Avaliação da taxa de prenhez de vacas Nelore lactantes e não lactantes submetidas à inseminação artificial em tempo-fixo a base de progesterona injetável

Most fixed-time artificial insemination (FTAI) protocols utilize progesterone (P4) as a hormonal source to achieve synchronization of estrus in cattle. The use of an injectable P4 source to control estrus would be an interesting pharmacological strategy owing to the practicality of parenteral application. However, the effects of injectable P4 on estrus cycle control in cattle remain poorly studied. In particular, no existing studies have investigated the effect of injectable P4 on the fertility of cows subjected to FTAI. The aim of this study was to evaluate the pregnancy rate of lactating and non-lactating Nelore cows subjected to FTAI with injectable P4. Of the 422 non-lactating cows in this study, 162 (38.3%) became pregnant by 60 days post-FTAI. In the lactating group (n = 516), 166 (32.1%) were pregnant by 60 days after treatment with injectable P4. The proportions of lactating and non-lactating cows becoming pregnant were compared using the chi-square test, adopting a significance level of P < 0.05. It was found that the pregnancy rate of the cows subjected to FTAI with injectable P4 was influenced by lactation status. Lactating cows had lower reproductive performance, possibly because of their higher nutritional requirements. However, the use of injectable P4 shows promising results and may prove to be a useful strategy in large-scale livestock production.Em bovinos a maioria dos protocolos de inseminação artificial em tempo-fixo (IATF) utiliza a progesterona (P4) como base hormonal para a sincronização do estro. A utilização de uma fonte de P4 injetável para este controle do ciclo estral poderia ser uma estratégia farmacológica interessante, devido à praticidade de uma aplicação parenteral. O efeito da P4 injetável no controle da dinâmica folicular em bovinos tem sido pouco estudado, e até o momento, não há estudos que investigaram o efeito da P4 injetável na fertilidade de vacas submetidas à IATF. O objetivo do presente estudo foi avaliar a taxa de prenhez de vacas Nelore em lactação e não lactantes, submetidas a um protocolo de IATF a base de P4 injetável. Neste estudo, foi demonstrado que das 422 vacas não lactantes, 162 (38,3%) tornaram-se gestantes 60 dias após a IATF. No lote de vacas lactantes (N = 516), 166 (32,1%) estavam prenhes após a sincronização com protocolo hormonal à base de P4 injetável. A proporção de vacas gestantes lactantes e não lactantes foram comparadas pelo teste do Qui-Quadrado adotando nível de significância quando p < 0,05. A taxa de prenhez de vacas submetidas à IATF com P4 injetável foi influenciada pelo status lactacional. Vacas lactantes apresentaram menor desempenho reprodutivo, possivelmente devido a maior exigência nutricional. No entanto, o uso da P4 injetável mostrou resultados promissores, podendo ser uma estratégia interessante em rebanhos em larga escala

Avaliação da taxa de prenhez de vacas Bos indicus submetidas a sincronização da ovulação com diferentes protocolos a base de progesterona injetável ou dispositivo intravaginal

