Epigenetics and developmental programming of welfare and production traits in farm animals.

The concept that postnatal health and development can be influenced by events that occur in utero originated from epidemiological studies in humans supported by numerous mechanistic (including epigenetic) studies in a variety of model species. Referred to as the 'developmental origins of health and disease' or 'DOHaD' hypothesis, the primary focus of large-animal studies until quite recently had been biomedical. Attention has since turned towards traits of commercial importance in farm animals. Herein we review the evidence that prenatal risk factors, including suboptimal parental nutrition, gestational stress, exposure to environmental chemicals and advanced breeding technologies, can determine traits such as postnatal growth, feed efficiency, milk yield, carcass composition, animal welfare and reproductive potential. We consider the role of epigenetic and cytoplasmic mechanisms of inheritance, and discuss implications for livestock production and future research endeavours. We conclude that although the concept is proven for several traits, issues relating to effect size, and hence commercial importance, remain. Studies have also invariably been conducted under controlled experimental conditions, frequently assessing single risk factors, thereby limiting their translational value for livestock production. We propose concerted international research efforts that consider multiple, concurrent stressors to better represent effects of contemporary animal production systems.

Improve intra-uterine insemination in rabbits using ultra-high temperature skim milk as extender to keep semen at room temperature.

Two experiments were carried out to examine in vitro quality and in vivo fertility of rabbit semen diluted in ultra-high temperature (UHT) skim milk. In the first experiment, pooled ejaculates of 10 adult rabbits were divided in three aliquots. Each aliquot was diluted in saline solution, TrisC or UHTm extender and kept at room temperature for 24 h. Sperm quality assessment was performed during all the incubation periods. In the second experiment, 27 adult rabbit does were inseminated with semen incubated for 5 h. Embryo recovery was performed 96 h after insemination. Results showed that treatments diluted in UHTm registered the highest values of spermatozoon with total motility, intact and functional plasma membrane and greater number of embryos recovered in rabbit does. We conclude that UHT skim milk would be a good extender for improved intra-uterine insemination in rabbits and to keep sperm cells for several hours at room temperature.

Vaginal histological changes after using intravaginal sponges for oestrous synchronization in anoestrous ewes.

To characterize the histological and cytological vaginal changes generated by the use of intravaginal sponge (IS) applied in oestrous synchronization treatments in ewes during mid-non-breeding season. Thirty-five multiparous ewes were allocated to three experimental groups according to the moment in which the samples were taken: (i) ewes treated with IS containing 60 mg of medroxyprogesterone acetate for 14 days, sampled the day of IS removal (group ISR; n = 10), (ii) or after sponge removal at time of oestrus or 72 h after removal (group AR; n = 14) and (iii) ewes without sponge treatment that were sampled at the day of IS removal of the other groups (group CG; n = 11). Vaginal biopsies and cytological samples were taken from the anterior vaginal fornix area. The vagina of the CG group had a stratified squamous epithelium with a moderate degree of cellular infiltration with lymphocytes and plasma cells in the lamina propia. Treated ewes (ISR and AR) had epithelial hyperplasia and hypertrophy. ISR ewes had haemorrhage and perivascular infiltrate and an increased number of epithelial cells, neutrophils, macrophages and erythrocytes at IS removal. The use of IS generated histological and cytological alterations in the vaginal wall when used for oestrous synchronization in anoestrous ewes.

Effect of seminal plasma on post-thaw quality and functionality of corriedale ram sperm obtained by electroejaculation and artificial vagina.

We have already shown that seminal collection method affects seminal plasma composition and sperm quality in Corriedale rams. In this study, we evaluated the effect of seminal plasma collected by electroejaculation or artificial vagina on sperm resistance to cryodamage. Seminal plasma of five rams of the Corriedale breed collected by artificial vagina or electroejaculation was added before freezing to sperm cells collected by the two methods, and post-thaw quality parameters were evaluated. We found that seminal plasma has no effect on sperm resistance to cryodamage. However, we observed significantly higher percentages of sperm with intact and functional plasma membrane, intact acrosome and greater fertilizing potential after thawing in samples obtained by electroejaculation. This study demonstrates that sperm collected by electroejaculation are more resistant to damage caused by cryopreservation than those collected by artificial vagina.

Electroejaculation increases low molecular weight proteins in seminal plasma modifying sperm quality in Corriedale rams.

