Effect of ovary preservation period on recovery rate and categories of dromedary camel oocytes

The aim of this study was to evaluate the effect of different periods of ovary preservation at 25–30 °C for 5, 6, 7, 9, 12 and 24 h on recovery rate and oocyte categories of dromedary camel oocytes. Camel ovaries were collected from El-Bassatein slaughterhouse, Cairo. The collected ovaries were placed immediately after slaughtering into thermos in saline solution (0.9% NaCl) supplemented with antibiotics (100 IU penicillin and 100 μg streptomycin/ml) at 25–30 °C and transported to the laboratory within 4–5 h. Ovaries were washed three times with warmed (30 °C) phosphate buffer solution (PBS) and one time with ethanol (70%). All visible follicles on the ovarian surface (2–8 mm in diameter) were counted. Oocytes were aspirated using a 20-gauge hypodermic needle. Oocyte yield was recorded and the number of oocytes/ovary was calculated. Oocytes were classified into five categories (compact, partial denuded, denuded, shrunken and cleaved oocytes). Results show that average number of follicles on each ovary was not significantly affected by preservation period, although tended to reduce only after 5 h of ovary preservation. However, this number was insignificantly reduced by increasing period of ovary preservation more than 5 up to 24 h. Average number of oocytes on each ovary was significantly (P < 0.05) reduced only between 5 and 6 h of ovary preservation. Average number of oocytes showed higher reduction rate between 5 and 6 h from 12.4 to 9.3/ovary as well as between 9 and 12 h. Oocyte recovery rate showed insignificant decrease from 88.1% at 5 h to 78.6% at 9 h of preservation. However, it showed significant (P < 0.05) reduction to 62.0% between 9 and 12 h, then insignificantly decreased to 58.6 at 24 h of preservation of the ovaries. Frequency distribution and recovery rate of each category was the highest for compact oocytes and the lowest for cleaved oocytes at all periods of preservation. Increasing preservation period significantly (P < 0.05) decreased frequency distribution of compact and cleaved oocytes, while increased frequency distribution of partial denuded, denude and shrunken oocytes.It might be concluded from the present results that the preservation of dromedary camel ovaries at 25–30 °C for 5–6 h was effective for maintaining the oocytes quality and recovery rate compared with the other preservation periods.     

Generation of Live Piglets from Cryopreserved Oocytes for the First Time Using a Defined System for In Vitro Embryo Production

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.     

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 10⁶ spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.     

Comparison of methods to stimulate ovarian follicular growth in cynomolgus and African green monkeys for collection of mature oocytes

The objective was to compare various gonadotropin-based methods to stimulate ovarian follicular growth in female cynomolgus (n=16) and African green monkeys (n=8) for collection of mature oocytes. On the 1st day of menstruation, the monkeys were treated with 3.75 mg leuprorelin acetate (a GnRH agonist). Starting 2-3 weeks later, ovarian follicular growth was stimulated as follows: (a) 25 IU/kg of human FSH (hFSH) in a glycerol solution given once daily for 9 d; (b) 200 IU of eCG given six times during a 9-d interval; (c) 75 IU/kg hFSH in a glycerol solution given three times (72 h intervals) during a 6-d interval. In addition, the monkeys were given 1200 or 4000 IU of hCG 36 h (Methods A and B) or 60 h (Method C) after the last gonadotropin treatment, and oocyte collection was attempted 36-38 h after hCG. Although there were no significant differences among methods in the number of oocytes collected, in cynomolgus monkeys, hFSH (Methods A and C) was better than eCG (Method B; 12 and 10 versus 7 mature oocytes, respectively), whereas in African green monkeys, eCG (Method B) was more effective than hFSH (Method A; 12 versus 7 mature oocytes). Furthermore, in cynomolgus monkeys, Method C was nearly as effective as Method A; using a glycerol solution as a solvent decreased the frequency of hFSH administration from nine to three times. In conclusion, in cynomolgus and African green monkeys, ovarian response depended on the species and on the individual, and in cynomolgus monkeys, hFSH in a glycerol solvent was effective.     

