Ovarian stimulation with commercial preparations of equine chorionic gonadotropin (eCG) produces extremely variable responses in domestic animals, ranging from excessive stimulation to practically no stimulation, when applied on the basis of their declared unitage. This study was conducted to analyze four commercial preparations from different manufacturers via reversed-phase HPLC (RP-HPLC) in comparison with a reference preparation and an official International Standard from the World Health Organization. The peaks obtained by this qualitative and quantitative physical–chemical analysis were compared using an in vivo bioassay based on the ovarian weight gain of prepubertal female rats. The RP-HPLC data showed one or two peaks close to a main peak (tR = 27.9 min), which were related to the in vivo bioactivity. Commercial preparations that have this altered peak showed very little or no in vivo activity, as demonstrated by rat ovarian weight and in peripubertal gilts induced to ovulate. Overall, these findings indicate that RP-HPLC can be a rapid and reliable tool to reveal changes in the physicochemical profile of commercial eCG that is apparently related to decreased biological activity of this hormone.
This study aimed to evaluate the use of low-or high-dose intravaginal progesterone (P4)-releasing devices on pregnancy rates of Zebu heifers submitted to fixed-timed artificial insemination (TAI). Pubertal Bos indicus Nellore heifers were injected at day 0 with 2 mg estradiol benzoate (EB) and assigned randomly to three groups. An intravaginal device containing 1.9 or 0.75 g P4 was inserted in animals of groups 1 (n=80) and 2 (n=79), while group 3 (n=76) received no device. The intravaginal devices were removed on day 8 and one luteolytic dose (125 µg) of d-cloprostenol was administered. Half of the animals in each group additionally received 300 IU of eCG. Twenty-four hours later, the heifers received a second injection of 1 mg EB and TAI was performed 54 hours after the injection of cloprostenol. Pregnancy was diagnosed 40 days after TAI by transrectal ultrasonography. Data were analyzed by logistic regression. Pregnancy rates were 27.5%, 30.4% and 28.9% in groups 1, 2 and 3, respectively (P>0.05). This result was not influenced by body score, farm, eCG, or presence of CL at the beginning of the treatments. In conclusion, the use of intravaginal devices containing either a high (1.9 g) or low (0.75 g) dose of P4 resulted in similar pregnancy rates after TAI in pubertal Nellore heifers.
RESUMO: Devido à semelhança na estrutura e função com o hormônio luteinizante (LH), a gonadotrofina coriônica humana (hCG) tem sido convencionalmente o principal hormônio utilizado para induzir o estro e a ovulação nas fêmeas suínas. Recentemente, foi demonstrado que o neuropeptídio kisspeptina é o precursor da descarga de LH em diferentes espécies de mamíferos, incluindo a suína. Dessa forma, o presente estudo objetivou determinar se uma das formas dessa substância (kisspeptina-10) associada à gonadotrofina coriônica equina (eCG) pode ser um método eficiente para induzir a ovulação de marrãs pré-púberes. Trinta marrãs de 5 meses de idade, com peso médio de 78,0 ± 8,4 kg, foram distribuídas aleatoriamente em cinco grupos de seis animais cada. Os grupos 1, 2 e 3 receberam 750 UI de eCG, enquanto que os grupos 4 e 5 receberam solução fisiológica. Setenta e duas horas após, o grupo 2 recebeu 380 nmol de kisspeptina-10, os grupos 3 e 4 receberam 500 UI de hCG e os grupo 1 e 5 receberam solução fisiológica. As injeções foram aplicadas em dose única, utilizando a via intramuscular. Os animais foram abatidos sete dias após e a ovulação foi constatada pela presença de corpos lúteos nos ovários recuperados. Os dados foram analisados pelo teste de Kruscal-Wallis e teste exato de Fisher. O número de fêmeas que ovulou foi semelhante nos grupos 2 e 3. Essa resposta foi superior ao grupo 5 (P<0,01) e aos grupos 1 e 4 (P=0,08). O peso e o tamanho dos ovários não foram influenciados pelos tratamentos, enquanto que o número de ovulações foi maior para os grupos 2 e 3 (P<0,01). Esses resultados sugerem que a kisspeptina-10 tem atividade biológica comparável à hCG para induzir a ovulação de marrãs pré-púberes.Palavras-chave: eCG, hCG, kiss-10, ovulação, puberdade, suínos. INDUCTION OF OVULATION AND OVARIAN CHARACTERISTICS OF PREPUBERTAL GILTS TREATED WITH HUMAN CHORIONIC GONADOTROPIN OR KISSPEPTINABSTRACT: In view of its similar structure and function to luteinizing hormone (LH), human chorionic gonadotropin (hCG) has conventionally been used as the main hormone to induce estrus and ovulation in sows. Recently, it has been demonstrated that the neuropeptide kisspeptin is associated with pituitary LH release in different mammalian species, including swine. Thus, the aim of this study was to determine whether kisspeptin-10 combined with equine chorionic gonadotropin (eCG) could be an efficient method to induce ovulation in prepubertal gilts. Thirty 5-month-old gilts weighing 78.0 ± 8.4 kg were randomly divided into five groups of six animals each. Groups 1, 2 and 3 received 750 IU eCG, while groups 4 and 5 received saline solution. After
Superovulation in ruminants can be induced with a single injection of equine chorionic gonadotropin (eCG). However, ovarian response is sometimes lower than expected because of, among other factors, the source of eCG used. This study aimed at establishing the physical-chemical profile of commercial eCG, in order to find differences which can be related to their biological activity. Four different commercial eCG products for veterinary use (A, B, C, D) and one eCG chemical reagent from Sigma (St. Louis, MO, USA), here used as reference preparation, were analysed by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C4-Grace Vydac 214 TP 54-column (25 cm × 4.6 mm I.D.), with UV detection at 220 nm. All eCG preparations presented at least three peaks with retention times (tR) of ~27(I), 34(II), and 36(III) minutes, with a peak at tR = 27 min common to A, C, D, and Sigma eCG, whereas preparation B did not present this peak. A bioassay test was carried with all of these preparations. Immature 21- to 25-day-old Wistar female rats received the equivalent to 10 IU of eCG of each one of these preparations. Autopsy was performed 48 h later and ovaries were removed and weighed. The average ovarian weight for preparations A, C, D, and Sigma were ~0.0795 ± 0.0107 g, whereas preparation B was 0.035 ± 0.007 g (P < 0.01). Preparation B was not different from saline (0.034 ± 0.002 g). In order to establish which one of these three peaks presented the highest biological activity, a mass equivalent to 10 IU of eCG from peaks I, II, and III of Sigma and of product A were studied. The average ovarian weight of animals injected with material from peak II and III (~0.0285 ± 0.003 g) were similar to that of the control whereas peak I produced ovarian weights of 0.059 ± 0.007 g and 0.075 ± 0.010 g for Sigma and product A, respectively (P < 0.01). These results suggest that the lack of ovarian response to eCG treatments can be related to differences in the physical-chemical profile of commercial eCG products and that RP-HPLC is a fast and reliable tool for detecting these differences. Supported by FAPESP (Grant 11/13096-0).
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