This study was conducted to evaluate the effect of seminal collection method (artificial vagina or electroejaculation) on the protein composition of seminal plasma and sperm quality parameters in Corriedale rams. To address this question, we assessed the effect of seminal collection method on motility, plasma membrane integrity and functionality, mitochondrial functionality and the decondensation state of nuclear chromatin in sperm cells. Volume, pH, osmolarity, protein concentration, total protein content and protein profile using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 2-D polyacrylamide electrophoresis of seminal plasma collected with artificial vagina and electroejaculation were also analysed. The main findings from this study were that ejaculates obtained with electroejaculation had (i) a higher number of spermatozoa with intact plasma membrane and functional mitochondria and (ii) a higher proportion of seminal plasma, total protein content and relative abundance of low molecular weight proteins than ejaculates obtained with artificial vagina. Five of these proteins were identified by mass spectrometry: binder of sperm 5 precursor; RSVP14; RSVP22; epididymal secretory protein E1 and clusterin. One protein spot with molecular weight of approximately 31 kDa and isoelectric point of 4.8 was only found in the seminal plasma from electroejaculation.

Review: A barnyard in the lab: prospect of generating animal germ cells for breeding and conservation.

In vitro gametogenesis (IVG) offers broad opportunities for gaining detailed new mechanistic knowledge of germ cell biology that will enable progress in the understanding of human infertility, as well as for applications in the conservation of endangered species and for accelerating genetic selection of livestock. The realisation of this potential depends on overcoming key technical challenges and of gaining more detailed knowledge of the ontogeny and developmental programme in different species. Important differences in the molecular mechanisms of germ cell determination and epigenetic reprogramming between mice and other animals have been elucidated in recent years. These must be carefully considered when developing IVG protocols, as cellular kinetics in mice may not accurately reflect mechanisms in other mammals. Similarly, diverse stem cell models with potential for germ cell differentiation may reflect alternative routes to successful IVG. In conclusion, the fidelity of the developmental programme recapitulated during IVG must be assessed against reference information from each species to ensure the production of healthy animals using these methods, as well as for developing genuine models of gametogenesis.

Recent advances in stem and germ cell research: implications for the derivation of pig pluripotent cells.

Pluripotent stem cells have the unique capacity to contribute to all the tissues of an adult animal after transfer into a host embryo. How pluripotency is acquired during early development and how it is maintained in stem cells have attracted the interest of many scientists for over three decades. Much progress in our understanding of how stem cells arise in culture and the signals required for homoeostasis has enabled the derivation of pluripotent cells in multiple species. Here, we discuss recent developments in stem cell biology that will impact the generation of pluripotent cells from different embryonic origins and will contribute to increase our capacity for generating transgenic animals.

Influence of the length of progestagen treatment and the time of oestradiol benzoate application on the ovulatory follicle size and ovulation time in anoestrous and cyclic beef cows.

Previous research from our laboratory in beef cattle suggests that the pre-ovulatory follicle size, maturity and subsequent susceptibility to gonadotropin are influenced by the length of progestagen treatment in artificial insemination programme in beef cows. To test this hypothesis, two experiments were conducted. In experiment 1, 35 anoestrous beef cows received an intravaginal sponge containing 200 mg of medroxyprogesterone acetate. The treatment lasted for 7 (n = 12), 8 (n = 11) or 9 (n = 12) days. Half of the animals in each group were injected with 0.7 mg of oestradiol benzoate (EB) at device removal (0 h) and the other half 24 h later. In experiment 2, 38 cycling beef cows were treated with the same protocols as in experiment 1. Ultrasound examinations were performed to determine the follicular diameter at device removal (dominant follicle), interval to ovulation and ovulatory follicle diameter. The dominant follicle of anoestrous cows with progestagen for 7 days (8.4 ± 1.6 mm) resulted smaller (p < 0.05) than the cows treated for 8 (10.5 ± 1.6 mm) and 9 days (10.6 ± 1.2 mm). However, regardless of the length of the treatments, ovulation time after device removal was longer (p < 0.05) when EB was injected 24 h after withdrawal than at 0 h in anoestrous cows (EB0 = 52.7 ± 4.0 h; EB24 = 70.8 ± 6.2 h) and in cyclic cows (EB0 = 50.0 ± 21.0 h; EB24 = 73.0 ± 20.0 h). In anoestrous cows, the treatment with progestagen for 9 days and EB at 24 h increased the diameter of the ovarian follicle (p = 0.033) but did not affect the diameter of the ovulatory follicle in cyclic cows. In conclusion, increasing the length of progestagen treatment for 8 or 9 days compared to 7 days increased the diameter of the dominant follicle, in anoestrous and cyclic beef cows. Oestradiol benzoate administered at device removal resulted in a shorter interval from device removal to ovulation compared with EB injection 24 h after the end of a progestagen treatment.

