Oocyte cryopreservation is a strategic tool for assisted reproduction, but has limited use due to the complex cellular structure of oocytes, which leads to sub-optimal survival rates. In this study, we used the SPOM in vitro maturation system, which is based on supplementation of cAMP modulators in order to extend meiotic arrest and improve oocyte maturation. cAMP modulators (Forskolin and IBMX) were administered in a short term culture (STC) before or after vitrification, followed by an extended maturation with cilostamide. We hypothesized that a STC with cAMP modulators would improve immature oocyte health and enhance cryotolerance. We found vitrification caused oocyte damage in a great extent, impairing nuclear maturation rates in all vitrified groups (Percentage of matured oocytes: CONT FRESH 77.8 c ; CONT VIT 31.4 ab ; STC/VIT 39.5 b ; VIT/STC 18.6 a). Vitrification also promoted degradation of cytoskeletal actin filaments (Percentage of
Context In vivo embryo production, also called multiple ovulation and embryo transfer, can accelerate genetic gain, and thus improve animal production. However, there are issues limiting a wider use of this biotechnology in sheep livestock. Aims This study aimed to determine (1) whether a previous response to superovulation (SOV) can be used as a criterion to select ewes for in vivo embryo production, (2) whether the intensity of the SOV response (number of corpora lutea, CL) can affect the embryo recovery rate, and (3) whether the number of CL quantified by colour Doppler ultrasonography can be used to calculate the recovery rate. Methods Twenty-five Santa Inês ewes underwent SOV three times (SOV1, SOV2 and SOV3), with 200 mg FSH and natural mating. The number of CL after each SOV was determined by laparoscopy and by colour Doppler ultrasonography. Key results The number of CL significantly decreased (P < 0.05) after SOV1 (7.5 ± 4.8) to 3.0 ± 5.0 at SOV 2 and 2.2 ± 3.5 at SOV3. Strong correlations were observed between SOV2 and SOV3 in terms of numbers of CL (r = 0.86, r2 = 0.74; P < 0.0001) and viable embryos (r = 0.79, r2 = 0.63; P < 00001). However, no correlations were observed between SOV1 and SOV2 or between SOV1 and SOV3. Recovery rate did not differ with the intensity of the SOV response (≤6, 7–10, >10 CL) or between the methods used to quantify CL. Conclusions Ewes did not show the same pattern of response when submitted to successive FSH-based SOV. The intensity of the SOV response did not affect the recovery rate, and the number of CL estimated by colour Doppler ultrasonography can be used to calculate the recovery rate. Implications Selecting sheep embryo donors by a previous SOV response is not always feasible. The recovery rate is homogeneous and it is not affected by the intensity of the SOV response. A nonsurgical technique can be used to assess the recovery rate, improving animal welfare in MOET programs.
The present study aimed to evaluate the effect of the simulated physiological oocyte maturation (SPOM) system on F1 Gyr × Holstein oocytes and embryos by evaluating the meiotic arrest, embryo production rates, total number of cells and lipid score. Three experiments were conducted and the following three experimental groups were formed according to in vitro maturation (IVM) treatments: CONTROL 1 (TCM 199 medium without FBS), CONTROL 2 (commercial medium) and SPOM (TCM 199 medium with forskolin and 3-isobutyl-1-methylxanthine (IBMX) in pre-IVM and extended IVM with cilostamide). In the first experiment (ovum pick-up), a significant (P < 0.05) reduction in the percentage of matured oocytes in SPOM group was observed. In the second (slaughterhouse ovaries) and third (ovum pickup) experiments, the cleavage and blastocyst rates were reduced (P < 0.05) in the SPOM group. There was no significant (P > 0.05) difference in total number of cells among the groups. No difference (P > 0.05) was found on lipid score among the groups at Day 7 of development, in both Experiments 2 and 3. At Day 9 (Experiment 2), only the CONTROL 2 showed a significant increase (P > 0.05) compared with the other treatments. It was concluded that under our conditions, the SPOM system was efficient in prolonging meiotic arrest on Gyr × Holstein oocytes, offering the oocytes in vitro conditions more similar to those found in vivo; however, it adversely affected embryo production rates and promoted no beneficial effect on the total number of cells and the lipid score.
RESUMOO objetivo deste estudo foi avaliar o efeito da adição de probiótico com ou sem Ca e Zn na mistura mineral sobre a concentração sérica de Zn em ovinos. A adição de probióticos contendo Ca e Zn resultou na redução da concentração sérica de Zn em relação aos grupos Gc e GP, respectivamente. Não houve diferença significativa (p<0,05) entre os grupos e dias de observação, portanto a adição de elementos minerais aos probióticos torna-se prejudicial à saúde dos ovinos, podendo predispor os animais a apresentar deficiências subclínicas e/ou clínicas de um determinado mineral ao longo do tempo da utilização destes produtos.
