LH pulsatility, the LH surge and ovarian responses were analysed. Low BCS ewes had lower glucose and insulin (P<0.001) and supplementation increased both (P≤0.001). The increase in LH induced by GnRH was reduced in low BCS ewes (P=0.015) but it was not affected by supplementation. Similarly, LH pulsatility was reduced in low BCS ewes (P<0.05). The LH surge and ovarian cyclicity were not affected but the follow-up cycle was delayed (P=0.034) and progesterone was reduced (P=0.029) in low BCS ewes. There was an effect of BCS on ovulation rate (P<0.05). These results show that the BCS can modify the response to the 'ram effect' and that supplementation has little effect on this response.

Effects of prolonged nutrient restriction on baseline and periprandial plasma ghrelin concentrations of postpubertal Holstein heifers.

Objectives of this study were to measure both daily and periprandial plasma ghrelin concentrations of postpubertal Holstein heifers during prolonged undernutrition. Following an acclimation period, Holstein heifers [n=10; 339.5 ± 8.6 kg of body weight (BW)] were fed ad libitum [well fed (WF); n=5] or restricted to 50% of ad libitum intake [underfed (UF); n=5) for 8 wk. Body condition scores (BCS) were recorded at the beginning and end of the treatment period, and weekly measurements of BW, plasma ghrelin, progesterone, and nonesterified fatty acids (NEFA) concentrations were obtained. Ovarian follicular and luteal structures were measured twice weekly via transrectal ultrasonography. Plasma ghrelin concentrations were also measured during a periprandial window bleed conducted at the end of the experiment. During the window bleed, samples were collected every 15 min between 0500 and 0900 h, with feed offered at 0700 h. Underfed heifers lost BW and BCS, whereas WF heifers gained weight and either increased or maintained BCS. Chronic underfeeding increased circulating ghrelin and NEFA concentrations. By wk 4 of the treatment period, circulating ghrelin concentrations of the UF heifers reached a plateau. Periprandial fluctuations in ghrelin concentrations were apparent as plasma ghrelin concentrations changed over time. Overall differences in periprandial plasma ghrelin concentrations were primarily due to prefeeding effects of plane of nutrition. Plasma ghrelin concentrations and change in BCS were negatively correlated such that heifers that lost the most BCS had the highest concentrations of circulating ghrelin. Two of the 5 UF heifers became anestrus by wk 3 of the treatment period. Despite being of similar age, the heifers that became anestrus had lower BW and plasma ghrelin concentrations than the UF heifers that continued to ovulate. In the current experiment, long-term undernutrition elicited ghrelin responses similar to those reported for shorter durations of nutrient restriction in cattle and other ruminants. These results demonstrate that plane of nutrition is a chronic regulator of plasma ghrelin concentrations, and that these concentrations can be experimentally manipulated in postpubertal heifers for up to 8 wk with no evidence of an adaptive response.

The effect of equine chorionic gonadotropin on follicular size, luteal volume, circulating progesterone concentrations, and pregnancy rates in anestrous beef cows treated with a novel fixed-time artificial insemination protocol.

The objective was to determine the effects of eCG given on the day of, or 2 days before removal of an intravaginal progestin device, on ovarian follicle diameter, luteal volume, serum progesterone (P4) concentrations, and pregnancy per insemination in a fixed-time AI (FTAI) protocol. Lactating, anestrous, multiparous Bos taurus cross beef cows, 40 to 60 days postpartum, were given estradiol benzoate (2 mg im) and a progestin intravaginal device containing 250 mg of medroxyprogesterone acetate on Day 0 and cloprostenol (0.265 mg) on Day 6. Intravaginal devices were removed on Day 8 and GnRH (100 µg im) was given on Day 9, with timed AI 16 hours later. In experiment 1, cows were randomly assigned to receive 400 IU im eCG on Day 6 (eCG6; N = 8) or Day 8 (eCG8; N = 8), or to not receive eCG (control; N = 8). Dominant follicle diameter on Day 9 in the eCG6 group (10.0 ± 0.5 mm) was larger (P < 0.05) than in the eCG8 (8.6 ± 0.2 mm) or control (8.5 ± 0.4 mm) groups. Corpora lutea (CL) in all cows in the control group underwent premature luteolysis within 10 days after ovulation. Luteal volumes and P4 concentrations 10 and 15 days after ovulation were higher (P < 0.05) in the eCG6 group than in the eCG8 group. In experiment 2, the eCG6 (N = 121) and eCG8 (N = 125) protocols were compared in lactating anestrous cows that underwent FTAI. Pregnancy rate was higher (P < 0.05) in the cows that received eCG on Day 6 (27.3%; 33/121) than on Day 8 (16.0%; 20/125). Furthermore, CL volumes and P4 concentrations were higher (P < 0.05) in the eCG6 group (5784.0 ± 857.3 mm(3) and 8.1 ± 1.3 ng/mL, respectively) than in the eCG8 group (3220.9 ± 505.1 mm(3) and 4.5 ± 0.7 ng/mL, respectively). We concluded that eCG given 2 days before progestin removal in this FTAI protocol for anestrous beef cows increased diameter of the dominant follicle, luteal volume, serum P4 concentrations, and pregnancy rates.