The Sda/GM2-glycan is a carbohydrate marker of porcine primordial germ cells and of a subpopulation of spermatogonia in cattle, pigs, horses and llama.

Spermatogonia are a potential source of adult pluripotent stem cells and can be used for testis germ cell transplantation. Markers for the isolation of these cells are of great importance for biomedical applications. Primordial germ cells and prepubertal spermatogonia in many species can be identified by their binding of Dolichos biflorus agglutinin (DBA). This lectin binds to two different types of glycans, which are α-linked N-acetylgalactosamine (GalNac) and ß-linked GalNac, if this is part of the Sda or GM2 glycotopes. We used the MAB CT1, which is specific for the trisaccharides motif NeuAcα2-3(GalNAcß1-4)Galß1-, which is common to both Sda and GM2 glycotopes, to further define the glycosylation of DBA binding germ cells. In porcine embryos, CT1 bound to migratory germ cells and gonocytes. CT1/DBA double staining showed that the mesonephros was CT1 negative but contained DBA-positive cells. Gonocytes in the female gonad became CT1 negative, while male gonocytes remained CT1 positive. In immunohistological double staining of cattle, pig, horse and llama testis, DBA and CT1 staining was generally colocalised in a subpopulation of spermatogonia. These spermatogonia were mainly single, sometimes paired or formed chains of up to four cells. Our data show that the Sda/GM2 glycotope is present in developing germ cells and spermatogonia in several species. Owing to the narrower specificity of the CT1 antibody, compared with DBA, the former is likely to be a useful tool for labelling and isolation of these cells.

Effect of serum on the mitochondrial active area on developmental days 1 to 4 in in vitro-produced bovine embryos.

Certain morphological changes at the subcellular level caused by the current techniques for in vitro embryo production seem to affect mitochondria. Many of these, including dysfunctional changes, have been associated with the presence of serum in the culture medium. Thus, the aim of the present work was to assess the mitochondrial dynamics occurring in embryos during the first 4 days of development, in order to analyze the most appropriate time for adding the serum. We used transmission electron microscopy (TEM) micrographs to calculate the embryo area occupied by the different morphological types of mitochondria, and analyzed them with Image Pro Plus analyzer. The results showed hooded mitochondria as the most representative type in 1- to 4-day-old embryos. Swollen, on-fusion, orthodox and vacuolated types were also present. When analyzed in embryos cultured without serum, the dynamics of the different mitochondrial types appeared to be similar, a fact that may provide evidence that the developmental changes control the mitochondrial dynamics, and that swollen mitochondria may not be completely inactive. In contrast, in culture medium supplemented with serum from estrous cows, we observed an increased area of hooded mitochondria by developmental day 4, a fact that may indicate an increased production of energy compared with previous days. According to these results, the bovine serum added to the culture medium seems not to be responsible for the functional changes in mitochondria.

Simplified superovulatory treatments in Corriedale ewes.

Two experiments were designed to evaluate the possibility of simplifying superovulatory treatments in Corriedale ewes with use of ovine FSH (oFSH). Ewes received intravaginal progestogen sponges for 14 days. In Experiment 1, several simplified schedules were tested. Ewes were treated with 176 NIH-FSH-S1 units' oFSH given as a single injection in saline, along with 500 IU eCG 48 h before sponge removal (Group A1), in four equal doses (B1), or given as a single injection in a polyvinylpyrrolidone vehicle (C1) 24 h before sponge removal. In Experiment 2, the simplified protocol that exhibited the most desirable results in Experiment 1 (A2) was compared with the same protocol, but using less oFSH (132 units) (B2) and with the most conventional protocol (176 units of oFSH in eight decreasing doses; C2). Estrus was detected and ewes were naturally mated. The ovarian response and embryo production were assessed on Day 6 after estrus. LH was measured at 6h intervals from pessary withdrawal. The onset of estrus and the pre-ovulatory LH surge were advanced (P<0.05) in ewes treated with FSH and eCG. In Experiment 1, protocol A1 produced a greater percentage of superovulated ewes compared to C1 (100.0 compared with 58.3%; P<0.05), increased ovulation rate (13.8 corpora lutea compared with 6.2 and 4.7 for B1 and C1, respectively; P<0.05), and tended to increase the number of transferable embryos compared to B1 (P=0.08). In Experiment 2, percentages of superovulated ewes and ovulation rates were similar among groups; however, Group A2 tended to have more large follicles (P=0.07) than C2. The number of transferable embryos was similar among the three treatments. In conclusion, the reduced-dose oFSH given once along with eCG is the most appropriate superovulatory treatment because it combines simplicity and a lesser dose of gonadotropin, which also implies a reduction in cost, without reducing embryo production.

