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.

Effect of Bovine Viral Diarrhea Virus on the ovarian functionality and in vitro reproductive performance of persistently infected heifers.

The aim of this study was to study the effect of Bovine Viral Diarrhea Virus on the reproductive female tract by means of analyzing the ovarian follicular population of persistently infected (PI) heifers, and evaluating the performance of oocytes procured form those heifers in in vitro fertilization procedures. Seven BVDV PI Aberdeen Angus and British crossbred heifers ranging from 18 to 36 months of age were spayed and their ovaries used for viral isolation, microscopic examination, and in vitro fertilization procedures. Bovine Viral Diarrhea Virus was detected from the follicular fluid and sera of all PI heifers. Microscopic examination of the ovaries from PI heifers showed a significant drop in the number of follicles cortical regions, compared with controls. A comparative analysis of the stages of follicular development showed a significant decrease in the number of primordial and tertiary follicles in the cortical regions of ovaries from PI heifers. Viral antigen was detected by immunohistochemistry, and was widely distributed throughout the ovarian tissues. There were differences in the rate of cleavage and embryo development between oocytes obtained from the ovaries of control animals and PI heifers. Furthermore, two developed embryos obtained from oocytes from one of the PI heifers were positive to BVDV, as well as two media from in vitro fertilization (IVF) procedures. The results of this study demonstrate that BVDV PI heifers exhibit alterations in follicular population through of the early interaction between the virus and germ cell line affecting directly the mechanisms involved in the ontogenesis of the ovary.

Clinical and reproductive consequences of using BVDV-contaminated semen in artificial insemination in a beef herd in Argentina.

The current report was prompted by an atypical outbreak of mucosal disease that occurred in a beef herd in the southwestern part of Buenos Aires Province, Argentina, where a total of 9/41 (21.9%) yearling bulls died. Blood samples from 73 bulls and 189 heifers were tested for evidence of persistent BVDV infection with Bovine Viral Diarrhea Virus (BVDV). Non-cytopathic BVDV was isolated from 7 (9.6%) 24- to 36-month-old bulls, and 3 (1.6%) 36-month-old heifers. Non-cytopathic BVDV was also detected in the seminal plasma of three of six persistently infected (PI) bulls. Furthermore, a 171 bp genomic fragment of BVDV was consistently detected by nested RT-PCR in one of the two samples of the commercial semen used for artificial insemination, indicating that this semen could be a possible source of infection for the whole herd. To evaluate the possible reproductive consequences of PI heifers and bulls, ovaries and semen were obtained from PI cattle for in vitro assays. The in vitro fertilization of oocytes with semen from PI bulls was associated with decreased cleavage and embryo development rates. Additionally, non-cytopathic BVDV was isolated from the follicular fluid of PI heifers. Genetic typing revealed that all isolates BVDV from the present study had a high percentage of homology and that all of the fragments from the RT-PCR clearly fit with the BVDV 1b cluster. These findings confirm the negative impact that BVDV can have on the reproductive performance of cattle and the importance of applying the proper sanitary controls to minimize the risk of BVDV infection.

Effect of repeated eCG treatments and ovum pick-up on ovarian response and oocyte recovery during early pregnancy in suckling beef cows.

This study was designed to evaluate in suckling early pregnant beef cows with and without eCG-pre-stimulation: (i) the influence of day gestation (from 40 to 101 days) and the consecutive eCG treatments on the follicular growth induced by means of ultrasound-guided transvaginal follicle ablation (FA; all follicles ≥ 5 mm) and the number and quality oocytes recovered by ovum pick-up (OPU) and (ii) the possible effects of repeated hormonal stimulation and FA/OPU on pregnancy outcome. Twelve suckling early pregnant Angus cows (40 days post fixed-time artificial insemination) were randomly assigned to each of two groups (n=6 group(-1)). Group 1 treatments included: FA (Day 0), eCG (1600 IU; Day 1) and OPU (Day 5). Group 2: as cited Group 1 with no eCG treatment. In both groups, OPU was repeated five times (Days 45, 59, 73, 87 and 101 of gestation). The numbers (mean ± SEM) of class II (5-9 mm; 4.3 ± 0.9) and class III (≥10 mm; 2.5 ± 0.4) follicles visualized per cow per OPU session in eCG-treated cows were greater (P<0.05) than for non-treated cows (0.9 ± 0.1 and 0.9 ± 0.1, respectively). In contrast, the number (mean ± SEM) of class I (<5mm) follicles per cow per OPU session was lower for cows with eCG treatment (2.8 ± 0.4) than for non-treated cows (5.7 ± 0.5). The mean number of aspirated follicles was not significantly different (P<0.05) between eCG-treated cows and non-treated cows at 45 and 59 days of pregnancy. However, the mean number of aspirated follicles was greater (P=0.03) in eCG-treated cows than non-treated cows from 73 day of pregnancy onwards. The numbers (mean ± SEM) of recovered oocytes and viable oocytes/cow/session were greater (P<0.05) for eCG-treated cows (2.2 ± 0.2 and 1.6 ± 0.4, respectively) than for non-treated cows (1.0 ± 0.2 and 0.9 ± 0.2, respectively). No donor pregnancies were lost either during or following OPU procedure. We can conclude that (1) eCG-treated pregnant suckled cows can be a source of oocytes for IVF at least to 100 days of gestation and (2) repeated FA/eCG treatment/OPU procedures did not affect the pregnancy outcome.