Test accuracy of metabolic indicators in predicting decreased fertility in dairy cows.

Negative energy balance is a known risk factor for decreased fertility in dairy cows. This study evaluated the accuracy of plasma concentrations of nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHBA), and insulin-like growth factor 1 (IGF-1)-factors related to negative energy balance-in predicting decreased fertility. One plasma sample per cow was collected from 480 cows in 12 herds during the period from d 4 to 21 in milk and analyzed for NEFA, BHBA, and IGF-1. For each cow, data on breed, parity, calving date, gynecological examinations, and insemination dates were obtained. Milk samples from 241 cows in 7 of the participating herds were analyzed for progesterone concentration to define the first day of luteal activity. The diagnostic sensitivity (Se) and specificity (Sp) at different cut-off concentrations of NEFA, BHBA, or IGF-1 were calculated and related to individual cow fertility status, measured as anestrus (ANEST), delayed first artificial insemination (DFAI), or delayed conception. Positive and negative predictive values (PV+; PV-) were calculated considering different levels of (within-herd) prevalence. Strata (i.e., subgroup)-specific Se and Sp and associations between test results and fertility parameters were investigated using logistic regression. The NEFA and BHBA tests for ANEST and DFAI had the highest combined Se and Sp and were thus evaluated further. Cut-off values with Sp around 80% were used in this step to provide a reasonable number of test-positive cows, representing a practical situation. This corresponded to a cut-off value for the NEFA test of 400 µEq/L (Se 0.27-0.45) and for the BHBA test of 1.8mM (Se 0.15-0.30) across all cows included in the study. The estimated Sp was generally higher than the original 80%, but the corresponding Se was further decreased when the test was used in heifers compared with older cows. The true prevalence of ANEST in the study population was 27%, which gave a PV+ of 0.36 to 0.45 and a PV- of 0.76 to 0.79. With 35% true prevalence of DFAI, PV+ was 0.29 to 0.38 and PV- was 0.64 to 0.66. Thus, overall test performance was low when metabolic indicators measured as single values in early lactation were used to predict fertility in dairy cows, but accuracy was influenced by cow-level factors such as parity. The prevalence of the target condition (in this case, decreased fertility) also influences test usefulness and should be considered when planning test systems and interpreting test results.

Male goat vocalizations stimulate the estrous behavior and LH secretion in anestrous goats that have been previously exposed to bucks.

We investigated whether live vocalizations emitted by bucks interacting with anestrous females stimulate secretion of LH, estrous behavior and ovulation in anestrous goats. In experiment 1, bucks rendered sexually active by exposure to long days followed by natural photoperiod were exposed in a light-proof-building to five anestrous females. Buck vocalizations were reproduced through a microphone-amplifier-loudspeaker system to an open pen where one group of goats (n=6) was exposed for 10 days to these live vocalizations. Another group of females (n=6) was isolated from males and vocalizations. The proportion of goats displaying estrous behavior was significantly higher in females exposed to buck vocalizations than in females isolated from males. The proportion of goats that ovulated did not differ between the 2 groups (exposed to males versus isolated). In experiment 2, female goats that either had previous contact with males (n=7), or no previous contact with males (n=7) were exposed to live buck vocalizations, reproduced as described in experiment 1, for 5 days. The number and amplitude of LH pulses did not differ between groups before exposition to buck vocalizations. Five days of exposure to male vocalizations significantly increased LH pulsatility only in females that had previous contact with males, while LH pulse amplitude was not modified. We concluded that live buck vocalizations can stimulate estrous behavior and LH secretion in goats if they have had previous contact with bucks.