Cell-free DNA in Human Follicular Microenvironment: New Prognostic Biomarker to Predict in vitro Fertilization Outcomes

Cell-free DNA (cfDNA) fragments, detected in blood and in other biological fluids, are released from apoptotic and/or necrotic cells. CfDNA is currently used as biomarker for the detection of many diseases such as some cancers and gynecological and obstetrics disorders. In this study, we investigated if cfDNA levels in follicular fluid (FF) samples from in vitro fertilization (IVF) patients, could be related to their ovarian reserve status, controlled ovarian stimulation (COS) protocols and IVF outcomes. Therefore, 117 FF samples were collected from women (n = 117) undergoing IVF/Intra-cytoplasmic sperm injection (ICSI) procedure and cfDNA concentration was quantified by ALU-quantitative PCR. We found that cfDNA level was significantly higher in FF samples from patients with ovarian reserve disorders (low functional ovarian reserve or polycystic ovary syndrome) than from patients with normal ovarian reserve (2.7 ± 2.7 ng/μl versus 1.7 ± 2.3 ng/μl, respectively, p = 0.03). Likewise, FF cfDNA levels were significant more elevated in women who received long ovarian stimulation (> 10 days) or high total dose of gonadotropins (≥ 3000 IU/l) than in women who received short stimulation duration (7–10 days) or total dose of gonadotropins < 3000 IU/l (2.4 ± 2.8 ng/μl versus 1.5 ± 1.9 ng/μl, p = 0.008; 2.2 ± 2.3 ng/μl versus 1.5 ± 2.1 ng/μl, p = 0.01, respectively). Finally, FF cfDNA level was an independent and significant predictive factor for pregnancy outcome (adjusted odds ratio = 0.69 [0.5; 0.96], p = 0.03). In multivariate analysis, the Receiving Operator Curve (ROC) analysis showed that the performance of FF cfDNA in predicting clinical pregnancy reached 0.73 [0.66–0.87] with 88% specificity and 60% sensitivity. CfDNA might constitute a promising biomarker of follicular micro-environment quality which could be used to predict IVF prognosis and to enhance female infertility management.     

Superovulatory response to a low dose single-daily treatment of rhFSH dissolved in polyvinylpyrrolidone in rhesus monkeys

To simplify the procedure for superovulation in the rhesus monkey, this study was designed using polyvinylpyrrolidone (PVP) solution as a solvent for gonadotropins. Thirty-five cycling females (aged 5-8 years old) were divided into six groups during the breeding season (November- February). The groups were as follows: Group I, animals received twice-daily 35 IU recombinant human follicle-stimulating hormone (rhFSH) dissolved in 0.5 ml saline for 8 days as the control; Groups II and III, animals received single-daily 35 IU and 17 IU rhFSH in 0.5 saline, respectively, for 9 days; Groups IV, V and VI, received single-daily injection of 35 IU rhFSH, 17 IU rhFSH and 8.5 IU rhFSH dissolved in 0.5 ml 30% PVP (w/v) solution, respectively, for 9 days. After human chorionic gonadotropin was administered to induce the nuclear maturation of oocytes, oocytes were retrieved and the development competence of recovered oocytes treated with in vitro fertilization were observed. The plasma concentrations of follicle-stimulating hormone and ovarian responses were monitored during the treatment. The results showed that the number of recovered oocytes and the in vitro developmental competence of mature oocytes was equivalent among monkeys when treated with a single-daily treatment of 17 and 35 IU rhFSH with PVP preparation in Groups IV and V compared with the twice-daily 35 IU rhFSH treatments received by Group I. However, almost all animals in Groups II, III and VI responded poorly to corresponding stimulations. These findings indicate that a single-daily low dose of rhFSH dissolved in PVP solution can induce the satisfactory ovarian responses in rhesus monkeys. This has the potential to reduce treatment distress, stress to the animals, the labor of the operator as well as the amount of rhFSH used in ovarian stimulation, compared with traditional superovulation methods.     

