Eficácia da associação dupla dose PGF2 alfa-eCG no proestro de vacas leiteiras mestiças submetidas à IATF / Efficacy of the double PGF2 alpha dose-eCG association in proestrus of crossbred dairy cows submitted to IATF

Objetivou-se avaliar o efeito de uma ou duas doses de prostaglandina F2α (PGF2α) associada ou não a gonadotrofina coriônica equina (eCG) sobre a dinâmica folicular, a função luteal pré-ovulatória, assim como as características morfofuncionais pós-ovulatórias do corpo lúteo (CL) em fêmeas mestiças cíclicas submetidas a um protocolo de inseminação artificial em tempo fixo (IATF). Para tanto, 29 vacas 3/4 Gir x Holandês multíparas foram submetidas ao exame de ultrassonografia (US) transretal e após a detecção do CL iniciou-se um protocolo de IATF em um dia denominado zero (D0), por meio da inserção do implante de progesterona (P4) associado à aplicação de 2,0mg de benzoato de estradiol. No D7 esses animais receberam 12,5mg de dinoprost trometamina. No D9 realizou a remoção dos dispositivos de P4 e aplicou 0,6mg de cipionato de estradiol. Nesse momento, as fêmeas foram subdivididas nos seguintes tratamentos: Grupo Controle (n=7), foi administrado 2,5mL de solução fisiológica; Grupo 2PGF (n=7), aplicou 12,5mg de dinoprost trometamina; Grupo eCG (n=7), administrou-se 300UI de eCG; Grupo 2PGF+eCG (n=8), realizou a aplicação de 300UI de eCG e 12,5mg de dinoprost trometamina. Para avaliar a dinâmica folicular foram realizados exames de US em modo B e power doppler (Mindray Z5, Shenzhen, China) a cada 12h do D7 até o momento da ovulação ou 96h após a remoção dos implantes de P4, mensurando-se o diâmetro folicular (DFOL), a área da parede folicular (AFOL) e a área de perfusão sanguínea da parede folicular (VFOL). Concomitante a cada exame, foram coletadas amostras de sangue sendo determinada a concentração sérica de P4 pré-ovulatória por meio da metodologia de quimioluminescência. No D24 foi realizada a US modo B e doppler analisando-se o diâmetro luteal (DCL), área luteal (ACL) e área de perfusão sanguínea do CL (VCL), assim como, foi coletada amostra de sangue para averiguar a concentração sérica de P4 pós-ovulatória. Os dados foram avaliados pelo Two-way ANOVA e análise de medidas repetidas considerando os efeitos do eCG, 2PGF e interação eCG*2PGF, P<0,05. Não houve diferença significativa entre os protocolos de sincronização para as variáveis DFOL, AFOL e VFOL ao longo do tempo da dinâmica folicular. Os grupos experimentais apresentaram uma concentração sérica de P4 pré-ovulatória semelhante em cada momento da avaliação. Não foi observada distinção da ACL e VCL entre os tratamentos hormonais, contudo o Grupo eCG demonstrou tendência (P=0,08) a apresentar maior DCL em relação ao Grupo 2PGF e 2PGF+eCG. Adicionalmente a estes achados, também foi constatado tendência (P=0,07) a maiores concentrações de progesterona no dia 24 do protocolo nos animais do Grupo eCG (11,00±3,32ng/mL) em relação ao Grupo 2PGF (6,37±1,31ng/mL), enquanto o Controle e 2PGF+eCG demonstraram resultados intermediários que se assemelham a ambos os grupos, com concentrações de 8,43±3,85 e 9,18±2,82ng/mL, respectivamente. As tentativas de ajustes no proestro foram incapazes de melhorar a qualidade folicular e minimizar a função luteal pré-ovulatória, assim como não incrementaram a morfologia do CL e a função luteal pós-ovulatória, sugerindo que em animais cíclicos mestiços protocolos de IATF com a utilização de uma única dose PGF2α e sem o suporte gonadotrófico da eCG parece promover adequada resposta folicular e luteal.(AU)

