An in vitro model of embryo production was used to examine the effects of insulin-like growth factor (IGF)-I on maturation and developmental competence of oocytes exposed to heat shock. Cumulus–oocyte complexes were matured at 38.5°C or exposed to acute heat shock (HS; 41.5°C), with or without 100 ng/ml IGF-I, for 22 h through in vitro maturation. The experimental groups were control (C), C + IGF-I, HS, and HS + IGF-I. Oocytes were fertilized at the end of maturation, and the proportion of cleaved embryos was recorded 44 h later. HS during maturation increased the proportion of TUNEL-positive oocytes (P < 0.05). HS did not have any effect on cortical granule translocation but impaired resumption of meiosis, expressed as a decreased proportion of oocytes with nuclei in metaphase I (P < 0.05) and metaphase II (MII; P < 0.05). HS decreased the proportion of oocytes that cleaved (P < 0.05), in particular those oocytes that further developed to 4-cell-stage embryos (P < 0.05). IGF-I alleviated, to some extent, the deleterious effects of HS on the oocytes as reflected by a reduced proportion of TUNEL-positive oocytes (P < 0.03). While not significant, IGF-I tended to increase the proportion of MII-stage oocytes (P < 0.08) and 4-cell-stage cleaved embryos (P < 0.06). Further examination is required to explore whether IGF-I also affects the developmental competence of oocytes exposed to HS.

Much effort has been focused on establishing optimal conditions for obtaining in vitro maturation of oocytes from different species with results comparable to those achieved after in vivo development (reviewed by Brackett, 1992). However, even though extraordinary progress has been made, the in vitro technology for oocyte maturation lags far behind that in vivo and improvements are needed to increase the quantity and quality of the embryos produced from these matured oocytes.

Aiming to improve in vitro production of bovine embryos and to obtain supplements to replace serum for in vitro maturation (IVM), this study evaluated the effects of macromolecular supplementation of IMV medium (bovine serum albumin – BSA, polyvinyl alcohol – PVA, polyvinyl pyrrolidone – PVP, Ficoll, KnockoutSR, or fetal calf serum – FCS) and oxygen tension [5% CO2 in air (20% O2) or 5% CO2, 5% O2 and 90% N2 (5% O2)] on oocyte maturation and embryo development. Nuclear progression to germinal vesicle breakdown, metaphase I and metaphase II stages were evaluated and overall results revealed that undefined (FCS) and semi-defined (BSA) media gave better results at 20% O2 and defined media (PVA, PVP and Ficoll) at 5% O2. Independent of macromolecule supplement, IVM at 20% O2 was considered optimal for nuclear maturation. To evaluate embryo development, oocytes matured in the previously described conditions were fertilized and cultured at the same oxygen tension used for IVM and assessed for cleavage (43.0 to 74.8%) and development to morulae (16.4 to 33.8%), blastocyst (7.7 to 52.9%) and hatched blastocyst (9.6 to 48.1%). Apart from oxygen tension, all treatments, except Knockout (22.7%), gave similar results for blastocyst development (26.5 to 38.7%). Independently of macromolecule supplement, higher development rates were obtained in an oxygen tension of 20% O2 (67.4% cleavage, 29.2% morulae, 40.8% blastocyst and 34.0% hatched blastocyst) when compared with 5% O2 (52.5, 21.8, 18.2 and 15.6%, respectively). This study indicates that BSA, PVA, PVP and Ficoll can replace serum during IVM and that the optimal atmospheric condition for in vitro production of bovine embryos is 5% CO2 and 20% O2.

This study aimed to examine the gonadal morphology of diploid and triploid fish through stereological analysis. Triploid individuals were obtained after temperature shock (40°C for 2 min) at 2 min post-fertilization and reared until 175 days post-fertilization (dpf). Intact eggs were used to obtain the diploids. Gonads were collected for histological analysis at 83, 114, 144 and 175 dpf. Diploid females and males presented normal oogenesis and spermatogenesis through all the experimental period. Conversely, stereological analysis revealed that triploid females were sterile and oogonia were the prevalent cell type in the ovaries. Triploid males presented increased amounts of spermatocyte cysts and a large area of lumen when compared with diploids and in addition the amount of spermatozoa was lower than that observed for diploids. However, some triploid males presented spermatogenesis similar to diploids. Therefore, we concluded that triploidization is an interesting alternative to produce sterile individuals in A. altiparanae.

