Lucas Carvalho Siqueira

Angiotensin II, progesterone, and prostaglandins are sequential steps in the pathway to bovine oocyte nuclear maturation.

Oocyte meiotic resumption is triggered by the ovulatory gonadotropin surge; in cattle, angiotensin II (AngII) and prostaglandins (PG) are key mediators of this gonadotropin-induced event. Here, we tested the hypothesis that progesterone (P(4)) is also involved in oocyte meiotic resumption induced by the gonadotropin surge. In Experiment I, P(4) induced nuclear maturation in a dose-dependent manner using a coculture of follicular hemisections and cumulus-oocyte complexes. In the second experiment, using an in vivo model, an injection of mifepristone (MIFE; P(4) receptor antagonist) at the antrum of preovulatory follicles prevented GnRH-induced oocyte meiotic resumption in vivo. In Experiment III (coculture system similar to that of Experiment I), MIFE prevented stimulatory effects of AngII on resumption of meiosis, but saralasin (AngII receptor antagonist) did not inhibit P(4) actions. In Experiments IV and V, fibroblast growth Factor 10 (FGF10; known to suppress steroidogenesis in granulosa cells), blocked AngII-but not P(4)-induced oocyte meiotic resumption. Therefore, we inferred that AngII is upstream to P(4) in a cascade to induce meiotic resumption. Previously, we had reported that AngII acted throughout the PGs pathway to modulate nuclear progression. In Experiment V, indomethacin inhibited resumption of meiosis induced by P(4), providing further support to the AngII-P(4) sequential effect on meiotic resumption. In conclusion, we inferred that AngII, P(4) and PGs are sequential steps in the same pathway that culminates with bovine oocyte maturation.

Molecular characterization and regulation of the angiotensin-converting enzyme type 2/Angiotensin-(1-7)/MAS receptor axis during the ovulation process in cattle:

The objective of this study was to characterize the profiles of Ang-(1-7), MAS receptor, ACE2, NEP and PEP during the ovulatory process in cattle. For this study, 40 synchronized cows with follicular diameter ≥ 12 mm were ovariectomized at different time-points (0, 3, 6, 12 and 24 h) after i.m. application of gonadotropin-releasing hormone (GnRH) to induce a luteinizing hormone surge. Follicular fluid was collected for measuring Ang-(1-7) by radioimmunoassay. Theca and granulosa cells were isolated from the preovulatory follicles to evaluate the gene expression of MAS receptor, ACE2, NEP and PEP by qRT-PCR assay. Cross-contamination between theca and granulosa cells was tested by RT-PCR to detect cytochrome P450 aromatase (CYP19A1) and 17α-hydroxylase (CYP17A1) mRNA. Ang-(1-7) levels were constant until 12 h and then increased (p < 0.05) at 24 h after GnRH. Messenger RNA expression of MAS, ACE2, NEP and PEP was detected in theca and granulosa cells at all time-points after GnRH. In granulosa cells, ACE2, NEP and PEP were differentially expressed after GnRH treatment (p < 0.05). In conclusion, the Ang-(1-7), MAS receptor, ACE2, NEP and PEP profiles in preovulatory follicles indicate that Ang-(1-7) plays a role in the regulation of the ovulatory process in cattle.

Trypanosoma evansi: cholinesterase activity in acutely infected Wistar rats.

The aim of this study was to evaluate cholinesterase activity during the early acute phase of Trypanosoma evansi infection in rats. Fifteen male Wistar rats were randomly distributed into three groups (n=5 animals per group): two trypanosome-infected groups (T3 and T5) and uninfected controls (C). The animals were inoculated intraperitoneally with 10(6) trypanosomes. The blood was collected by cardiac puncture on the 3rd (T3) or 5th day post-infection (T5 and C). Cerebrum and cerebellum were removed for the evaluation of acetylcholinesterase (AChE) activity. AChE activity was also evaluated in whole blood and butyrylcholinesterase activity (BUChE) in plasma samples. Parasitemia were progressive increase and parasites were observed in the peripheral blood of all infected animals one day post-inoculation. AChE activity was not altered in cerebrum and cerebellum tissues. AChE activity in blood significantly decreased in the T3 and T5 groups (26.63 and 25.86mU/lmolHb) compared with the control (37.84mU/lmolHb). In addition BUChE activity in plasma was lower in the T3 (7.01micromol BTC hydrolyzed/h/mL) than the T5 and C groups (9.84 and 12.00micromol BTC hydrolyzed/h/mL). This study therefore, shows that reductions in the activity of cholinesterase occur in acute infection by T. evansi in rats and this demonstrates an important change occurring in animals infected by the protozoan and may indicate a potential role the enzymes play in the mechanism of disease.

Preovulatory changes in the angiotensin II system in bovine follicles.