Effect of container, vitrification volume and warming solution on cryosurvival of in vitro-produced bovine embryos.

The aim of the present research was to develop a low cost and easy to perform vitrification method for in vitro-produced cattle embryos. Effect of container material was evaluated (plastic straw compared to glass capillary, experiment 1), two volume sample (1 compared to 0.5 microL, experiment 2) and warming solution composition medium (Tissue Culture Medium 199 (TCM-199) compared to phosphate buffered saline (PBS), experiment 3) as modifications of the open pulled straw (OPS) system in order to reduce embryo damage caused by exposure to cold. In all experiments, day 7 and expanded blastocysts of cattle were exposed to the vitrification solution 1 for 3 min and 30s in solution 2. After this, embryos were placed in a droplet and loaded in a narrow end container, and immediately submerged into liquid nitrogen. For warming, vitrified embryos were plunged into warming solution 1 for 3 min, and transferred into warming solution 2 for 1 min. Fresh embryos kept in culture were used as control group. Hatching rates were recorded in all cases at day 13. In experiment 1 there was no significant effect of container material on hatching rates. Postwarming survival rate of vitrified embryos was lower than control (27.5% plastic straws, 18.9% glass capillary and 80.5% control, P<0.05). In experiment 2, there was no significant effect of volume in hatching rates (58.3% 1 microL, 61.3% 0.5 microL and 80.5% control, P<0.05). In experiment 3, the composition of the holding medium of warming solution influenced hatching rates (84.1% TCM-199, 74.8% PBS and 91.1% control P<0.05). These data suggest that neither glass capillaries nor reduced sample volume could improve hatching rates after vitrification-warming with open pulled straw (OPS) procedure, and that PBS can replace TCM-199 in warming solutions, but lesser hatching rates should be expected.

Transvaginal follicular aspiration and embryo development in superstimulated early postpartum beef cows and subsequent fertility after artificial insemination.

This study was conducted to investigate in early postpartum suckled beef cows with and without FSH pre-stimulation: (i) the influence of the postpartum period on the number and quality of oocytes recovered by ovum pick-up (OPU), (ii) the overall efficiency of the OPU/IVP embryos from days 30 to 80 postpartum and (iii) if repeated OPU negatively affect fertility following a fixed-time artificial insemination protocol. After parturition suckled Angus cows (n = 30) were divided in three groups (n = 10 group(-1)). All cows were anestrous at the commencement of experimental treatments (30.0 +/- 3.2 days postpartum, mean +/- SD; range 25-34 days). Group 1 treatments included: dominant follicle ablation (DFA), FSH treatment and OPU procedure 5 days after DFA. A total of 9 mg FSH (Ovagen) was administered s.c. once a day over 2 days at equal doses (4.5 + 4.5mg). For fertility test the cows received an intravaginal progesterone treatment from Days 78 to 86 postpartum and were fixed-time artificially inseminated (FTAI) at 56 and 72 h after device removal. Group 2: as cited for Group 1 with no FSH treatment. In both groups, OPU was repeated four times (Days 35, 49, 63 and 77 postpartum) and the collected oocytes classified as viable were in vitro matured, fertilized and presumptive embryos cultured for 8 days. Group 3 (Control FTAI): cows that had not previously aspirations were FTAI as Groups 1 and 2. Pregnancy was diagnosed by means ultrasonography 39 days after FTAI. The numbers (mean +/- SEM) of follicles visible and aspirated at the time of OPU in FSH-treated cows were greater (P < 0.05) than in non-treated cows (10.6 +/- 0.6 and 8.4 +/- 0.4 vs. 8.0 +/- 0.5 and 4.6 +/- 0.3, respectively). Following FSH treatment, the number (mean +/- SEM) of recovered oocytes per cow per OPU session and percentage of viable oocytes were greater in the treated (P < 0.05) than in non-treated animals (3.0 +/- 0.1 and 39.5% vs. 1.5 +/- 0.1 and 30.0%). The cleavage and embryo development rates were similar (P > 0.05) for both groups (14.8 and 6.4% vs. 16.6 and 5.5%). After FTAI the pregnancy rates were not different (P > 0.05) among groups (70, 60 and 90% for Groups 1, 2 and 3, respectively). We can conclude that (1) FSH-treated suckled postpartum cows can be a source of oocytes for in vitro fertilization and (2) repeated DFA/OPU applied during postpartum period did not affect the subsequent fertility following FTAI.