The study aimed to evaluate the effect of one or two prostaglandin doses F2α (PGF2a) with or without equine chorionic gonadotropin (eCG) in the follicular dynamics, the preovulatory luteal function, as well as the structural and functional characteristics post-ovulatory of the corpus luteum (CL) in cyclic crossbred females subjected to a fixed time artificial insemination (FTAI) protocol. For this, 29 multiparous 3/4 Gyr x Holstein cows were subjected to transrectal ultrasound examination (US) and upon detection of CL initiated a FTAI protocol on day called zero (D0) by the insertion of progesterone implant (P4) associated with the application of 2.0mg estradiol benzoate. On D7, these animals received 12.5mg of dinoprost tromethamine. At D9 happened the removal of the P4 devices and was applied 0.6mg of estradiol cypionate. At that time, the females were divided into the following treatments: control group (n=7) - which received 2.5mL of saline solution, 2PGF group (n=7) - received 12.5mg of dinoprost tromethamine, eCG group (n=7) - was administered 300IU eCG and eCG+2PGF group (n=8) - which received 300 IU eCG and 12.5mg of dinoprost tromethamine. To assess follicular dynamics were performed US scans B-mode and power doppler (Mindray Z5, Shenzhen, China) each 12h on D7 until the time of ovulation or until 96h after removal of the P4 implants, considering the follicular diameter (DFOL), the area of the follicular wall (AFOL) and the blood perfusion area of the follicular wall (VFOL). Concomitant with each test, blood samples were collected to determine the serum concentration of P4 preovulatory by chemiluminescence methodology. In D24 had held US B-mode and doppler to analyse the luteal diameter (DCL), luteal area (ACL) and blood perfusion area CL (VCL). Also, a blood sample was collected to determine the serum concentration of P4 post-ovulatory. All data was evaluated by Two-way ANOVA and repeated measures analysis considering the effects of eCG, 2PGF and eCG*2PGF, P<0.05. There was not significant difference between the synchronization protocols for DFOL, AFOL and VFOL variables over time of follicular dynamics. Experimental groups had a serum concentration of P4 preovulatory similar in every moment of evaluation. There wasn't distinction of ACL and VCL between hormone treatments. However, the eCG group showed a tendency (P=0.08) to present higher DCL compared to the 2PGF and 2PGF+eCG groups. In addition to these findings, there was also a tendency (P=0.07) to higher concentrations of P4 on D24 of the protocol in the animals of the eCG group (11.00±3.32ng/mL) compared to the 2PGF group (6,37±1.31ng/mL), meanwhile the Control and 2PGF+eCG showed intermediate results that resembled both groups, with concentrations of 8.43±3.85 and 9.18±2.82ng/mL, respectively. Attempts to adjust proestrus were unable to improve follicular quality and minimize preovulatory luteal function, nor did they increase CL morphology and post-ovulatory luteal function, suggesting that in cyclic animals, FTAI protocols using a single PGF2α dose and without the gonadotrophic support of eCG seems to promote adequate follicular and luteal responses.(AU)

The influence of nutrition on the insulin-like growth factor system and the concentrations of growth hormone, glucose, insulin, gonadotropins and progesterone in ovarian follicular fluid and plasma from adult female horses (Equus caballus).