The present study evaluated whether the gonadotrophin surge modulates components of the renin-angiotensin system and whether angiotensin II (Ang II) plays a role in the production of hormones by follicular cells during the ovulatory process. In Experiment 1, cows were ovariectomised at various times (0, 3, 6, 12 and 24 h) after GnRH injection to obtain preovulatory follicles. The concentration of Ang II in follicular fluid increased after GnRH and reached a peak at 24 h, concomitant with the peak of angiotensinogen (AGT) mRNA expression in granulosa cells. AGT mRNA was not expressed in theca cells. Ang II receptor type 2 and angiotensin-converting enzyme mRNA levels were transiently upregulated in theca cells. In Experiment 2, an in vitro culture was used to determine whether Ang II could modulate hormone production by healthy dominant follicles. In the absence of LH, Ang II did not alter hormonal production by either theca or granulosa cells. Ang II plus LH increased progesterone and prostaglandin secretion by granulosa cells. In summary, the renin-angiotensin system is actively controlled during the preovulatory period and Ang II amplifies the stimulatory effects of LH on the secretion of progesterone and prostaglandins by granulosa cells.

Effects of estradiol and progestins on follicular regression before, during, and after follicular deviation in postpartum beef cows

The objective was to evaluate the effect of estradiol benzoate (EB), in association with three progestin protocols, on ovarian follicular regression of suckled beef cows treated at three stages of follicular development (pre-deviation, deviation, or post-deviation). Thirty-six suckled beef cows (60-90 d postpartum, given 125 microg cloprostenol on two occassions, 12h apart). Forty-eight hours after the first cloprostenol treatment, all follicles >5mm were ablated and transrectal ultrasound scanning (8 MHz) was performed every 24h until Day 7 (Day 0=treatment). When the largest follicle reached a designated diameter of 5-7, 8-10 or >10mm, cows were randomly allocated to receive 2mg of EB im in association with an intravaginal device containing 250 mg of medroxyprogesterone acetate (MPA) with or without 100mg of progesterone (P(4)) given im, or an intravaginal device containing P(4) (3 x 3 factorial design). Treatments induced follicular regression in all cows, independent of follicular stage or treatment. There was no interaction between progestin treatment and follicular stage, nor was there any difference in the time of follicular regression or new wave emergence among follicular stages. Treatment with MPA plus P(4) delayed follicular regression. In conclusion, EB in association with various progestins induced regression of growing follicles and emergence of a new follicular wave in postpartum beef cows, regardless of the stage of follicular development.

Sistemas de inseminação artificial em dois dias com observação de estro ou em tempo fixo para vacas de corte amamentando

O objetivo do presente experimento foi investigar se a realizacao exclusiva da inseminacao artificial em tempo fixo (IATF), empregando como indutor da ovulacao o benzoato de estradiol (BE), proporciona taxas de prenhez semelhantes a uma associacao de IA convencional e IATF com GnRH, em vacas de corte no pos-parto. Duzentos e cinquenta vacas amamentado receberam um pessario vaginal contendo 250mg de acetato de medroxi-progesterona (MAP) e uma injecao intramuscular (IM) de 5mg de BE no dia 0. O pessario vaginal permaneceu por sete dias. No dia 6, foram aplicadas 400UI de gonadotrofina corionica equina por via IM e 5mg de analogo de prostaglandina na submucosa vulvar, realizando nesse momento o desmame por 96h. Apos a retirada dos pessarios (dia 7), as vacas foram distribuidas em dois grupos. No grupo BioRep (n=150), as femeas foram observadas duas vezes por dia para deteccao de estro por 48h e inseminadas 12h apos sua manifestacao. Os animais que nao manifestaram estro nesse periodo receberam uma injecao IM de 100mg de GnRH, sendo submetidas a IATF, 16 a 18h apos. No grupo BE (n=100), as vacas receberam uma injecao de 1mg de BE IM no dia 8 e foram inseminadas em tempo fixo no dia 9. A porcentagem de prenhez no grupo BioRep (54,7%) foi maior (P<0,01) do que no grupo BE (33,3%). Em vaca amamentando, a observacao de estro por dois dias associada a IATF, utilizando GnRH para induzir a ovulacao, proporcionou taxas de prenhez superiores ao uso exclusivo de IATF com BE.

Assessment of bovine spermatozoa viability using different cooling protocols prior to cryopreservation

The aim of our study was to evaluate the effect of different cooling rates on the post-thawing quality of bovine spermatozoa. Ejaculated semen from a 24-month-old Jersey bull was collected using an artificial vagina and diluted in a commercial extender to evaluate spermatozoan concentration and motility subjectively before cooling and freezing and after thawing. Straws were allocated to four cooling curves: rapid (RD), semi-rapid (SRD), semi-slow (SSLW) and slow (SLW). The temperature was decreased from 25°C to 4°C in 10, 50, 110 and 135 min, which represents a cooling rate of 2.06, 0.40, 0.18 and 0.15°C/min, respectively. Then straws were frozen and stored at −196°C. After thawing, one aliquot of each straw was used for evaluation. Spermatozoan integrity and mitochondrial function were evaluated using a combination of fluorescent probes containing 100 mg/mL FITC-PSA, 0.5 µg/mL PI and 153 µM JC-1. At the end of cooling, spermatozoan motility did not differ among RD (63.3%), SRD (66.7%), SSLW (66.7%) and SLW (80.0%). However, normal spermatozoan morphology was lower in SRD (84.8%) compared to RD (91.7%), SSLW (91.7%) and SLW (90.3%) (P<0.05). In thawed semen, spermatozoan motility and normal morphology did not differ among RD (40.0%; 88.8%), SRD (43.3%; 82.5%), SSLW (40.0%; 87.2%) and SLW (36.7%; 88.0%). The percentage of damaged spermatozoa, including plasma and acrosome membrane damage and low mitochondrial potential, was higher in RD compared to the others (P<0.05). In conclusion, a rapid cooling curve is detrimental to the spermatozoa and affects the post-thaw spermatozoan integrity of bovine frozen semen.