Neurokinin B acts via the neurokinin-3 receptor in the retrochiasmatic area to stimulate luteinizing hormone secretion in sheep.

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Pulsatile LH secretion and ovarian follicular wave emergence and growth in anestrous ewes.

The objective of this study was to determine if pulsatile LH secretion was needed for ovarian follicular wave emergence and growth in the anestrous ewe. In Experiment 1, ewes were either large or small (10 x 0.47 or 5 x 0.47 cm, respectively; n = 5/group) sc implants releasing estradiol-17 beta for 10 d (Day 0 = day of implant insertion), to suppress pulsed LH secretion, but not FSH secretion. Five sham-operated control ewes received no implants. In Experiment 2, 12 ewes received large estradiol-releasing implants for 12 d (Day 0 = day of implant insertion); six were given GnRH (200 ng IV) every 4 h for the last 6 d that the implants were in place (to reinitiate pulsed LH secretion) whereas six Control ewes were given saline. Ovarian ultrasonography and blood sampling were done daily; blood samples were also taken every 12 min for 6 h on Days 5 and 9, and on Days 6 and 12 of the treatment period in Experiments 1 and 2, respectively. Treatment with estradiol blocked pulsatile LH secretion (P < 0.001). In Experiment 1, implant treatment halted follicular wave emergence between Days 2 and 10. In Experiment 2, follicular waves were suppressed during treatment with estradiol, but resumed following GnRH treatment. In both experiments, the range of peaks in serum FSH concentrations that preceded and triggered follicular wave emergence was almost the same as control ewes and those given estradiol implants alone or with GnRH; mean concentrations did not differ (P < 0.05). We concluded that some level of pulsatile LH secretion was required for the emergence of follicular waves that were triggered by peaks in serum FSH concentrations in the anestrous ewe.

Pulsed GnRH secretion and the FSH secretory peaks that initiate ovarian antral follicular wave emergence in anestrous ewes.

In ewes, immunization against GnRH blocks LH pulses but mean serum FSH concentrations are only partly reduced; the fate of the FSH peaks that precede ovarian follicular waves has not been studied. In this study, we used immunization against GnRH to examine the need for pulsed GnRH secretion in the genesis of FSH peaks in the anestrous ewe. Six anestrous ewes were given a GnRH immunogen on Day 0 and a booster injection on Day 28. Control ewes (n=6) received adjuvant only. Transrectal ultrasonography was performed daily for 2 days prior to and 10 days following both the primary (Days -2 to 10) and booster (Days 26-38) injections and for a 13-day period beginning 26 days after booster injection (Days 54-66). Blood samples were collected daily. Intensive bleeding (every 12min for 7h) was performed on Days 9, 37, and 65 of the experimental period to characterize the pulsatile pattern of LH secretion. GnRH antibody titers were increased and LH pulses were abolished immediately after booster immunization (P<0.05). The number of FSH peaks, FSH peak concentration and amplitude and basal FSH concentrations were only decreased in immunized ewes in the period of observations starting 26 days after booster immunization (P<0.05); however, some peaks were still seen. The number of follicular waves was decreased in the period around booster immunization and no follicular waves were seen during the period starting 26 days after booster immunization in immunized ewes (P<0.05). In summary, in anestrous ewes, when pulsed LH secretion was abolished by immunization against GnRH, the peaks in serum concentrations of FSH that trigger ovarian follicular waves continued for a period of time. We concluded that although blocking the effects of GnRH gradually causes a diminution of FSH secretion, there is no acute requirement for GnRH in the regulation of FSH peaks. The existence of FSH peaks in the absence of follicular waves, and pulsed LH secretion, suggests that some endogenous rhythm may drive the occurrence of FSH peaks, independent of both changes in negative feedback by secretory products from ovarian antral follicles and GnRH.

Efficacy of Ovsynch protocol in cyclic and acyclic Egyptian buffaloes in summer.