BACKGROUND: Feed intake affects the GH-IGF system and may be a key factor in determining the ovarian follicular growth rate. In fat mares, the plasma IGF-1 concentration is high with low GH and a quick follicular growth rate, in contrast to values observed in thin mares. Nothing is known regarding the long-term effects of differential feed intake on the IGF system. The objective of this experiment was to quantify IGFs, IGFBPs, GH, glucose, insulin, gonadotropin and progesterone (P4) in blood and in preovulatory follicular fluid (FF) in relation to feeding levels in mares. METHODS: Three years prior to the experiment, Welsh Pony mares were assigned to a restricted diet group (R, n = 10) or a well-fed group (WF, n = 9). All mares were in good health and exhibited differences in body weight and subcutaneous fat thickness. Follicular development was scanned daily and plasma was also collected daily. Preovulatory FF was collected by ultrasound-guided follicular aspiration. Hormone levels were assayed in FF and plasma with a validated RIA. RESULTS: According to scans, the total number of follicles in group R was 53% lower than group WF. Insulin and IGF-1 concentrations were higher in WF than in R mares. GH and IGF-2 concentrations were lower in plasma from WF mares than from R mares, but the difference was not significant in FF. The IGFBP-2/IGFBP-3 ratio in FF was not affected by feeding but was dramatically increased in R mare plasma. No difference in gonadotropin concentration was found with the exception of FSH, which was higher in the plasma of R mares. On the day of puncture, P4 concentrations were not affected by feeding but were higher in preovulatory FF than in plasma. CONCLUSIONS: The bioavailability of IGF-1 or IGF-2, represented by the IGFBP2/IGFBP3 ratio, is modified by feed intake in plasma but not in FF. These differences partially explain the variability in follicular growth observed between well-fed mares and mares on restricted diets.

Bioassay for follicle stimulating activity of equine gonadotropic hormone in mare serum using frozen/thawed transiently transfected reporter cells.

The objective was to establish a cell line-based bioassay for FSH in horse serum for screening samples with high eCG bioactivity. A cell line (HEK293) was transiently cotransfected with an FSH reporter expression plasmid and a cAMP-responsive ß-galactosidase reporter plasmid. Cells were bulk frozen, and thawed for assay purposes. This assay was specific for FSH, with no cross-reaction with LH or insulin-like growth factor-1. Standard curves (eCG) and serum samples from pregnant mares passed parallel line bioassay validity tests (linearity and parallelism). Estimates of bioactivity with this bioassay were highly correlated with estimates obtained with the Steelman-Pohley hCG augmentation assay. The colorimetric end point permitted the use of this assay as a rapid screen for FSH bioactivity without the need for animal use or complex cell culture facilities.

Sincronização da ovulação utilizando FSH em substituição à eCG em cabras / Ovulation synchronization using FSH replacing eCG in goats

The effect of substitution of equine chorionic gonadotropin (eCG) by follicle stimulating hormone (FSH) in synchronization protocols of ovulation in dairy goats was evaluated. Twelve goats received intravaginal sponges impregnated with 60mg of medroxyprogesterone acetate (MAP) for 10 days. The sponges were removed and the animals were distributed into two groups (G): G1 (n=6) treated with 0.5mL of a synthetic analogue of PGF2 α and 100 IU of eCG for each 10kg weight, intramuscular injection (IM); and G2 (n=6) treated with 0.5mL of a synthetic analogue of PGF2α and 20mg of FSH (IM). All animals were monitored for estrus detection with aid of a ruffian after sponge removal. The ovarian dynamics were analyzed by ultrasound, since six hours after sponge removed. Each animal was analyzed in time elapsed of six hours until 12 hours after ovulation detection. For data analyses, the Wilcoxon test and variance analyses were used. There was not difference between the analyzed parameters (P>0.05). In this way, eCG can be replaced by FSH in synchronization protocols of ovulation in dairy goats.

Enzyme immunoassay (EIA) for equine chorionic gonadotropin/pregnant mare serum gonadotropin (eCG/PMSG).

A simple, accurate, sensitive enzyme immunoassay (EIA) has been developed that permits the measurement of equine Chorionic Gonadotropin activity in pregnant mare plasmas or serums as well as in commercial and highly-purified preparations. This assay is specific for eCG and eLH which share the same polypeptide structure but differ in their oligosaccharidic chains. The more important result is that this EIA has been found to be give data in very close agreement with the in vivo assay. Therefore this very rapid and convenient assay can be used to measure the activity of eCG/PMSG in pregnant mares serums in in-field conditions as well as in crude or highly-purified preparations.