The antioxidant properties of Amburana cearensis extract may be a useful substitute for standard cell culture medium. Thus, the aim of this study was to evaluate the effect of this extract, with or without supplementation, on in vitro survival and development of sheep isolated secondary follicles. After collection of the ovaries, secondary follicles were isolated and cultured for 18 days in α-MEM+ supplemented with bovine serum albumin, insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (control medium) or into medium composed of different concentrations of A. cearensis extract without supplements (Amb 0.1; 0.2 or 0.4 mg/ml) or A. cearensis extract supplemented with the same substances described above for α-MEM+ supplementation. The A. cearensis supplemented medium was named Amb 0.1+; 0.2+ or 0.4+ mg/ml. There were more morphologically normal follicles in Amb 0.1 or Amb 0.4 mg/ml than in the control medium (α-MEM+) after 18 days of culture. Moreover, the percentage of antrum formation was significantly higher in Amb 0.1 or Amb 0.2 mg/ml than in α-MEM+ and Amb 0.1+ mg/ml, and similar to the other treatments. All A. cearensis extract media induced a progressive and significant increase in follicular diameter throughout the culture period. In conclusion, this study showed that 0.1 mg/ml of this extract, without supplementation, maintains follicular survival and promotes the development of ovine isolated secondary follicles in vitro. This extract can be an alternative culture medium for preantral follicle development.

The nuclei of spermatozoa leaving the testies are transcritionally inactivated and are structurally stabilised by extensive association of sperm DNA with protamines, highly basic proteins that replace somatic histones during spermiogenesis. During sperm matureation within teh epididymis, protamine–SH is oxidised to -SS-(Bedford & Calvin, 1974).

To enhance the probability of reprogramming somatic cell nuclei, fibroblast cells from an adult male rabbit and a 12-day-old fetus were fused with oocytes at the second metaphase. The chromosomes of recipient oocytes were then removed by treatment with demecolcine for 1 or 2 h after fusion. Demecolcine treatment of fused oocytes induced membrane protrusions that contained all the maternal chromosomes, thus making it possible to remove the chromosomes. The potential of nuclear-transferred oocytes to develop into blastocysts was high (48% and 59%) and the average cell number of the blastocysts was large (149 and 159) 96 h after in vitro culture. The proportions of nuclear-transferred oocytes enucleated 1 h after fusion and implanted after transfer to pseudopregnant recipients were relatively high (2.8% and 4.9%) compared with our previous reports (1.7%: Yin et al., 2000; 0.6% and 1.0%: Yin et al., 2002a) where donor cells were fused with previously enucleated oocytes. Of 34 adult somatic cell implantation sites, 6 had fetuses on day 12 or 14 of pregnancy, but none of the fetuses had a heart beat or developed to term. None of the nuclear-transferred oocytes whose chromosomes were removed 2 h after demecolcine treatment implanted after transfer to recipients. The possible reasons why the high-quality nuclear-transferred oocytes did not develop to term are discussed.

Maturation of porcine oocytes was examined after oocytes were cooled at the germinal vesicle stage. Cumulus-oocyte complexes (COCs) collected from medium-sized follicles were cooled at 24 °C or 4 °C for 5, 30 or 120 min in a solution with or without 1.5 M dimethylsulfoxide (DMSO). After rewarming, COCs were cultured in maturation medium at 39 °C, 5% CO2 in air for 44 h. Meiotic spindle organisation (by immunostaining and confocal microscopy), nuclear maturation (by orcein staining) and cytoplasmic maturation (by intracellular glutathione assay) of oocytes were examined after maturation. When COCs were cooled at 24 °C for various times in the medium without DMSO, a tendency to decreased spindle formation, nuclear maturation and cytoplasmic maturation was observed, but there was no statistical difference compared with controls. Addition of DMSO during cooling inhibited subsequent nuclear maturation and spindle formation. When COCs were cooled at 4 °C, both nuclear and cytoplasmic maturation as well as spindle formation were inhibited in most oocytes in a time-dependent manner. DMSO during cooling did not have any beneficial effect on subsequent oocyte maturation and spindle formation. These results suggest that porcine oocytes are very sensitive to a drop in the temperature before exposure to culture. Cooling oocytes before maturation inhibits their subsequent spindle organisation, nuclear and cytoplasmic maturation. Addition of DMSO to the cooling solution did not protect porcine oocytes from cooling-induced damage.

Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

Sperm preparation is an important step in the in vitro production of embryos. Centrifugation through colloids has been used to select normal sperm for assisted reproduction in several species. Animal models can sometimes be used as a preliminary step to investigate sperm preparation methods that are potentially of use for human fertility treatments. In this study bovine semen was prepared using three variants of the single-layer centrifugation sperm selection technique (Small, Mini, Mini-EP) with Bovicoll (Androcoll-B). Computer-assisted sperm motility analysis, the hypo-osmotic swelling test, and the sperm chromatin structure assay were performed on unselected (control) and SLC-selected sperm samples. Mini and Mini-EP gave the highest yield of motile spermatozoa, progressive motility and membrane integrity. In vitro fertilization trials were performed to investigate the fertilizing ability of the frozen–thawed bovine spermatozoa selected with Bovicoll. Mini-SLC (single-layer centrifugation) and swim-up (Control) were performed and cleavage rate and blastocyst rate did not differ significantly between groups. As there was a trend to an increased number of cells in blastocysts in the SLC group, the Mini-SLC method is at least as good as swim-up for selecting frozen–thawed bull spermatozoa for in vitro fertilization (IVF). This method could potentially be used to prepare human sperm for assisted reproduction.

The aim of the study was to examine the effects of insulin-like growth factor I (IGF-I) on ram sperm traits after hypothermic storage. Sperm ejaculates from Lacaune rams were diluted in a Tris extender, pooled, divided into groups of IGF-I doses tested (0, 10, 100 or 200 ng.ml−1) and stored (0–5°C) for 96 h. IGF-I elevated whole sperm motility as measured by a Computer-assisted Sperm Analyser (CASA) system, by 24 h (10 ng.ml−1) and 48 h (200 ng.ml−1) of storage, and by progressive movement on each day of storage. After 72 h the sperm samples were analysed for plasma membrane integrity (peanut agglutinin–fluorescein isothiocyanate), membrane stability (annexin V–Fluos) and apoptosis (Yo-Pro®-1) using fluorescence microscopy. The addition of IGF-I (at 100 or 200 ng.ml−1) reduced the ratio of sperm with disrupted membranes and the ratio of annexin V-labelled sperm. The ratio of apoptotic sperm was reduced by IGF-I given at 10 or 100 ng.ml−1 compared with control. Sperm fertilizing ability, determined at 48 h by an in vitro fertilization (IVF) test on bovine oocytes, was increased by IGF-I given at 100 ng.ml−1 from 47.0 to 67.7%. In conclusion, IGF-I maintained ram sperm functions following cooling storage and its effects were reflected in sperm fertilizing ability in vitro.

This study was conducted to verify the effect(s) of melatonin treatment on frog Leydig cells. Morphological observation after melatonin treatment indicates that many frog Leydig cells show degenerative changes (i.e. heterochromatic nuclei, loss of cellular adhesion) while in adjacent germinal tubules several Sertoli cells show heterochromatic nuclei, confirming the presence of a paracrine effect between interstitial and germinal compartments. The effect of melatonin on frog Leydig cell steroidogenesis was investigated in in vitro experiments; after 6 h of incubation melatonin severely inhibits both control and GnRH-induced testosterone secretion. In addition, in order to verify the effect of indolamine on frog Leydig cell activity, we investigated, by in situ hybridization, the presence of frog relaxin (fRLX, a transcript specifically expressed by these cells) in the testes of melatonin-injected animals after 48 h. fRLX signal completely disappeared from the testis of melatonin-injected frogs. The results of the present study indicate that melatonin treatment provokes Leydig cell morphological changes, blocks GnRH-antagonist-induced testosterone secretion and decreases fRLX expression. Taken together these results strongly indicate that melatonin acts on Leydig cells in the testis of the frog Rana esculenta.