The Impact of Endometrial Thickness on the Day of Human Chorionic Gonadotrophin (hCG) Administration on Ongoing Pregnancy Rate in Patients with Different Ovarian Response

In order to explore the impact of endometrial thickness on hCG administration day on ongoing pregnancy rate (OPR) in IVF-ET cycles, we retrospectively analyzed data from 10,406 patients undergoing their first IVF cycles with standard gonadotropin releasing hormone analogue (GnRH-a) long protocol. Firstly, patients were divided into poor (≤ 5 oocytes), medium (6–14 oocytes), and high (≥ 15 oocytes) ovarian responders based on the number of oocytes retrieved. In each group, patients were sub-divided into three groups according to the endometrial thickness on the day of hCG administration: Group A, thin endometrial thickness (≤ 7 mm); Group B, medium endometrial thickness (8–13 mm); Group C, thick endometrial thickness (≥ 14 mm). (1) For poor responders, OPRs were significantly different in the three endometrial thickness groups (28.57%, 44.25%, and 51.34%; P = 0.008). The association between thin endometrial thickness and OPR was significant after controlling for age, number of embryos transferred by multivariate logistic regression analysis (adjusted OR: 0.408; 95% CI: 0.186–0.898; P = 0.026. Reference = thick endometrial thickness). (2) For medium responders, OPRs were 31.58%, 55.56%, and 63.01% (P = 0.000) in the three groups. Adjusted OR for thin endometrial thickness was 0.284 (95% CI: 0.182–0.444; P = 0.000). (3) For high responders, OPRs were also significantly different in the three groups (28.13%, 52.63%, and 63.18; P = 0.000). Adjusted OR for thin endometrial thickness was 0.233 (95% CI: 0.105–0.514; P = 0.000). For patients undergoing IVF with different ovarian response, a thin endometrium on the day of hCG administration adversely affects ongoing pregnancy rate.     

Improvement in in?vitro fertilization outcome following in?vivo synchronization of oocyte maturation in mice

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2–4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.     

Improvement of in vitro fertilisation after treatment with buserelin,an agonist of luteinising hormone releasing hormone

Treatment with buserelin, an agonist of luteinising hormone releasing hormone, and human menopausal gonadotrophin was compared with the conventional treatment of clomiphene citrate and human menopausal gonadotrophin in the outcome of in vitro fertilisation. Seventy seven infertile women had 83 cycles of treatment with buserelin and human menopausal gonadotrophin, and concurrently another 328 infertile women were treated with clomiphene citrate and human menopausal gonadotrophin. Seven (8%) cycles were cancelled owing to inadequate superovulation or ovarian hyperstimulation in the women receiving buserelin and 103 (31%) were cancelled because of poor follicular development in those receiving clomiphene citrate. The mean number of oocytes recovered was significantly higher with buserelin (9·5 (SD 4·5) v 5·5 (2·2)) as was the mean number of embryos obtained (4·3 (2·4) v 2·9 (1·7)). Significantly more women who had an embryo transfer became clinically pregnant after treatment with buserelin (53% (30/57) v 30% (48/159), or 36% v 14% of treatment cycles). Altogether 33% (10) of pregnancies in women treated with buserelin were multiple compared with 23% (11) in those treated conventionally. Of the 17 completed pregnancies in women treated with buserelin, 11 resulted in the birth of live babies (eight singletons, two sets of twins, and one set of triplets) and six failed, five before 12 weeks'' gestation and one at 22 weeks. The 13 continuing pregnancies (32 weeks) were eight singletons, two sets of twins, and three sets of triplets. Of the 48 completed pregnancies in women treated with clomiphene citrate, 35 resulted in the birth of live babies (26 singletons, five sets of twins and four sets of triplets) and 13 failed, eleven before 12 weeks'' gestation and two by 27 weeks.Buserelin increased the chance of pregnancy after in vitro fertilisation compared with conventional treatment, but the risk of multiple pregnancy may be increased.     