Effect of progesterone administration on the ovarian response to superovulatory treatments in cattle.

To evaluate ovarian response in Angus cows previously treated with progesterone (P4), animals were randomly assigned to two groups: T600 group (n=14), 600 mg of P4/day. P4 was injected from days 3 to 7 of the estrous cycle. On day 7, superovulatory treatments began. The control group (n=12) was given vehicle only. The superovulatory treatments in the control group began on days 7-9 of the estrous cycle. The superovulatory total treatment dose of 400mg NIH FSH P1 was given twice a day over a 4-day period. Ultrasonography of the ovaries was conducted 3 days preceding the initiation of superovulatory treatment, every 24h. In both groups, an additional ultrasonographic evaluation was made at 24h after the end of superovulatory treatment. Blood samples were collected 4 days preceding the initiation of superovulatory treatment, every 24h. Additional samples were taken from the P600 group for 12 day after of initiation of superovulatory treatment every 24h, except on the fifth day after the initiation of superovulatory treatment. In the P600 group, P4 concentrations were greater than in the control group (P<0.01) and remained over 1 ng/ml up to day 11 after beginning of superovulatory treatment. The diameter of the dominant follicle was larger in the animals of the control group (P<0.01). Cows of the P600 group had a greater number of Class I (3-4mm) follicles (P<0.01). A significant day and treatment effect (P<0.01) were observed in Class II (5-9 mm) follicles. Effects due to treatment on the number of Class III follicles (P<0.05) were observed. In the P600 group, no estrous post-superovulatory was observed and there were no ovulations that occurred. Conversely, 100% of the cows of the control group showed estrous. In the P600 group, there were a greater number of Class III follicles (P<0.01) and a lesser number of Class II follicles (P<0.05) at 24h after the end of superovulatory. In the control group, 66.7% of the cows responded to superovulatory treatments. In conclusion, the daily administration of 600 mg of P4, from days 3 to 7 of the estrous cycle, produces an increase of plasma concentrations of this hormone from day 4, resulting in changes in follicular dynamics (absence of follicles greater than 10mm of diameter and an increase of the population of Class I follicles). As to the ovarian stimulation using Folltropin V in animals receiving a daily injection of 600 mg of P4 from days 3 to 7 of the estrous cycle, a greater population of follicles>or=10mm developed by 24h after superovulatory treatments were completed.

Seasonal variations in the composition of ram seminal plasma and its effect on frozen-thawed ram sperm.

It has been proposed that seminal plasma (SP) in the extender or in post-thaw media can prevent and revert cold-shock damage in cryopreserved ram sperm; however, this was dependent on season. We evaluated sperm parameters from Frisian ram semen incubated for various intervals with SP from all seasons and stored at -18 or -196 degrees C. At both temperatures, SP from autumn or winter increased (P<0.05) sperm motility, whereas no SP, or SP from spring or summer, had no effect. However, neither viability nor membrane or acrosomal status were modified by SP. Thirteen SP proteins were bound to the sperm surface (16.1, 16.7, 17.4, 23.3, 25.2, 27.5, 35.0, 40.0, 49.0, 53.5, 55.5, 61.0, and 86.0kDa). The SP proteins that bound to sperm were affected by season, but not by conservation temperature. Sperm incubated with SP from autumn had increased concentrations of five proteins; two were identified (with specific antibodies) as RSVP14 and RSVP20. In conclusion, SP from autumn and winter improved sperm motility of frozen-thawed ram sperm, and storage of ram SP at -18 or -196 degrees C did not affect protein composition. The SP proteins that bound to the sperm surface may be responsible for sperm membrane stabilization and should be further investigated.