This study evaluated the pregnancy rate in Nelore cows (Bos indicus) that were subjected to fixed-time artificial insemination (FTAI) using different protocols consisting of injectable progesterone (P4) or an intravaginal device (impregnated with P4). Multiparous cows 72-84 months in age, 30-45 days postpartum, were selected on the basis of the absence of a corpus luteum (CL) and follicles < 8 mm after transrectal palpation and ultrasound examinations. On a random day of the estrus cycle (D0), the selected animals (n = 135) were randomly assigned to one of three experimental groups (n = 45 each). Group I (injectable P4/FTAI 36 hours) received 250 mg of injectable P4 and 2 mg EB on D0; on D7, they received 500 µg of cloprostenol; on D8, 300 IU of eCG and 1 mg of EB were administered; and finally, FTAI was performed 36 hours after the application of EB. Group II (injectable P4/FTAI 48 hours) received the same protocol as Group I, except that the FTAI was performed 48 hours after ovulation induction. The animals of Group III (Control/CIDR) received a conventional protocol for FTAI using an intravaginal device (D0: P4 and 2 mg EB; D8: device removal, 500 µg cloprostenol, 300 IU eCG, 1 mg EB; and FTAI performed 48 hours after removal of the device). The results showed that cows synchronized with the conventional protocol for FTAI (Control/CIDR) had a higher pregnancy rate (60 %, 27/45) than those synchronized with an injectable P4/FTAI 36 hours (33.33 %; 15/45, P = 0.010). However, the group receiving injectable P4 group/FTAI 48 hours had a similar pregnancy rate (48.9 %; 22/45; P = 0.290) when compared to both the group receiving the conventional protocol and that receiving injectable P4/FTAI 36 hours (P = 0.134). Although the injectable P4 may affect pregnancy rate with the FTAI performed in 36 hours, we found similar pregnancy rates from cows inseminated 48 hours after induction ovulation, considering injectable or intravaginal P4. Therefore, we suggest that injectable P4 represents an alternative source of progesterone for synchronization of cattle for FTAI.Este estudo avaliou a taxa de prenhez de vacas Nelore (Bos indicus) submetidas a diferentes protocolos de IATF a base de progesterona (P4) injetável ou dispositivo intravaginal impregnado com P4. Vacas multíparas entre 72 e 84 meses de idade, com 30 a 45 dias pós-parto foram previamente selecionadas com base na ausência de corpo lúteo (CL) e folículos < 8 mm após palpação e exame ultrassonográfico transretal. Em um dia aleatório do ciclo estral (D0) os animais selecionados (N = 135) foram aleatoriamente distribuídos em um dos três grupos experimentais (N = 45 / grupo). O grupo I (P4 injetável/IATF 36 horas) recebeu 250 mg de P4 injetável e 2 mg BE no D0. No D7 aplicou-se 500 µg de Cloprostenol. No D8 300 UI de eCG e 1 mg de BE foram administrados, sendo que a IATF foi realizada 36 horas após a aplicação do BE. O grupo II (P4 injetável/IATF 48 horas) recebeu o mesmo protocolo de sincronização da ovulação, exceto pela IATF que foi realizada 48 horas após indução da ovulação. Os animais do grupo III (Controle/CIDR) receberam um protocolo convencional de IATF com dispositivo intravaginal (D0 - P4 e 2 mg BE, D8 - remoção do dispositivo, 500 µg Cloprostenol, 300 UI eCG, 1 mg BE e IATF realizada 48 horas após a remoção dos dispositivos). Os resultados foram analisados pelo teste do Qui-Quadrado (p ? 0,05). No estudo, as vacas sincronizadas com protocolo convencional de IATF (Controle/CIDR) apresentaram maior taxa de prenhez (60%; 27/45) do que aquelas submetidas à sincronização da ovulação com P4 injetável/IATF 36 horas (33,33%; 15/45; p = 0,01). Porém, o grupo P4 injetável/IATF 48 horas demonstrou uma taxa de prenhez semelhante (48,9%; 22/45; p = 0,290) ao grupo com protocolo convencional e ao grupo de P4 Injetável/IATF 36 horas (p = 0,134). Embora a P4 injetável demonstrou afetar a taxa de prenhez, as vacas inseminadas 48 horas após a indução da ovulação apresentaram taxas semelhante às vacas que receberam dispositivo intravaginal. Portanto, sugerimos que a P4 injetável representa mais uma fonte progesterônica para sincronização da ovulação em vacas

Effect of fixative type and fixation time on the morphology of equine preantral ovarian follicles