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatoryrelated pathways in apolipoprotein E knockout (apoE^−/−) mice with diabetic atherosclerosis. Forty-five male apoE^−/− mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorizedtreadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339±75.613×10³µm²) compared to the control (325.485±72.302×10³ µm²) and sedentary (340.188±159.108×10³µm²) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE^−/− mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.Key words: diabetes, atherosclerosis, exercise, matrix metalloproteinases, plaque stability.     

Ovarian surface epithelium receptors during pregnancy and estrus cycle of rats with emphasis on steroids and gonadotropin fluctuation

The present study is designed to demonstrate the ovarian surface epithelial cells’ (OSE) estrogen receptor α (ERα) and progesterone receptor (PR) during pregnancy and estrous cycle in rat. Moreover, determination of the levels of plasma progesterone, estradiol, FSH and LH was also made. The levels of plasma progesterone, estradiol, FSH and LH concentrations were determined on days 7 (n = 5), 14 (n = 5), and 21 (n = 5) of pregnancy in three groups of rats and during the estrous cycle (n = 5) using an ELISA kit. Immunohistochemical method for PR and ERα expressions was also made on the ovary. During pregnancy, FSH and LH remained low except at term when LH levels began to increase from 16 ng/ml to 47 ng/ml. Progesterone levels significantly exceeded estradiol values in all pregnant rats with a peak value of 202 ng/ml on day 14. Elevated progesterone levels were associated negatively with LH and estradiol levels during pregnancy. The levels of estradiol surged significantly on day 21. Immunohistochemistry of the ovary showed low levels of OSE cells staining positive for ERα expression. ERα positive cells were absent on day 7 and 14 of pregnancy, only day 21 recorded a very low percentage of immunostaining (0.5%) within the nuclei of OSE cells. On the contrary, immunostaining of PR was not observed within the nuclei of OSE cells in all groups of study. In conclusion, these results may suggest that the progesterone effect during pregnancy seems to be overriding the positive effect of estrogens on OSE cells. High progesterone levels may have a direct negative effect on gonadotropin production and thereby it might inhibit events leading to both follicular development and OSE proliferation. Understanding the factors affecting OSE proliferation may help elucidating the mechanism(s) of assisted diseases such as ovarian cancer.Keyword: OSE pregnancy rat steroid receptors gonadotropins     

Transforming growth factor-α in a defined medium during in vitro maturation of porcine oocytes improves their developmental competence and intracellular ultrastructure

We examined the effects of transforming growth factor-α (TGF-α) to develop a defined medium for in vitro maturation (IVM) of porcine (Sus scrofa domesticus) oocytes. Cumulus-oocyte complexes (COCs) were matured in porcine oocyte medium containing 3 mg/mL polyvinyl alcohol (POM) and TGF-α (0, 1, 10, or 100 ng/mL) in the presence or absence of the gonadotropins equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG). In the absence of gonadotropins, adding 10 ng/mL TGF-α increased (P < 0.05) the percentage of oocytes that reached metaphase II (24.2%) compared with that of the control (no TGF-α addition; 5.6%). In the presence of gonadotropins, although maturation rate did not differ among TGF-α treatments (75.4% to 84.8%), the rate of blastocyst formation (28.1%) was higher (P < 0.05) in the TGF-α group (28.1%) than that in the control group (15.9%) after in vitro fertilization and embryo culture. An electron microscope study revealed that TGF-α–treated oocytes contained more homogenous lipid droplets than did control oocytes. Moreover, mitochondria surrounded by the endoplasmic reticulum were observed only in the TGF-α–treated oocytes. In blastocysts derived from the latter oocytes, mitochondria with numerous cristae were frequently observed compared with that in blastocysts from control oocytes. When the Day-5 blastocysts obtained from oocytes matured with TGF-α were surgically transferred into four recipients, a total of 29 piglets were farrowed. We concluded that the addition of TGF-α to the defined IVM medium of porcine oocytes improved the subsequent blastocyst formation and that the blastocysts produced by the defined in vitro production system have developmental competence to full term after embryo transfer.     