Biological and immunoactive substances resembling chorionic gonadotropin are present in full-term horse and zebra placentas.

This study describes the presence of immunoactive and bioactive eCG-like material in full-term placentas of both domestic horses and zebras. Term placental extracts were immunoreactive in an LH monoclonal antibody RIA, and methods successfully used previously for the purification of eCG and eLH were employed to further concentrate the immunoreactive materials to the point where additional characterization studies could be performed. Sufficient equine material was obtained to perform a final fractionation on a concanavalin A Sepharose column yielding an unadsorbed fraction, e17A, and an adsorbed fraction, e17B. There was insufficient zebra material, z5D, for this step. HPLC gel filtration coupled with LH immunoassays of the column eluates showed all the final placental fractions to be highly heterogeneous, but a discrete peak of immunoactivity was found in one of the two equine fractions (e17B) and in the zebra fraction (z5D). The HPLC gel filtration elution volumes for e17B and z5D suggest that they have a smaller molecular size than either eCG or eLH but almost the same size as ovine LH. Both e17B and z5D were bioactive in the rat Leydig cell assay for LH but low in potency compared to eCG or eLH; e17A was inactive at very high doses (5 micrograms). This latter fraction, however, cross-reacted in an eCG alpha RIA to a much greater extent (6 times) than e17B, suggesting that it may be an incompletely formed or degraded alpha subunit. RIAs for LH, eCG, and eCG beta suggest that epitopes distinctive for these molecules are also present or similar to those in the term placental materials.(ABSTRACT TRUNCATED AT 250 WORDS)

Equine chorionic gonadotropin.

Cells from the chorionic girdle of the equine trophoblast invade the maternal endometrium at day 36 of gestation and become established as secretory elements known as the endometrial cups. These structures, which persist for 40-60 days, produce a gonadotropin which can be found in circulation until about day 130 of gestation. This glycoprotein has been identified in the horse and the donkey, with the former having received much better characterization. It consists of 2 noncovalently linked peptide chains; an alpha-subunit of 96 amino acids, which is common to that found in other horse glycoprotein hormones. The beta-subunit of 149 amino acids is identical to horse LH beta. Horse CG is the most heavily glycosylated of the known pituitary and placental glycoprotein hormones. The alpha-subunit has two and the beta-subunit one N-linked glycosylation site, and the beta-chain has in excess of four O-linked glycosylation sites. The N-linked glycans have some oligosaccharides that are not found on other glycoprotein hormones. The sialic component of glycosylation confers an exceptionally long half-life on CG compared to other glycoprotein hormones. Horse CG has LH-like activity in horse receptor and in vitro bioassays. In spite of the amino acid homology, it has lower LH activity than does horse LH. Its most intriguing, and as yet unexplained, characteristic is its pronounced FSH and LH activity in species other than the horse. Horse CG binds to FSH receptors of virtually all mammalian species, other than the horse, in which it has been tested and will produce biological effects peculiar to FSH. It has similar and potent interaction with LH receptors. The structural basis of this duality is not known but may be related to the region 90-110 of the beta-chain. Horse CG is believed to be constitutively expressed by the trophoblastic cells until the endometrial cups degenerate. The role of CG in equine gestation is not completely understood. It is believed to act as an LH-like hormone to induce supplementary ovulation and/or luteinization of follicles in the mare. It has not been established whether CG or the accessory corpora lutea are necessary for successful horse pregnancy. They may serve as a redundant system to assure that there is sufficient secretion of the primary corpus luteum to maintain pregnancy until the placenta assumes its role as the principal steroidogenic organ of gestation.

Intra-follicular and peripheral steroid characteristics during vernal transition in the pony mare.