The aim of the present study was to evaluate the efficacy of an Ovsynch protocol in the cyclic and non-cyclic Egyptian buffaloes in the summer. The present study was carried out on 21 Egyptian pluriparous buffalo cows (11 cyclic and 10 acyclic) and 8 heifers (5 cyclic and 3 acyclic). All animals were administered 100 microg GnRH i.m. at Day 0, 500 microg of PGF2 proportional, variant i.m. at Day 7 and a second dose (100 microg) of GnRH i.m. at Day 9. Ovarian structures of the animals were recorded by means of transrectal ultrasonography (6-8 MHz linear-array transducer) daily from Day 0 (before treatment) to Day 9 (the second GnRH administration) and thereafter, at 12-h interval till ovulation or for a maximum of 48 h. After ultrasound examination, a blood sample was taken daily from each animal for progesterone analysis. The buffalo cows were inseminated at 16-20 h after 2nd GnRH administration. After the first administration of GnRH, 46% (5 of 11) of cyclic and 50% (5 of 10) of acyclic cows and 40% (2 of 5) of cyclic and 33% (1 of 3) of acyclic heifers responded by ovulation or luteinization of the follicles. The mean diameters (+/-S.E.M.) of the follicles in the responding cows and heifers were significantly higher than those in non-responding animals (9.7 +/- 0.4 mm vs. 6.7 +/- 0.6 mm, P < 0.0001; 11.1 +/- 1.5 mm vs. 7.3 +/- 0.7 mm, P < 0.05, respectively). After the second administration of GnRH, 81% (9 of 11) of cyclic and 60% (6 of 10) of acyclic cows and 80% (4 of 5) of cyclic and 33% (1 of 3) of acyclic heifers ovulated, respectively. Ovulations occurred earlier (P = 0.059) and over a wider range of time in the non-cyclic cows (26 +/- 4.8 h; range: 12-36 h) and in a heifer (12 h,) than in the cyclic cows (34.7 +/- 1.3 h; range 24-36 h) and heifers (33 +/- 3 h; range: 24-36 h). The conception rate was 18% (2 of 11) and 0% (0 of 10) in cyclic and non-cyclic cows, respectively. In conclusion, the Ovsynch protocol could be used effectively for synchronization of ovulation in cyclic cows and heifers in summer.

Male sexual behavior contributes to the maintenance of high LH pulsatility in anestrous female goats.

The objective of this study was to determine the importance of male sexual behavior in stimulating LH secretion in anovulatory female goats. Two groups of females (n=10 per group) were each exposed to a buck in sexual rest and submitted to natural daylength. In one group, the buck was awake, whereas in the other group, it was sedated to prevent its sexual behavior. Two other groups of goats (n=10 per group) were exposed to sexually active bucks that had been exposed to 2.5 months of long days. In one group, the buck was awake, and in the other group, it was sedated. LH secretion was determined every 15 min from 4 h before introducing the bucks to 8 h after, then every 15 min again from 20 to 24 h after introducing the bucks. The bucks submitted to natural daylength did not stimulate LH secretion (P>0.05), whether they were sedated or not. In contrast, both the awake and the sedated light-treated bucks induced an increase (P<0.05) of LH pulsatility in the first 4 h following their introduction. However, pulsatility remained elevated until 24 h in the females exposed to the light-treated awake buck, whereas in the group with the light-treated sedated buck, pulsatility diminished (P<0.05) after the first 4 h of stimulation by the buck. In conclusion, the sexual behavior of males contributes to the maintenance of a high LH pulsatility up to 24 h after introduction into a group of anovulatory goats.

Use of gonadotropin releasing hormone to improve reproductive responses of ewes introduced to rams during seasonal anestrus.