Maturation and fertilization of porcine oocytes from primordial follicles by a combination of xenografting and in vitro culture

Our objective was to develop a method of endowing oocytes from porcine primordial follicles with full maturation and fertilizing ability as a model for ovarian xenografting of large mammals. Ovarian tissues from 20-day-old piglets, in which most of the follicles were primordial, were transplanted under the capsules of both kidneys of ovariectomized athymic mice. The host mice were treated with 5 IU of equine chorionic gonadotropin (eCG) for 10 days (eCG-10), 30 days (eCG-30), or 60 days (eCG-60) after detection of cornified epithelial cells in their vaginal smears. Cumulus-oocyte complexes, ovarian grafts, and blood samples were obtained 48 h after eCG treatment. Forty-five to 70 days after grafting, the host mice in all groups for the first time showed vaginal cornification, accompanied by the formation of a small number of antral follicles in the grafts. However, we recovered large numbers of full-sized oocytes only from mice in the eCG-60 group; the numbers of full-sized oocytes in the other groups were low. Peripheral levels of total inhibin were highest in the eCG-60 group; this supports our finding that the most enhanced growth of antral follicles occurred in this eCG-60 group. Of 573 oocytes obtained from the eCG-60 group, 98 (17%) were at the metaphase II stage after in vitro culture for maturation. Moreover, 55% of matured oocytes with the first polar body (n = 20) were fertilized in vitro. These results clearly demonstrate that fertilization of oocytes from porcine primordial follicles is achievable by a combination of xenografting and in vitro culture.     

Effects of Different Maturation Systems on Bovine Oocyte Quality,Plasma Membrane Phospholipid Composition and Resistance to Vitrification and Warming

The objective of this study was to evaluate the effects of different maturation systems on oocyte resistance after vitrification and on the phospholipid profile of the oocyte plasma membrane (PM). Four different maturation systems were tested: 1) in vitro maturation using immature oocytes aspirated from slaughterhouse ovaries (CONT; n = 136); 2) in vitro maturation using immature oocytes obtained by ovum pick-up (OPU) from unstimulated heifers (IMA; n = 433); 3) in vitro maturation using immature oocytes obtained by OPU from stimulated heifers (FSH; n = 444); and 4) in vivo maturation using oocytes obtained from heifers stimulated 24 hours prior by an injection of GnRH (MII; n = 658). A sample of matured oocytes from each fresh group was analyzed by matrix associated laser desorption-ionization (MALDI-TOF) to determine their PM composition. Then, half of the matured oocytes from each group were vitrified/warmed (CONT VIT, IMA VIT, FSH VIT and MII VIT), while the other half were used as fresh controls. Afterwards, the eight groups underwent IVF and IVC, and blastocyst development was assessed at D2, D7 and D8. A chi-square test was used to compare embryo development between the groups. Corresponding phospholipid ion intensity was expressed in arbitrary units, and following principal components analyses (PCA) the data were distributed on a 3D graph. Oocytes obtained from superstimulated animals showed a greater rate of developmental (P<0.05) at D7 (MII = 62.4±17.5% and FSH = 58.8±16.1%) compared to those obtained from unstimulated animals (CONT = 37.9±8.5% and IMA = 50.6±14.4%). However, the maturation system did not affect the resistance of oocytes to vitrification because the blastocyst rate at D7 was similar (P>0.05) for all groups (CONT VIT = 2.8±3.5%, IMA VIT = 2.9±4.0%, FSH VIT = 4.3±7.2% and MII VIT = 3.6±7.2%). MALDI-TOF revealed that oocytes from all maturation groups had similar phospholipid contents, except for 760.6 ([PC (34:1) + H]⁺), which was more highly expressed in MII compared to FSH (P<0.05). The results suggest that although maturation systems improve embryonic development, they do not change the PM composition nor the resistance of bovine oocytes to vitrification.     