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at −20 °C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).

The present study was to investigate if a completely chemically defined medium (PZM-4) could support the early development of porcine embryos derived from parthenogenetic activation (PA) and cloning (somatic cell nuclear transfer, SCNT), and to lay the foundation for determining the physiological roles of certain supplements in this medium. Porcine embryos derived from PA and SCNT were cultured in media: PZM-3 (a chemically semi-defined medium), PZM-4 (a fully defined medium), and PZM-5 (an undefined medium). Early embryo development was observed. We found that the three medium groups (PZM-3, PZM-4 and PZM-5) exhibited no significant differences in cleavage rates of PA embryos (p > 0.05), while the blastocyst rate in PZM-3 was significantly higher than in PZM-4 and PZM-5 (78.9% vs. 36.0% and 52.3%) (p < 0.05). Moreover, total cell number per blastocyst in PZM-3 was clearly higher than in PZM-5 but similar to that in PZM-4. As for SCNT embryos, no significant differences were observed for the cleavage rates or the blastocyst rates among the three groups (p > 0.05). However, total cell number per blastocyst in PZM-3 was notably higher than in PZM-5, but was similar to that in PZM-4. In conclusion, our results suggested that the completely chemically defined medium PZM-4 can be used to efficiently support the early development of porcine PA and SCNT embryos.

Competent oocyte selection remains a bottleneck in the in vitro production (IVP) of mammalian embryos. Among the vital assays described for selecting competent oocytes for IVP, the brilliant cresyl blue (BCB) test has shown consistent results. The aim of the first experiment was to observe if oocytes directly submitted to IVM show similar cleavage and blastocyst rates as those obtained with oocytes maintained under the same in vitro conditions as the oocytes that undergo the BCB test. Bovine cumulus–oocyte complexes (COCs) were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised grouped into three groups: (1) directly submitted to IVM; (2) oocytes submitted to the BCB test without the addition of BCB stain (BCB control group); and (3) submitted to the BCB test. The results showed that oocytes directly submitted to IVM reached similar cleavage (48/80 – 60%) and embryonic development rates to the blastocyst stage (10/48 – 21%) as the results obtained with the BCB control group oocytes (45/77 – 58% and 08/45 – 18%, respectively). The aim of the second experiment was to determine the cleavage and blastocyst rates obtained from BCB+ oocytes undergoing IVM in the presence of BCB– oocytes at a ratio of 10:1. COCs were recovered from slaughterhouse-derived ovaries and, after morphological evaluation, were randomised into two groups that were submitted to IVM either directly (1: control group) or submitted to the BCB test prior to IVM. After the BCB test, the COCs were classified as either BCB+ (blue cytoplasm) or BCB– (colourless cytoplasm) and then divided into four experimental groups: (2) BCB+; (3) BCB–; and (4) BCB+ matured in same IVM medium drop as (5) BCB– at a ratio of 10:1. After IVM (24 h), oocytes from the different experimental groups were submitted to in vitro fertilisation (IVF) and in vitro culture (IVC) under the same culture conditions until they reached the blastocyst stage (D7). With regards to the cleavage rate (48 h after IVF), only group 3 (102/229 – 44%) differed (P < 0.05) from the other groups [1 (145/241 – 60%); 2 (150/225 – 67%); 4 (201/318 – 63%) and 5 (21/33 – 63%)]. On day 7, the embryos from group 2 (BCB+) achieved the highest blastocyst rate (46/150 – 31%) (P < 0.05) when compared with the embryo development capacity of the other experimental groups (1: 31/145 – 21%; group 3: 17/102 – 17%; group 4: 46/201 – 23%; and group 5: 2/21 – 10%). In conclusion, submitting BCB+ oocytes that were separated from BCB– oocytes to IVM increases the rate of embryonic development to the blastocyst stage when compared to the control group, BCB– oocyte group, BCB+ paracrine group and BCB– paracrine group. The presence of non-competent oocytes during IVM, even in low proportion (1:10), reduces the capacity of competent oocytes to undergo embryo development and achieve blastocyst stage during IVC.