Artificial insemination system without estrous observation in suckled beef cows

O objetivo deste estudo foi desenvolver um protocolo de inseminacao artificial com tempo fixo (IATF) em vacas de corte durante periodo de amamentacao, avaliando o intervalo entre a retirada do progestageno e a aplicacao de GnRH sobre a dinâmica folicular e a prenhez. Para tanto, vacas (n=227) em pos-parto de 60 a 80 dias receberam benzoato de estradiol (5mg) e um pessario vaginal de acetato de medroxiprogesterona (250mg MAP; dia 0). No dia seis, os animais receberam cloprostenol sodico (125µg), gonadotrofina corionica equina (400UI) e desmame temporario (88h). O MAP foi retirado no dia sete (Grupo BioRep) ou no dia oito (Grupo IATF). Todas as vacas do grupo TAI e aquelas que nao manifestaram cio do grupo BioRep receberam GnRH (100µg) no dia nove. No experimento I, o monitoramento das estruturas ovarianas de 14 vacas foi realizado a cada 24h, desde o dia seis ate 36h apos a aplicacao de GnRH em ambos os grupos. O tamanho medio do foliculo dominante no dia nove foi de 11,1±0,99mm (BioRep n=7) e 11,5±0,65mm (IATF n=7) e todos os animais ovularam. No experimento II, no grupo BioRep (n=106), apos a retirada do MAP, as femeas foram inseminadas com deteccao de estro durante 48 horas. O restante dos animais do grupo BioRep e todos do grupo IATF (n=107) receberam 100µg de GnRH (dia nove) e IATF 16h depois. As taxas de prenhez foram de 57,6% (BioRep) e de 52,3% (IATF). O intervalo de 24h entre a retirada de MAP, mantido por oito dias, e a aplicacao de GnRH nao interfere na dinâmica folicular e na prenhez, permitindo inseminar vacas de corte amamentando sem observacao de estro.

Controle sobre GnRH durante o anestro pós-parto em bovinos

The bovine postpartum period is characterized as a moment when the ovulation is suppressed, mainly in consequence of insufficient release of gonadotropins. Concepts and regulatory mechanisms of gonadotropin-releasing hormone (GnRH) had been described independently. This review covers the influence of nutrition and suckling with emphasis on GnRH regulation, and provides up to date concepts of neuroendocrine control of GnRH secretion during postpartum in cattle. Current knowledge of gonadotropin-inhibitory hormone (GnIH), leptin, estrogens and kisspeptin during this period are presented in order to provide a better understanding of the subject.

Indução hormonal da ovulação e desmame precoce na fertilidade pós-parto de vacas de corte homozigotas e heterozigotas para o microssatélite BMS3004

The aim of this experiment was to compare the efficiency of a hormonal protocol, associated to the temporary weaning for 96 hours, with the definitive weaning at 60 days in beef cows, for the induction of estrus and ovulation. One hundred and eighty-three suckled beef cows were used. The breeds of the cows were Charolais (C) and Nellore (N) and their crosses. The animals were genotyped as homozygous (HOM) and heterozigous (HET) for the microsatellite BMS3004, that is localized in the same chromossome of the LH b chain gene. The cows were distributed in two groups between 60 and 80 days postpartum (day 0). In the hormonal induction group (HI), the cows (n=87) received (day 0) 250 mg of medroxiprogesteron acetate for 8 days, 2.5 mg of estradiol benzoate (day 1) and 500 UI of eCG (day 7). On day 8, the calves were weaned for 96 hours. In the same day (day 8), the cows (n=96) of the other group were just submitted to early weaning (group EW). Twelve hours after weaning, artificial insemination (AI) was done during four days. After this period, they were mated. The first diagnosis of pregnancy (DP) was performed 60 days after the AI period and, the second, 60 days after the end of mating. The estrus rates were higher in cows from HI group than in those of EW group. In the HI group, the cows with body condition 2.5 and 3.0 presented lower pregnancy rates at the 1st DP (29.6 and 46.4%) than in the EW group (56.0 and 72.2%). The rates of pregnancy in cows with body index 65-73 did not differ between the HI and EW groups. The N cows of HI group presented lower pregnancy rate at 1st DP than the F1 (27.7 vs. 64.2%), but was not different than the C cows (40.0%). In the HI group, the pregnancy rate at the 2nd DP was lower in HOM cows than in the HET ones. The cows in the early definitive weaning group showed to be more efficient than in the hormonal induction group to improve the pregnancy rate.