This experiment investigated steroid production by ovarian tissues, in vitro, of pony mares during vernal transition from anoestrus to the breeding season. Follicular dynamics were monitored to detect the first, second, third or fourth transition follicle, greater than or equal to 30 mm diameter or the first large post luteal follicle of the breeding season. Twenty-four hours after a large follicle was detected, theca (T) and granulosa (G) tissues were harvested. Separate and co-incubations of these tissues were conducted to determine steroid production in early transition (ET), late transition (LT) and pre-ovulatory (OV) follicles. Peripheral plasma and follicular fluid steroids and gonadotrophins also were assayed. Peripheral plasma oestradiol concentrations increased from ET to LT and again from LT to OV in parallel with tissue production and follicular fluid content. Androgen production increased from LT to OV whereas progesterone production showed no change, thereby indicating a possible failure of 17-alpha steroid hydroxylase in ET follicles. Examination of tissue steroid secretion rates revealed that granulosa was the major site of oestrogen production, whereas theca secreted greater amounts of androgen.

Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin.

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.

[Determination of the activity of luteinizing hormone in PMSG preparations using testosterone synthesis in vitro]. / Stanovení aktivity luteinizacního hormonu v preparátech PMSG podle syntézy testosteronu in vitro.

The activity of luteinizing hormone (LH) in serum gonadotropin (PMSG) was studied by a bioassay in vitro, using the cells of mouse testes prepared by the combination of enzymatic and mechanical diffociation. The activity of individual preparations was calculated according to the amount of produced testosterone measured by radioimmunoassay. Comparison with the 2nd international standard of PMSG (WHO) showed that in the PMSG substandard (Dessau) the activity of LH was twice lower, and in three charges of the commercial PMSG preparation (Bioveta, Ivanovice in Haná) four-and-a-half times lower than declared. No great variability of LH activity was observed among the charges of the commercial PMSG preparation.

Radioimmunoassay for PMSG and its application to in-vivo studies.

A double-antibody radioimmunoassay for PMSG, especially for meauring PMSG in cattle blood after exogenous application, has been developed. A rabbit antiserum against PMSG and pure PMSG for radioiodination were used. There was a strong cross-reaction against equine LH and FSH, but the slight cross-reaction against bovine LH and FSH could be eliminated by adding bovine LH to each tube during the assay. Unspecific, interfering influences of equine or cow serum could be eliminated by adding a constant amount of PMSG-free serum to each tube. PMSG added to 200 microliter of serum could be recovered by this method with a mean of 90 . 5 +/- 9 . 9%. Inhibition curves obtained with pregnant mare serum or cow serum after administration of PMSG were parallel to those obtained with the PMSG standard preparation. The intra-assay coefficient of variation (CV) was 6 . 9%. The inter-assay CV was 12 . 6%. Sensitivity of the assay was 1 mi.u. PMSG/tube. Values of PMSG measured in the serum of pregnant mares by this assay were comparable with those obtained by a bioassay on the same samples. PMSG was still measurable in blood serum about 10 days after injection of 1500-3000 i.u. PMSG. After infusion of 12,000 i.u. PMSG for 3 h (2 heifers), the half-life of PMSG was found to have two components, one of 51 or 40 h and a slower one of 123 or 118 h.

Influence of foetal genotype on the follicle-stimulating hormone:luteinizing hormone ratio of pregnant mare serum gonadotrophin.

Rat testicular radioreceptor assays specific for FSH and LH were used to determine the FSH:LH ratio of PMSG produced by horse, donkey, mule and hinny conceptuses. Measurements of FSH and LH activities in PMSG produced both in vivo and in vitro by the four types of conceptuses showed that the genotype of the foetus markedly influences the FSH:LH ratio of PMSG. The FSH:LH ratio of PMSG produced by the horse conceptus was around unity whereas the ratio of PMSG produced by the donkey conceptus was as low as 0-2. Furthermore, the hybrid mule and hinny conceptuses both produced PMSG with an FSH:LH ratio which was approximately midway between those of the horse and donkey.