Three experiments were conducted on anestrous ewes of Suffolk, Dorset, and Katahdin breeding to examine the potential value of GnRH to improve ovulation and pregnancy in response to introduction of rams. In Experiment 1, treatment with GnRH 2d after treatment with progesterone (P(4); 25mg i.m.) at introduction of rams was compared to treatment with P(4) alone at the time of introduction of rams. Treatment with GnRH did not increase percentages of ewes with a corpus luteum (CL) 14d after introduction of rams, pregnant 32d after treatment with PGF(2)alpha 14d after introduction of rams, or percent of treated ewes lambing to all services. In Experiment 2, treatments with GnRH on day 2, 7, or both after introduction of rams were compared. Treatments did not differ in mean estrous response, percentages of ewes with a detectable CL or number of CL present on day 11, or mean pregnancy and lambing rates. Therefore, neither one nor two injections of GnRH at these times appeared to be effective to induce anestrous ewes to breed. In Experiment 3, treatments compared included GnRH 4d before introduction of rams, GnRH 4d before and 1d after introduction of rams, ram introduction alone, and treatment with P(4) (25mg i.m.) at the time of introduction of rams. Percentages of ewes with concentrations of P(4) greater than 1ng/mL (indicating formation of CL had occurred) 7d after ram introduction tended to be greater (P<0.07) in ewes treated with GnRH or P(4) than in control ewes treated with ram introduction alone. However, there was no difference in P(4) concentrations between groups by day 11 or 12 after introduction of rams. Estrous response rates and percentages of ewes pregnant 95d after PGF(2)alpha was administered (on day 12 after introduction of rams) tended to be greater (P=0.08 and 0.06, respectively) in ewes treated with GnRH or P(4) than in ewes exposed to rams only. There was no difference in response variables between ewes treated with GnRH 4d before introduction of rams and ewes treated with GnRH 4d before and 1d after introduction of rams. In conclusion, treatment with GnRH 4d before ram introduction showed promise as an alternative to treatment with P(4) to improve the ovulatory response and reproductive performance of ewes introduced to rams during seasonal anestrus.

Early conception factor lateral flow assays for pregnancy in the mare.

The ECF lateral flow assay test is marketed to detect non-pregnancy in mares. The objectives of the present study were to determine the accuracy of the ECF test, the accuracy of the electronic reader accompanying the ECF test, and agreement between two human readers and the electronic reader. Serum samples were collected from anestrus, cycling but not inseminated, and inseminated mares, and were evaluated with the ECF test (EDP Biotech Company, Knoxville, TN, USA) at The Ohio State University and at the EDP Biotech Laboratory. Specificity ranged from 0.07 to 0.16, the negative predictive value ranged from 0.15 to 0.33, and accuracy ranged from 0.43 to 0.52. The electronic reader did not add improve the accuracy or predictive values of the test. Based on the electronic reader, 80.0% of the serum samples collected from the anestrus mares were false positives; Readers 1 and 2 had 60.0 and 33.3% false positives, respectively. For samples collected during the estrous cycle, 83.9% were false positives by the electronic reader, whereas Readers 1 and 2 had 43.7 and 26.4% false positives. We concluded that, regardless of whether the test strips were evaluated by a human or electronic reader, this assay was not accurate for determination of the non-pregnant mare.

Physiology of the canine anoestrus and methods for manipulation of its length.

Progression from early to late anoestrus is characterized by the appearance of a larger number of gonadotrophin-releasing hormone (GnRH) pulses with a higher amplitude, an increase in the sensitivity of the pituitary to GnRH, an increase in ovarian responsiveness to gonadotrophins, and an increase in basal plasma follicle-stimulating hormone (FSH) concentration. A period of increased luteinizing hormone (LH) pulsatility has been observed shortly before the onset of pro-oestrus. Apart from these changes in the hypothalamus-pituitary-ovary axis, the initiation of a new follicular phase in the bitch is also stimulated by dopaminergic influences other than the accompanying plasma prolactin decrease. Metergoline, a drug which in a low dosage lowers the plasma prolactin concentration via a serotonin-antagonistic pathway, does not shorten the anoestrus; while bromocriptine, in a dosage insufficient to cause a decrease in the plasma prolactin concentration, does prematurely induce a follicular phase. These observations indicate that it is not the decrease in the plasma prolactin concentration, but another dopamine-agonistic influence that plays a crucial role in the transition to a new follicular phase. The dopamine-agonist induced oestrus is associated with a rapid rise in the basal plasma FSH concentration, similar to what is observed during the physiological late anoestrus. Administration of GnRH, eCG and oestrogens may also be used to induce oestrus but with variable results. Oestrus can be prevented surgically or medically, for which purpose progestagens are the most important drugs. The mechanism is still unclear, although it has been demonstrated that with continuing medroxyprogesterone acetate (MPA) treatment the FSH response to GnRH stimulation decreases and changes occur in the pulsatile release of the gonadotrophins. In general, LH pulses coincide with a FSH pulse, but during MPA treatment, LH pulses were observed while there was such a small increase in FSH that it was not recognized as significant FSH pulse.