Multiple ovulation and embryo transfer with fresh, frozen and vitrified red deer (Cervus elaphus) embryos in Argentina.

Two multiple ovulation and embryo transfer (MOET) programs with fresh, frozen and vitrified red deer embryos were carried out during the reproductive season of 2005 and 2006 in a local breeding farm in Argentina. Multiparous (n=10 and 9, respectively) weaned hinds were used as donors for each year. The estrous synchronization treatment of donors and recipients consisted of inserting an ovine intravaginal sponge containing medroxiprogesterone acetate (MAP) for 12 days. Superovulation was conducted with a total dose of 180 mg of NIH-FSH-P1 (Folltropin-V, Bioniche, Belleville, Ontario, Canada), given i.m. in eight decreasing doses every 12h (40, 40; 27, 27; 15, 15; 8, 8 mg), from days 10 to 13. Donor females were mated with one stag of proven fertility. The recovery rate was 84.1% (122/145), obtaining 45.1% (55/122) of transferable embryos, 24.6% (30/122) of degenerated embryos and 30.3% (37/122) of unfertilized oocytes. Pregnancy rates after transfer of fresh, OPS vitrified/warmed and ethylene glycol (EG) frozen/thawed embryos were 64.3% (18/28), 53.3% (8/15) and 70.0% (7/10), respectively. Vitrification and freezing with ethylene glycol procedures constitute an interesting alternative for red deer embryo cryopreservation.

Timing of emergence of ovulatory follicles in polyovulatory goats.

The current study characterized the timing of emergence of ovulatory follicles during the follicular phase of the estrous cycle in polyovulatory does and assessed whether selection may influence ovulation rate through differences in ovarian follicular dynamics, by characterizing preovulatory follicular emergence and growth in two ecotypes of Neuquen-Criollo Argentinean goats (Short-Hair, n=11 and Long-Hair, n=9). During the breeding season, the time of estrus was synchronized in all does with two doses of a prostaglandin analogue. Ovarian laparoscopies were performed on days 17 and 19 after the induced estrus (day 0) and 7-15 h after the beginning of the subsequent estrus. Results indicate that both ecotypes of goats have common features in the ovarian follicular population and in the patterns of preovulatory follicular enlargement. In all the goats, most of the preovulatory follicles arose from the pool of follicles present in the ovary between days 17 and 19 of the estrous cycle. These follicles were all larger than 2mm at emergence, being the largest growing follicle present in the ovaries on days 17 and 19 in 56.5 and 78.6% of the does, respectively. The appearance of new follicles remained unaffected, while the mean number of small growing follicles decreased (P<0.05) during the follicular phase, indicating that preovulatory follicles do not suppress the emergence of new follicles but inhibit the growth of small follicles. A separate analysis of single and double ovulating does showed that 75% of the second ovulatory follicles in polyovulatory goats was present on the ovarian surface between days 17 and 19 of the estrous cycle, but appeared later in the other 25% of the estrous cycles. These findings support the hypothesis that follicular dominance effects are exerted during the preovulatory period, when the growth of follicles other than the ovulatory is inhibited, and that increases in ovulation rate in small ruminants are related to a reduced incidence of follicular atresia and an extended period of ovulatory follicle recruitment.

Influence of different doses of progesterone treatments on ovarian follicle status in beef cows.