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.

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.

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.

Effects of growth hormone on female reproductive organs.

During the last decade many experiments have been performed to study the effects of growth hormone (GH, somatotropin) on reproductive functions. Most of the studies found only slight or no effects of GH treatment, both on the oestrous cycle and on gonadotropin, progesterone. or oestrogen serum levels. In GH-treated animals, elevated levels of insulin-like growth factor I and GH in the serum could be correlated with an increased number of small (< 5 mm in diameter) ovarian follicles, possibly as a consequence of a reduction of apoptosis and follicular atresia. There is still controversy over the effects of GH on in vivo and in vitro embryo production and on the gestation period. Recent studies produced some evidence that GH-receptor is expressed in ovarian tissue, implying a direct role for GH in the ovary.

Ten-year follow-up of survival and myocardial infarction in the randomized Coronary Artery Surgery Study.

The Coronary Artery Surgery Study (CASS) randomized 780 patients to an initial strategy of coronary surgery or medical therapy. Of medically randomized patients, 6% had surgery within 6 months and a total of 40% had surgery by 10 years. At 10 years, there was no difference in cumulative survival (medical, 79% vs. surgical, 82%; NS) and no difference in percentage free of death and nonfatal myocardial infarction (medical, 69% vs. surgical, 66%; NS). Patients with an ejection fraction of less than 0.50 exhibited a better survival with initial surgery treatment (medical, 61% vs. surgical, 79%; p = 0.01). Conversely, patients with an ejection fraction greater than or equal to 0.50 exhibited a higher proportion free of death and myocardial infarction with initial medical therapy (medical, 75% vs. surgical, 68%; p = 0.04) although long-term survival remained unaffected (medical, 84% vs. surgical, 83%; p = 0.75). There were no significant differences either in survival and freedom from nonfatal myocardial infarction, whether stratified on presence of heart failure, age, hypertension, or number of vessels diseased. Thus, 10-year follow-up results confirm earlier reports from CASS that patients with left ventricular dysfunction exhibit long-term benefit from an initial strategy of surgical treatment. Patients with mild stable angina and normal left ventricular function randomized to initial medical treatment (with an option for later surgery if symptoms progress) have survival equivalent to those patients randomized to initial surgery.

Diagnosis of Mycoplasma pneumoniae pneumonia: sensitivities and specificities of serology with lipid antigen and isolation of the organism on soy peptone medium for identification of infections.

The sensitivities and specificities of isolation and serology for detection of Mycoplasma pneumoniae infections were determined for 3,546 pneumonia patients for whom both isolation and serological data were available. Soy peptone, fresh yeast extract, horse serum-supplemented agar, and diphasic medium were employed for isolation, and the lipid antigen of M. pneumoniae was used for serodiagnosis by complement fixation. The number of M. pneumoniae colonies most frequently detected was 200 to 600 per throat swab, with a range of less than or equal to 60 to greater than or equal to 2,000. The use of diphasic medium increased the number of isolates by 26% compared with direct isolation on agar plates. The organism was isolated from 360 of 525 patients who showed fourfold or greater antibody increases in their paired sera, resulting in a sensitivity of culture of 68%. When persons with titers of greater than or equal to 32 but no fourfold increase were used as the reference, the sensitivity of culture was 58%. The combined sensitivity of the culture method for persons with serological evidence of infection (fourfold increase and titer of greater than or equal to 32) was 64%. The specificity of the culture method was 97% for the 2,527 serologically negative persons. Fourfold antibody increases were found in 360 of 674 persons with isolates of the organism, resulting in a sensitivity of 53%. An additional 247 persons showed titers of greater than or equal to 32 (without a fourfold increase), resulting in a combined sensitivity of 90% for serology with the lipid antigen for the detection of antibodies in culture-positive persons. Fourfold antibody increases were found in 6% of culture-negative persons, resulting in a specificity of 94%. The quantitative culture results provide important base-line data for the development of rapid diagnostic tests for M. pneumoniae infection.