Importance of sperm genotype (indicus versus taurus) for fertility and embryonic development at elevated temperatures

Heat stress has negative effects on bovine reproduction, particularly for European breeds (Bos taurus taurus) that are less thermotolerant than zebu cattle (Bos taurus indicus). Here, the evidence that spermatozoa and oocyte both contribute to early embryonic resistance to heat shock is demonstrated. In addition, the use of reproductive biotechnologies to improve bovine thermotolerance, are outlined by comparing data from taurus, indicus and crossbred genotypes. (c) 2005 Published by Elsevier B.V.

Effects of fetuin on zona pellucida hardening, fertilization and embryo development in cattle

Mammalian oocytes can undergo spontaneous meiotic maturation when they are liberated from their follicles and cultured in vitro; however, the zona pellucida (ZP) becomes resistant to chymotrypsin digestion, or hardens, when spontaneous maturation occurs in serum-free medium. Schroeder et al. [Biol. Reprod. 43 (1990) 891] described that fetuin, a component of fetal calf serum (FCS), inhibits ZP hardening during oocyte maturation. The aim of this experiment was to study the effect of the presence of cumulus cells and addition of hormones to maturation media on bovine zona hardening and embryo development in medium with and without fetuin. In Experiment 1, different concentrations of fetuin were added to the maturation medium. The time necessary for digestion of 50% of the ZP (d50) was not different when oocytes were matured in presence of 10% FCS, 1 mg/ml polyvinyl alcohol (PVA), or 4, 1 and 0.25 mg/ml of fetuin; cleavage rates were also similar. However, significantly more blastocysts (P < 0.05) were formed when FCS was used compared to PVA and 0.25 mg/ml of fetuin. In Experiment 11, we examined the influence of the presence of cumulus cells and hormones during the maturation of oocytes in media with PVA, BSA, FCS and fetuin. The d50 was significantly higher (P < 0.05) when oocytes were matured in presence of cumulus cells. The cleavage rate of cumulus-intact oocytes was similar for all groups. However, when oocytes were partially stripped before maturation, the cleavage rate was significantly higher (P < 0.05) when FCS or fetuin was used. In both stripped and non-stripped groups, significantly more blastocysts (P < 0.05) were formed when oocytes were matured with FCS compared to BSA and PVA. These results indicate that zona hardening, as described for mouse and human oocytes, does not have a large effect on bovine cumulus-intact oocytes. Apparently fetuin can be used as a substitute for FCS during bovine oocyte maturation, since it leads to similar developmental rates as FCS in intact and partially stripped oocytes. (C) 2002 Published by Elsevier B.V. B.V.

The use of transmission electron microscopy and oocyte transfer to evaluate in vitro maturation of equine oocytes in different culture conditions

The current study evaluates the ability of equine oocytes matured in different conditions to undergo nuclear and cytoplasmic maturation.. After oocyte transfer, embryonic development was diagnosed at 1.5 and 90 days of gestation. For each group, immature oocytes obtained from slaughterhouse ovaries were matured in vitro (5 replicates). In experiment I, three different media were tested. HTF:BME, SOFaa, and TCM 199. In experiment 11, the HTF:BME was chosen as maturation medium containing pFSH, eFSH, or eFSH + eGH. Nuclear maturation was estimated after stripping the oocytes and staining with Hoechst 33342. The evaluation of cytoplasmic maturation was performed by transmission electron microscopy. For oocyte transfer, six non-cycling recipient mares were used, and 8 to 15 oocytes were transferred in each mare. In experiment I, the results showed no differences (P > .05) in nuclear maturation (MII) among experimental groups. The percentage of MII was 29.3 ( +/- 9.6), 23.4 ( +/- 8.4), and 13.5 ( +/- 12.4) for HTF:BME, SOF, and TCM, respectively. In experiment II, all media tested were efficient in inducing metaphase II. Also, no statistical differences (P > .05) were observed in percentages of nuclear maturation rates when porcine (37.1 +/- 22.4) or equine (25.8 +/- 8.2) FSH were used, or when eFSH + eGH was added to HTF:BME (29.4 +/- 12.3). The analysis of cytoplasmic morphology of oocytes cultured in TCM 199 and SOFaa showed signs of incomplete cytoplasmic maturation and premature cortical reaction. Meanwhile, oocytes cultured in HTF:BME medium presented cytoplasmic characteristics similar to those described by others for in vivo-matured oocytes. The addition of eFSH to the HTF:BME medium resulted in an improvement of cytoplasmic morphology. After oocyte transfer, two mares became pregnant, one from pFSH group and one from eFSH+eGH group. These results indicate that although in vitro matured equine oocytes are capable of fertilization and embryonic development, the percentage of competent oocytes is still low.