To determine a dose of progesterone (P4) that allow ovarian follicular wave control, Aberdeen Angus cows were randomly assigned into four groups: T600 (n=5), 600 mg of P4/day; T400 (n=5), 400 mg of P4/day; T200 (n=4), 200mg of P4/day and Control (n=4) (excipient only). Progesterone was injected from day 3 to 9 of estrous cycle. Ultrasonographies and blood sample collections were performed daily from day 2 to 10 and on day 15 of the estrous cycle. Additionally, an ultrasonographic study was conducted on day 13. Progesterone concentrations were different among all groups (P<0.01). The diameter of the dominant follicle was greater for control than for T200, T400 and T600 groups (P<0.01); there was no difference between T200 and T400 (P>0.05), but they had a greater diameter follicle than the T600 group (P<0.01). The growth rate of the dominant follicle between day 3 and 7 of estrous cycle was greater for control group (1.63+/-0.3 mmday(-1)) than for T200 (0.56+/-0.19 mmday(-1), P<0.05), T400 (0.6+/-0.23 mmday(-1), P<0.05) and T600 (0.11+/-0.13 mmday(-1), P<0.01) groups. The mean number of class I follicles (3-4mm) per day for the entire experimental period was less for the control group than for T200 (P<0.05), T400 and T600 (P<0.01) groups (3.7+/-1.3; 5.3+/-1.3; 6.6+/-1.8 and 8.1+/-1.9, respectively). The mean number for the T200 group was less than for T600 (P<0.05) and similar for T400 and T600 groups (P>0.05). The number of class III follicles was greater for control group than for the other groups (P<0.01). T200 and T400 groups had similar numbers of class III follicles (P>0.05) and both had greater numbers of follicles than the T600 group (P<0.05). The diameter of the corpus luteum of the T600 group (15.8+/-1.6 mm) was less than for control (21.0+/-2.5 mm, P<0.01), T200 (19.3+/-2.7 mm, P<0.01) and T400 (20.0+/-2.2 mm) groups (P<0.05). The mean diameter of corpus luteum of T200 was similar to T400 (P>0.05), but different from the control group (P<0.05). In conclusion, the daily intramuscular administration of 200mg or more of progesterone from day 3 to 9 of the estrous cycle indicates that plasma concentrations of progesterone can be used to modify the pattern of follicular development during the follicular wave. From day 5 of the estrous cycle, progesterone concentrations greater than 15 ng/ml (T600 group: 600 mg/day of progesterone from day 3 to 9 of the estrous cycle) inhibit dominant follicle development, increase the class I follicle populations (3-4 mm) and diminish the development of the corpus luteum.

Effect of estrous cow serum during bovine embryo culture on blastocyst development and cryotolerance after slow freezing or vitrification.

UNLABELLED: The present study investigated the effect of estrous cow serum (ECS) during culture of bovine embryos on blastocyst development and survival after cryopreservation by slow freezing or vitrification. Embryos were derived from in vitro maturation (IVM) and in vitro fertilization (IVF) of abbatoir-derived oocytes. At Day 3, embryos were cultured in three different media: Charles Ronsenkrans medium + amino acids (CR1aa; without bovine serum albumin (BSA)) + 5% estrous cow serum (CR1-ECS), CR1aa + 3 mg/mL BSA (CR1-BSA) or CR1aa + 5% ECS + 3 mg/mL BSA (CR1-ECS-BSA). At 7.5 d post-insemination (PI), blastocyst yield and quality were evaluated; blastocysts and expanded blastocysts from each media were cryopreserved by Open Pulled Straw (OPS) vitrification method or slow freezing (1.5 M ethylene glycol, EM). Total blastocyst yield did not differ among CR1-ECS, CR1-BSA and CR1-ECS-BSA (30.9, 33.1 and 32.9%, respectively, P < 0.05). Embryo survival (hatching rate) was higher in vitrified versus slow-frozen embryos (43% versus 12%, respectively, P < 0.01), and in embryos cultured in CR1-BSA (40.3%) compared with those cultured in serum-containing media (CR1-ECS, 21.5% and CR1-ECS-BSA, 19.8%; P < 0.01). IN CONCLUSION: (a) it was possible to produce in vitro bovine embryos in serum-free culture medium without affecting blastocyst yield and quality; (b) serum-free medium produced the best quality embryos (in terms of post-cryopreservation survival); and (c) vitrification yielded the highest post-cryopreservation survival rates, regardless of the presence of serum in the culture medium.

Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro.

Extensive work was done regarding the ability of Swim up and Percoll gradient to select functional sperm for in vitro embryo production (IVP) systems. The aim of this work was to compare Swim up and Percoll as methods of sperm selection by ultrastructural, biochemical and functional studies. Frozen-thawed semen from two bulls (Experiments 1 and 2, respectively) were treated using Swim up or Percoll discontinuous gradients. Motility, sperm membrane ultrastructure, sperm proteins, in vitro embryo production (insemination doses, cleavage, embryo yield and quality) and embryo sex ratio were scored and compared. Electron transmission microscopy of outer sperm membranes showed higher (P<0.05) percentage of sperm with lost acrosomes in Percoll treated samples compared to Swim up. A differential protein pattern was also detected. When in vitro embryo production was performed, Percoll gradient produced higher (P<0.05) number of fertilizing doses (7.6 versus 5.9, Bull 1; 13.5 versus 7.8, Bull 2) and higher sperm motility (90% versus 76.6%, Bull 1; 81.7% versus 68.3%, Bull 2) than Swim up. The percentage of cleavage (Day 3) was similar in both treatment groups, whereas embryo production rate (Day 7) was higher (39.4% versus 30.2%, Bull 1; 38% versus 32.4%, Bull 2; P<0.05) when Percoll gradient was used. The percentage of hatched embryos (Day 11) and sex ratio did not differ. Total cell counting and embryo differential staining (inner cell mass and trophoblast cells) of Day 7 embryos showed that Percoll treated sperm produced better quality embryos compared to Swim up. We concluded that Percoll had a better performance selecting sperm and an enhanced capacity for embryo production when compared with the Swim up procedure; this could be attributed to a better acrosome exocytosis, associated to the absence of certain membrane proteins.