Prediabetes Phenotype Influences Improvements in Glucose Homeostasis with Resistance Training

Purpose

To determine if prediabetes phenotype influences improvements in glucose homeostasis with resistance training (RT).

Methods

Older, overweight individuals with prediabetes (n = 159; aged 60±5 yrs; BMI 33±4 kg/m²) completed a supervised RT program twice per week for 12 weeks. Body weight and composition, strength, fasting plasma glucose, 2-hr oral glucose tolerance, and Matsuda-Defronza estimated insulin sensitivity index (ISI) were assessed before and after the intervention. Participants were categorized according to their baseline prediabetes phenotype as impaired fasting glucose only (IFG) (n = 73), impaired glucose tolerance only (IGT) (n = 21), or combined IFG and IGT (IFG/IGT) (n = 65).

Results

Chest press and leg press strength increased 27% and 18%, respectively, following the 12-week RT program (both p<0.05). Waist circumference (-1.0%; pre 109.3±10.3 cm, post 108.2±10.6 cm) and body fat (-0.6%; pre 43.7±6.8%, post 43.1±6.8%) declined, and lean body mass (+1.3%; pre 52.0±10.4 kg, post 52.7±10.7 kg) increased following the intervention. Fasting glucose concentrations did not change (p>0.05) following the intervention. However, 2-hr oral glucose tolerance improved in those with IGT (pre 8.94±0.72 mmol/l, post 7.83±1.11 mmol/l, p<0.05) and IFG/IGT (pre 9.66±1.11mmol/l, post 8.60±2.00 mmol/l) but not in those with IFG (pre 6.27±1.28mmol/l, post 6.33± 1.55 mmol/l). There were no significant changes in ISI or glucose area under the curve following the RT program.

Conclusions

RT without dietary intervention improves 2-hr oral glucose tolerance in individuals with prediabetes. However, the improvements in glucose homeostasis with RT appear limited to those with IGT or combined IFG and IGT.

Trial Registration

ClinicalTrials.gov: {"type":"clinical-trial","attrs":{"text":"NCT01112709","term_id":"NCT01112709"}}NCT01112709     

Deletion of SA β‐Gal+ cells using senolytics improves muscle regeneration in old mice

Systemic deletion of senescent cells leads to robust improvements in cognitive, cardiovascular, and whole‐body metabolism, but their role in tissue reparative processes is incompletely understood. We hypothesized that senolytic drugs would enhance regeneration in aged skeletal muscle. Young (3 months) and old (20 months) male C57Bl/6J mice were administered the senolytics dasatinib (5 mg/kg) and quercetin (50 mg/kg) or vehicle bi‐weekly for 4 months. Tibialis anterior (TA) was then injected with 1.2% BaCl₂ or PBS 7‐ or 28 days prior to euthanization. Senescence‐associated β‐Galactosidase positive (SA β‐Gal+) cell abundance was low in muscle from both young and old mice and increased similarly 7 days following injury in both age groups, with no effect of D+Q. Most SA β‐Gal+ cells were also CD11b+ in young and old mice 7‐ and 14 days following injury, suggesting they are infiltrating immune cells. By 14 days, SA β‐Gal+/CD11b+ cells from old mice expressed senescence genes, whereas those from young mice expressed higher levels of genes characteristic of anti‐inflammatory macrophages. SA β‐Gal+ cells remained elevated in old compared to young mice 28 days following injury, which were reduced by D+Q only in the old mice. In D+Q‐treated old mice, muscle regenerated following injury to a greater extent compared to vehicle‐treated old mice, having larger fiber cross‐sectional area after 28 days. Conversely, D+Q blunted regeneration in young mice. In vitro experiments suggested D+Q directly improve myogenic progenitor cell proliferation. Enhanced physical function and improved muscle regeneration demonstrate that senolytics have beneficial effects only in old mice.     