Parthenogenetic development of domestic cat oocytes treated with ionomycin, cycloheximide, roscovitine and strontium

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of melatonin on DNA damage of bovine cumulus cells during in vitro maturation (IVM) and on in vitro embryo development

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Morphological records of oocyte maturation in the parthenogenetic tick Amblyomma rotundatum Koch, 1844 (Acari: Ixodidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of strontium for bovine oocyte activation

Strontium efficiently activates mouse oocytes, however, there is limited information on its use in cattle. Thus, the objective of this study was to establish a suitable protocol for activating bovine oocyte with strontium. For pronuclear development, the absence of calcium and magnesium in the activation medium (TALP) with 10 and 50mM strontium (34.4 and 53.1%, respectively) was superior to the complete TALP (6.5 and 19.4%, respectively). In all activation media, better results were observed with 25 and 50 mM strontium (21.9-53.1 and 19.4-53.1%, respectively). Incubation for 4 h promoted similar results in all strontium concentrations. However, strontium at 15, 20, and 25 mM for 6 and 8 h (40.7, 46.7, and 48.3%, and 29.3, 48.3, and 40.7%, respectively) were superior to control (15.5 and 10%, respectively). After in vitro maturation for 26 h, strontium (S; 20 mM in Ca2+ and Mg2+-free TALP for 6 h), ionomycin + strontium (IS), and strontium + ionomycin (SI) (60, 63.3, and 65%, respectively) were similar in pronuclear development and superior to ionomycin (I; 5 mu M for 5 min; 36.7%). In treatments S and I, only 1 PN zygotes were observed. In treatment S, most of them had 1 and 2 PB (35.7 and 60.7%, respectively), and in treatment I, 0, 1, and 2 PB (14.3, 57.1, and 28.6%, respectively). Most of the zygotes in treatment IS and SI were 1 PN 2 PB (77.4 and 61.6%, respectively). The number of oocytes with clusters of cortical granules was similar in all treated groups (11-29%). Cortical granule exocytosis in treatment IS (68%) was similar to S (54%) and superior to 1, SI, and control (27, 45, and 5.0%, respectively). Cleavage and blastocyst rates were similar for S, I, IS, and SI treatments (61.7-76.7, and 8.3-13.3%, respectively) and the same was observed for ICM, TE, and total cell number, and ICM/total cell ratio (22-25, 64-69, and 86-95, and 0.26-0.27). In conclusion, strontium may be efficiently applied for bovine oocyte activation at 20 mM in Ca2+-and Mg2+-free TALP medium for 6 h.

Influence of sire breed (Bos indicus versus Bos taurus) and interval from slaughter to oocyte aspiration on heat stress tolerance of in vitro-produced bovine embryos

Based on in vitro experiments, Bos indicus embryos were more resistant to heat stress (HS) than Bos taurus embryos. To increase knowledge regarding differences between Bos indicus and Bos taurus in resistance to HS, the primary objective of this study was to determine if tolerance to HS is due to the breed, origin of the oocyte, sperm, or both. Additionally, the influence of the interval between ovary acquisition (in the abattoir) and oocyte aspiration in the laboratory, on early embryo development was ascertained. Oocytes were collected from Nelore and Holstein cows in an abattoir; 4.0 or 6.5 h later, oocytes were aspired in the laboratory, and then matured and fertilized using semen from Nelore (N), Gir (GIR), or Holstein (H) bulls. Ninety-six h post insemination (hpi), embryos with >= 16 cells were divided in two groups: control and HS. In the control group, embryos were cultured at 39 degrees C, whereas in the HS group, embryos were subjected to 41 degrees C for 12 h, and then returned to 39 degrees C. Rates of cleavage, and formation of morula and blastocysts were higher (P < 0.05) for oocytes aspirated at 4.0 versus 6.5 h after ovaries were acquired. Heat stress decreased rates of blastocyst formation for all breeds (N X N; H x H; and H X GIR) and in both time intervals (4.0 and 6.5 h). However, N X N had higher cleavage rate (P < 0.05) in both time intervals when compared with H X H and H X GIR. In addition, Nelore oocytes fertilized with Nelore semen (N X N) had higher blastocyst yields (P < 0.05) in the control and HS group, when compared with the other two breeds (H X H and H X GIR). We concluded that the breed of origin of the oocyte was more important than that of the sperm for development of thermotolerance, because bull breed did not influence embryo development after HS, and in vitro early embryonic development was impaired by increasing (from 4 to 6.5 h) the interval between ovary acquisition and oocyte aspiration. (C) 2011 Elsevier B.V. All rights reserved.

Follicular somatic cell factors and follicle development

Considerable attention is currently paid to oocyte-derived secreted factors that act upon cumulus and granulosa cells. Also important for follicle development are somatic cell-derived secreted factors. This is illustrated by the ability of granulosa cell-derived Kit ligand (KITL) to promote primordial follicle activation, and the loss of follicle development that accompanies KITL gene disruption. This review summarises our current understanding of somatic cell factors during both preantral and antral follicle growth, involving not only signalling from granulosa cells to the oocyte, but also signalling between granulosa and theca cells. Principal granulosa cell-derived factors include activin, anti-Mullerian hormone (AMH), bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Theca cells also secrete BMPs and FGFs. The interplay between these factors is equally important for follicle growth as the activity of oocyte-derived factors.