Mammalian oocyte activation: lessons from the sperm and implications for nuclear transfer.

Events after fertilisation have been carefully studied in the last decades. However, there are still several questions to be clarified in relation to the signalling pathway initiated by the sperm, the identification of proteins or factors involved in the activation of the arrested oocyte, and the inactivation of specific molecules involved in the meiotic arrest. The present state of knowledge in mammalian fertilisation allows the development of activation protocols that closely mimic the events initiated by the sperm according to certain major factors (MPF activity and MAPk activity). These protocols are successfully used for the activation of oocytes after NT giving rise to offspring. Few cloned animals have yet been produced. However, the pregnancy and the survival rates after birth are not significantly different when different activation protocols are compared. This fact argues fora major reason forthe low success in the efficiency of NT. Eventually, factors related to the recipient oocyte, the donor cell or the culture conditions are part of these major problems that the reconstructed embryo has to overcome to develop into a normal offspring. Nonetheless, the development of activation protocols that closely imitate the mechanism of activation initiated by the sperm are of special interest to improve the developmental potential of cloned embryos.

Combined epidermal growth factor and hyaluronic acid supplementation of in vitro maturation medium and its impact on bovine oocyte proteome and competence.

The conditions for in vitro oocyte maturation impact on cytoplasmic and nuclear processes in the oocyte. These events are differentially influenced by the nature of the maturation inducer and the presence of intact cumulus in cumulus-oocyte complexes. Epidermal growth factor is the main growth factor promoting oocyte maturation. Also, hyaluronic acid (HA) produced by cumulus cells is known to be responsible for the correct structural and functional organization of the cumulus during oocyte maturation. Therefore, we evaluated the developmental competence of bovine oocytes matured in vitro in a maturation medium supplemented with both EGF and HA, compared to FSH and fetal bovine serum (FBS). In addition, the impact of IVM conditions on the proteomic profile of metaphase II bovine oocytes was analyzed by two-dimensional electrophoresis. Cumulus-oocyte complexes were matured in two media: (1) 10 ng/mL EGF, 15 µg/mL HA, and 100-µM cysteamine and (2) 0.01 UI/mL rh-FSH and 10% FBS. The percentages of first polar body and embryo production and the kinetics of embryo development and oocyte proteomic profiles were analyzed. Oocytes matured in the presence of EGF-HA showed an increase (6%, P < 0.05) in the percentage of polar body extrusion. The blastocyst rate was 3% (P < 0.05) higher in the FSH-FBS group, but no differences were found in the rate of expanded blastocyst neither in total embryo production between IVM conditions. Cleavage rate of oocytes matured with FSH-FBS was 5% higher (P < 0.05) with respect to EGF-HA-matured oocytes when evaluated 30 hours after fertilization. However, at Day 7, those inseminated oocytes that underwent division at a correct timing showed that although there are still early blastocysts in the FSH-FBS condition, EGF-HA embryos have developed completely into blastocysts. Still, the production rate of those embryos that achieved expansion was similar between both maturation conditions. On the other hand, noncleaved presumptive zygotes at Day 7 developed into the different stages with similar rates (∼4%) independently of the medium condition. Modifications of IVM medium composition markedly affected protein profile of bovine oocytes in a differential manner. The proteomic approach revealed the presence of 68 spots in both treatments, 41 exclusively found in the FSH-FBS group and 64 exclusive for the EGF-HA group. Taken together, these results indicate that combined EGF-HA supplementation of in vitro maturation medium could be used to improve oocyte meiotic competence and ensure a better timing to develop into the blastocyst stage.