Os objetivos do presente trabalho foram: avaliar, na primavera e verão (novembro a março), a concentração plasmática de progesterona em ovelhas lanadas e deslanadas criadas no oeste paulista; avaliar a concentração plasmática de progesterona em função do tempo de permanência do progestágeno (6, 9 ou 12 dias) em protocolo de inseminação artificial em tempo fixo (IATF). No experimento 1 foram utilizadas 12 ovelhas mestiças: 6 padrão Texel (Te) e 6 Santa Inês (SI), nas quais foram feitas, entre novembro e março, sete colheitas de sangue (C1 a C7), por venopunção da jugular, para posterior dosagem de progesterona (P4)por radioimunoensaio (RIA). No experimento 2 foram utilizadas 38 ovelhas Te e SI divididas aleatoriamente em três grupos: G-6 (n= 13); G-9 (n= 13); e G-12 (n= 12). Inicialmente cada ovelha recebeu uma esponja intravaginal de progestágeno (D0) que permaneceu por 6 (G-6), 9 (G-9) ou 12 dias (G-12). Na retirada da esponja foram administrados, por via intramuscular, 0,1315 mg de prostaglandina F2α (PGF2α) e 300 UI de gonadotrofina coriônica equina (eCG). A IATF, por via laparoscópica, foi feita a partir de 50 horas após a retirada do progestágeno. Trinta dias após foi realizado diagnóstico de gestação através de ultrassonografia transabdominal. Exp. 1. Com exceção da colheita 7 (C7), em todas as outras o grupo SI apresentou concentração de P4 estatisticamente superior (P<0,05) ao grupo Te. Exp. 2. No momento da retirada do progestágeno o G-12 apresentou concentração de P4 significativamente (P<0,05)menor (0,342 ng/mL) que o G-6 (1,684 ng/mL) e G-9 (1,762 ng/mL). No entanto não houve diferença na taxa de prenhez entre os grupos G-6 (76,9%), G-9 (61,5%) e G-12 (91,6%). As ovelhas SI apresentaram concentração plasmática de P4 maior que as ovelhas Te nos períodos avaliados. A duração da permanência do progestágeno não afeta a taxa de prenhez em ovelhas.
Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modifications that render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium, heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PM and would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes in bull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quickly than buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similar whether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by mini-Percoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status and sperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed sperm after mini-Percoll processing followed by incubation with or without heparin supplementation.Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percoll and supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes), capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and 18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P < 0.05) in most of the sperm motility parameters after mini-Percoll. Conversely, ovine samples presented the highest (P < 0.05) rate of acrosome-reacted cells after mini-Percoll. Heparin supplementation did not affect most of the parameters evaluated (P > 0.05). In caprine and bovine species, a lower (P < 0.05) rate of sperm agglutination was observed in the presence of heparin at 18 h of incubation. In the absence of heparin, ovine samples showed a higher (P < 0.05) agglutination rate compared to the bovine species after long incubation period.Discussion: The present study compared sperm parameters (sperm kinematics, agglutination rate and capacitation status) of ruminant frozen-thawed sperm after mini-Percoll selection followed by in vitro incubation with or without heparin supplementation. In this study, it was observed the same rate of capacitated cells after the sperm selection (min-Percoll) between ruminant species. This indicate that the capacitation process occurs similarly between ruminant species, refuting the first hypothesis of this study. The presence of heparin did not influence the capacitation status of ruminant frozen-thawed sperm after mini-Percoll selection, it demonstrates that the second hypothesis was supported by this study making more economic and practical the use of ruminant frozen-thawed semen. The absence of heparin in the incubation medium did not harmed the capacitation status and sperm agglutination of ruminant frozen-thawed sperm. This supported the third hypothesis of the current study and indicate that the use of mini-Percoll technique regardless the presence of heparin could be a useful alternative for the preparation of ruminant frozen-thawed sperm. In conclusion, the capacitation status of ruminant frozen-thawed sperm is similar whether or not heparin is present after the mini-Percoll technique.
Holstein-Gyr crossbred cattle are strategic for dairy systems in tropical countries, since they combine milk yield genetics with adaptability to tropical climate. However, Holstein (Bos taurus) and Gyr (Bos indicus) breeds present remarkable differences regarding reproductive physiology. Brazil stands out as the world's largest user of embryo in vitro production (IVP) in bovine, and the use of this technique is increasing in dairy systems. As Holstein-Gyr crossbreds are important oocyte donors for IVP, the present work aimed at investigating whether increased Gyr or Holstein breed composition influences donor's performance. Sixteen Holstein-Gyr crossbred females presenting increased (HG, 71.4 to 87.5% Holstein; n = 9) or decreased (GH, 40.2 to 46.6% Holstein; n = 7) Holstein composition were submitted to three ovum pick up (OPU) sessions. We observed similar (P = 0.2946) antral follicle count between HG and GH donors (24.8 ± 3.2 vs 29.4 ± 2.8 respectively; mean ± SEM). Groups also display similar morphological oocyte grading (Grade I: 0.1 ± 0.1 vs 0.1 ± 0.1 -P = 0.9680; Grade II: 0.9 ± 0.5 vs 1.9 ± 0.5 -P = 0.1942; Grade III, 4.0 ± 1.2 vs 7.2 ± 1.4 -P = 0.1047, HG vs GH respectively; mean ± SEM). Additionally, the proportion of viable oocyte was similar between HG and GH groups (27.8% vs 31.9%, respectively, P = 0.3500) and oocyte lipid area fraction (6.8% vs 9.5%, respectively; P = 0.1539). Our results indicate that the individual variation has more influence than breed composition of crossbred oocyte donors.
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