Structural and ultrastructural characterization of the embryonic development of Pseudoplatystoma spp. hybrids

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bovine dominant follicular fluid promotes the in vitro development of goat preantral follicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Complete replacement of the mitochondrial genotype in a Bos indicus calf reconstructed by nuclear transfer to a bos taurus oocyte

Due to the exclusively maternal inheritance of mitochondria, mitochondrial genotypes can be coupled to a particular nuclear genotype by continuous mating of founder females and their female offspring to males of the desired nuclear genotype. However, backcrossing is a gradual procedure that, apart from being lengthy, cannot ascertain that genetic and epigenetic changes will modify the original nuclear genotype. Animal cloning by nuclear transfer using host ooplasm carrying polymorphic mitochondrial genomes allows, among other biotechnology applications, the coupling of nuclear and mitochondrial genotypes of diverse origin within a single generation. Previous attempts to use Bos taurus oocytes as hosts to transfer nuclei from unrelated species led to the development to the blastocyst stage but none supported gestation to term. Our aim in this study was to determine whether B. taurus oocytes support development of nuclei from the closely related B. indicus cattle and to examine the fate of their mitochondrial genotypes throughout development. We show that indicus:taurus reconstructed oocytes develop to the blastocyst stage and produce live offspring after transfer to surrogate cows. We also demonstrate that, in reconstructed embryos, donor cell-derived mitochondria undergo a stringent genetic drift during early development leading, in most cases, to a reduction or complete elimination of B. indicus mtDNA. These results demonstrate that cross-subspecies animal cloning is a viable approach both for matching diverse nuclear and cytoplasmic genes to create novel breeds of cattle and for rescuing closely related endangered cattle.

Effects of heat stress on development, quality and survival of Bos indicus and Bos taurus embryos produced in vitro

Heat stress is an important cause of poor development and low survival rates in bovine embryos. Experiments were conducted to test the hypothesis that Bos indicus embryos are more resistant to heat stress than are Bos taurus embryos. In experiment 1, Nelore and Jersey embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 6 hours), developmental ratios were assessed at Day 7 (Day 0 = day of fertilization), and blastocysts were frozen for RNA extraction. Experiment 2 evaluated expression of COX2, CDX2, HSF1, and PLAC8 in previously frozen blastocysts. In experiment 3, Nellore and Angus embryos from oocyte pick-up-derived oocytes were submitted to heat stress (96 hours post-insemination, 41 °C, 12 hours) and transferred to recipients on Day 7. In experiment 4, embryos developed as in experiment 3 were fixed for Terminal deoxynucleotidyl transferase dUTP nick end labeling labeling and total cell counting. In experiment 1, heat stress decreased the percentage of Jersey oocytes that became blastocysts, but had no effect on Nellore embryos (34.6%, 25.0%, 39.5%, and 33.0% for Jersey control, Jersey heat-stressed, Nellore control, and Nellore heat-stressed oocytes, respectively; P < 0.05). In experiment 2, heat stress decreased (P < 0.05) expression of CDX2 and PLAC8, with higher expression of these genes in Nellore embryos than in Jersey embryos. Heat stress also decreased (P < 0.05) expression of COX2 in Jersey embryos, but had no effect on Nellore embryos. Expression of HSF1 was decreased (P < 0.05) by heat stress in both breeds, with a greater effect in Nellore embryos. In experiment 3, heat stress tended (P = 0.1) to decrease the percentage of pregnancies among cows (Day 30 to 35) that received Angus embryos. In experiment 4, heat stress increased (P < 0.05) the percentage of apoptotic blastomeres, but had no breed-specific effects. In addition, Nellore embryos had fewer (P < 0.05) Terminal deoxynucleotidyl transferase dUTP nick end labeling- positive blastomeres than did Angus embryos. We concluded that the detrimental effects of heat stress were dependent upon embryo breed and were more evident in Bos taurus embryos than in Bos indicus embryos. © 2013 Elsevier Inc.

Effect of ricinoleic acid esters from castor oil (Ricinus communis) on the oocyte yolk components of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari: Ixodidae)

Rhipicephalus sanguineus are bloodsucking ectoparasites, whose main host is the domestic dog, thus being present in urban areas and closely located to people. Eventually, this tick species parasitize humans and can become a potential vector of infectious diseases. Methods to control this type of pest have been the focus of many research groups worldwide. The use of natural products is increasingly considered nowadays, due to the low toxicity levels to the host and low waste generation to the environment. This study tested the effect of ricinoleic acid esters from castor oil (as an potential acaricide) on the reproductive system of R sanguineus females, more specifically on the vitellogenesis process. For this, two groups were established: the control group (CG) and the treatment group (TG) with five rabbits in each (New Zealand White), used as hosts. NaCl and ester were added to rabbits' food and offered to the hosts. After full engorgement, the females were collected and had their ovaries extracted. The ticks ovaries were submitted to histochemical techniques so the effects of esters could be observed over polysaccharides, proteins and lipids yolk. Changes in the deposition of yolk components were observed. This caused modifications on elements of polysaccharide origin and on glycoprotein compounds, interfering in the final yolk synthesis and compromising the development of the future embryo. © 2012.