O objetivo deste estudo foi investigar a eficácia dos fixadores teciduais Bouin, Carnoy ou Formol 10% em fragmentos ovarianos equinos. Ovários (n=4) de éguas, sem raça definida, foram obtidos de abatedouro local e transportados em recipiente térmico a 20 ºC. Imediatamente após a coleta, os ovários foram lavados com solução de PBS modificado (Cultilab®, Campinas-SP, Brasil), e divididos em nove fragmentos com aproximadamente 5x5x1 mm, retirados do parênquima de cada ovário. Em seguida, os fragmentos ovarianos foram imersos em um dos três diferentes fixadores, Bouin (B), Carnoy (C) ou Formol 10% (F), por 6, 12 ou 24 horas. Cada fragmento foi acondicionado individualmente em um frasco contendo aproximadamente 20 vezes o volume da solução fixadora. Após este período, foram mantidos em álcool 70% por 24 horas. Para cada fixador e tempo foram realizadas quatro réplicas. No processamento histológico, os fragmentos foram desidratados em concentrações crescentes de álcool, diafanizados em xilol e incluídos em parafina. Em seguida, foram feitos cortes seriados de 5 ?m em micrótomo rotativo (Leica®, Wetzlar-Alemanha), seguidos da montagem de lâminas e coloração com ácido periódico de Schiff (PAS) e hematoxilina. Foram avaliadas 540 lâminas com 1.620 cortes histológicos, contendo 465 folículos pré-antrais que foram classificados como íntegros ou degenerados. A degeneração foi detectada pela presença de pelo menos um dos seguintes aspectos: retração do citoplasma, núcleo picnótico, vacúolos citoplasmáticos, deslocamento das células da granulosa e/ou rompimento da membrana basal. Um teste de regressão logística foi utilizado para a análise estatística, e as diferenças foram consideradas significativas quando P<0,05. O fixador Carnoy utilizado por 24 horas proporcionou as melhores condições de integridade morfológica (53,3%; 32/60) em relação aos demais, sendo Boiun por 24 h o tratamento menos eficaz (19,1%; 9/47). Os demais tratamentos apresentaram os resultados a seguir: C12h 50% (30/60), C6h 40% (24/60), F24h 37,8% (17/45), F12h 35,1% (13/37), F6h 32% (16/50), B12h 30,5% (18/59) e B6h 24,4% (11/45). Portanto, sugerimos que a fixação de tecido ovariano equino com Carnoy por 24 horas é o mais indicado para preservação morfológica de folículos pré-antrais.The aim of this study was to investigate the efficacy of the tissue fixatives Bouin, Carnoy and 10% Formaldehyde in equine ovarian fragments. Ovaries (n=4) from mares of mixed breeds were obtained at a local slaughterhouse and transported at 20 ºC in a thermo container. Immediately after collection, the ovaries were washed with a modified PBS solution (Cultilab®, Campinas-SP, Brazil) and divided into nine fragments with approximately 5x5x1 mm, removed from the parenchyma of each ovary. The ovarian fragments were then immersed in three different fixatives, Bouin (B) Carnoy (C) or 10% Formaldehyde (F) for 6, 12 or 24 hours. Each fragment was individually immersed in a 20 mL tube containing 20 times the volume of fixative solution. After this period, the fragments were held in 70% ethanol for 24 hours. Each procedure was performed in four replicates. For histological analysis, the specimens were dehydrated in increasing concentrations of alcohol, submitted to diaphanization in xylol and embedded in paraffin. Serial sections of 5 ?m were made with the use of a rotating microtome (Leica® type, Wetzlar, Germany), followed by slide mounting and staining with periodic acid-Schiff (PAS) and hematoxylin. A total of 540 slides with 1,620 sections were evaluated, which contained 465 preantral follicles that were classified as normal or degenerated. Follicles were considered as degenerated when presented at least one of the following aspects: cytoplasm retraction, pyknotic nucleus, cytoplasmic vacuoles, displacement of granulosa cells and/or disruption of the basal membrane. A logistic regression test was used for statistical analysis, and differences were considered significant when P<0.05. The Carnoy fixative, when used for 24 hours, provided the best conditions of morphological integrity (53.3%; 32/60) compared to all others, and the use of Boiun for 24 hours was considered the worst treatment (19.1%; 9/47). The other treatments lead to the following results: C12h 50% (30/60), C6 H 40% (24/60), F24h 37.8% (17/45), F12h 35.1% (13/37), F6h 32% (16/50), B12h 30.5% (18/59) and B6h 24.4% (11/45). Therefore, we suggest that fixation of equine ovarian tissue with Carnoy for 24 hours is the most suitable protocol for morphological preservation of pre-antral follicles

Recovery of equine oocytes by scraping of the follicular wall with different specifications of needles and morphological analysis of cumulus oophorus

In follicular aspiration, physical aspects are of high significance for the technique to succeed, such as vacuum pressure, caliber of the needle and the way the follicular wall curettage is performed. The aim of this study was to investigate the recovery rate of equine oocytes aspirated by scraping of the follicular wall, testing different calibers of disposable needles, as well as the morphological evaluation of the cumulus oophorus complexes (COCs). Mares ovaries (n=447) obtained at a local slaughterhouse were transported to the laboratory in a thermal container (20 °C) and had the tunica albuginea and connective tissues dissected. The aspirated follicles had 10 to 25 mm in diameter, and 30x8 (21G 1 ¼) or 40x12 (18G 1 ½) needles were used for the aspiration, forming group A (G-A) and group B (G-B), respectively. In G-A and G-B, 480 and 548 follicles were aspirated, respectively. Under the stereomicroscope, the oocytes were evaluated according to the quality of the ooplasm and characteristics of the cumulus cells (grade I, II, III and denuded). The statistical analysis was performed using the Student’s t-test, logistic regression and test of proportions, and differences were considered significant when P&lt;0.05. There was no difference between recovery rates of groups G-A (66.5%; 330/496) and G-B (65.5%; 359/548). In the G-A group, grade II oocytes were related to higher recovery rates (46.9%; 145/330) than grade I (23.6%; 72/330), grade III (20.6%; 59/330) and denuded oocytes (8.5%; 24/330; P&lt;0.05). However, in G-B, there was no statistical difference regarding the quality of the recovered oocytes: grade I (23.4%; 77/359), grade II (43.2%; 145/359), grade III (22.5%; 73/359) and denuded (11.1%; 32/359). The 30x8 (21G 1 ¼) needle provided a higher proportion of grades I and II oocytes than the 40x12 (18G 1 ½) needle, with 72.4% (239/330) and 65% (233/359; P&lt;0.05), respectively. Both calibers of needles tested in this study provide efficient oocyte recovery rates. Aspiration with 30x8 (21G 1 ¼) needles resulted in a higher proportion of morphologically good equine oocytes for use in reproductive biotechnologies. </p