Numerous factors affect vitrification success and post-thaw development of oocytes after in vitro fertilization. Therefore, elaboration of an optimal methodology ensuring higher cryotolerance of oocytes and subsequent blastocyst yield is still of great interest. This paper describes and evaluates critical factors affecting the success of oocyte vitrification. In particular, an appropriate oocyte stage such as maturation status (germinal vesicle stage, metaphase II stage), presence/absence of cumulus cells before vitrification, and the effect of follicle size, as well as different culture systems and media for in vitro production of embryos, the types and concentrations of cryoprotectants, and cooling and warming rates at vitrification are considered. Special attention is paid to various cryocarriers used for low-volume vitrification, which ensures safe storage of oocytes/embryos in liquid nitrogen and their successful post-thaw recovery. At the end, we focussed on how age of oocyte donors (heifers, cows) influences post-thaw development. This review summarizes results of recently published studies describing different methodologies of cryopreservation and post-thaw oocyte development with the main focus on vitrification of bovine oocytes.

Insulin, transferrin and selenium (ITS) supplementation to oocyte maturation medium improves the post-fertilization embryonic development in pigs. ITS is also commonly used as a supplement for the in vitro culture (IVC) of embryos and stem cells in several mammalian species. However, its use during IVC of pig embryos has not been explored. This study investigated the effect of ITS supplementation to IVC medium on the in vitro development ability of pig embryos produced by parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT). We observed that ITS had no significant effect on the rate of first cleavage (P > 0.05). However, the rate of blastocyst formation in ITS-treated PA (45.3 ± 1.9 versus 27.1 ± 2.3%), IVF (31.6 ± 0.6 versus 23.5 ± 0.6%) and SCNT (17.6 ± 2.3 versus 10.7 ± 1.4%) embryos was significantly higher (P < 0.05) than those of non-treated controls. Culture of PA embryos in the presence of ITS also enhanced the expansion and hatching ability (29.1 ± 3.0 versus 18.2 ± 3.8%; P < 0.05) of blastocysts and increased the total number of cells per blastocyst (53 ± 2.5 versus 40.9 ± 2.6; P < 0.05). Furthermore, the beneficial effect of ITS on PA embryos was associated with significantly reduced level of intracellular reactive oxygen species (ROS) (20.0 ± 2.6 versus 46.9 ± 3.0). However, in contrast to PA embryos, ITS had no significant effect on the blastocyst quality of IVF and SCNT embryos (P > 0.05). Taken together, these data suggest that supplementation of ITS to the IVC medium exerts a beneficial but differential effect on pig embryos that varies with the method of embryo production in vitro.

There is increasing evidence that 17β-estradiol is necessary for normal male fertility. The aim of the present study was to characterize estrogen receptor β (ERβ) expression in a non-mammalian vertebrate model, the lizard (Podarcis s. sicula) testis. Immunocytochemical analysis shows that ERβ proteins are present among germ cells in the nucleus of the spermatogonia, in primary spermatocytes and spermatids. Western blot analysis with antibodies against the ERβ gene product revealed an isoform with a specific weight of 55 kDa. In conclusion, the widespread expression of ERβ in the Podarcis s. sicula testis is consistent with a role for estrogens in modulating spermatogenesis in the male.

Livestock embryo production in in vitro systems has been highlighted due to the emergence of interest in embryo stem cells (ESC). ESC potency and their wide potential applications have been recognized in medicine, fundamental research fields and commercial markets due to ESC totipotency or pluripotency and self-renewal. Ovine ESC probably is a useful technical platform for transgenic livestock and animal cloning, but ESC lines have not yet been founded because of difficulties in ESC isolation and the lack of blastula materials. We have established an IVP (in vitro production) system in our laboratory, including in vitro maturation, in vitro fertilization and in vitro culture, to produce sheep blastula using fresh ovaries and testes collected from livestock production. This system can achieve rates of mature eggs and blastulas of 65 and 50% respectively, and can provide enough blastulas for ICM (inner cell mass) isolation. Furthermore, ESC-like clones were isolated from the ICM on ovine embryonic fibroblast (OEF) feeder cells and in ES-DMEM supplemented with the cell factors LIF and SCF, and these survived to the third passage, which was primarily identified by AKP staining and morphology. This work provides a basis for ovine ESC isolation and foundation of ESC lines.