Aphelenchoides microstylus n. sp. and Seinura onondagensis n. sp. (Nemata: Aphelenchina) from New York

Aphelenchoides microstylus n. sp. and Seinura onondagensis n. sp., a nematode predator, are described from dead Scots pine (Pinus sylvestris L.) in Onondaga County, New York. Females of A. microstylus are 370 to 485 µm long. The body is slender and tapers posteriorly to an amucronate, pointed terminus. The head is continuous with the body, and lips bear a stylet guide. Diagnostic characters of females are three incisures in the lateral field, a short stylet (6-7.5 µm) with small basal knobs, a single row of oocytes, and a long postuterine sac (25-50 µm). Males are characterized by small spicules (10-11µm); two pairs of post-anal, subventral papillae; and a single row of spermatocytes. A bursa and gubernaculum are absent. Seinura onondagensis females are characterized by a body of moderate length (475-595 µm), finely annulated cuticle, and a slightly set-off head. Diagnostic characters are four incisures in the lateral field, long stylet without basal knobs (17-22 µm), single row of oocytes, and presence of a postuterine sac (14-38 µm). Males are unknown. The monospecific genus Indaphelenchus is proposed as a synonym of Seinura, and S. siddiqii n. comb. is proposed for the only species, I. siddiqii.     

Effects of Ovarian Hormones and Oral Contraceptive Pills on Cardiac Vagal Withdrawal at the Onset of Dynamic Exercise

The purpose of this study was to investigate the effects of the ovarian hormones and the use of oral contraceptive pills (OCP) on cardiac vagal withdrawal at the onset of dynamic exercise. Thirty physically active women aged 19–32 years were divided into two groups: OCP users (n = 17) and non-OCP users (n = 13). Participants were studied randomly at three different phases of the menstrual cycle: early follicular (day 3.6 ± 1.2; range 1–5), ovulatory (day 14.3 ± 0.8; range 13–16) and midluteal (day 21.3 ± 0.8; range 20–24), according to endogenous (in non-OCP users) or exogenous (in OCP users) estradiol and progesterone variations. The cardiac vagal withdrawal was represented by the cardiac vagal index (CVI), which was obtained by the 4-s exercise test. Additionally, resting heart rate, systolic (SBP) and diastolic blood pressure (DBP) were obtained. The CVI was not significantly different between the three phases of the menstrual cycle in either the non-OCP users (early follicular: 1.58 ± 0.1; ovulatory: 1.56 ± 0.1; midluteal: 1.58 ± 0.1, P > 0.05) or OCP users (early follicular: 1.47 ± 0.1; ovulatory: 1.49 ± 0.1; midluteal: 1.47 ± 0.1, P > 0.05) (mean ± SEM). Resting cardiovascular responses were not affected by hormonal phase or OCP use, except that the SBP was higher in the OCP users than non-OCP users in all phases of the cycle (P < 0.05). In summary, our results demonstrate that cardiac vagal withdrawal at the onset of dynamic exercise was not impacted by the menstrual cycle or OCP use in physically active women.     

INTRAMITOCHONDRIAL YOLK-CRYSTALS OF FROG OOCYTES : I. Formation of Yolk-Crystal Inclusions by Mitochondria during Bullfrog Oogenesis

Electron microscope examination of thin sections of bullfrog (Rana catesbeiana) ovarian oocytes has shown the presence of mitochondria containing yolk-crystal inclusions in oocytes of all sizes, from 160 to 1500 µ mean diameter. The hexagonally shaped yolk-crystals have major periodicities of 73.8 ± 10.7 A (n = 100). Several forms of modified mitochondria, observed in the smaller oocytes, may be arranged into a series of structurally intermediate forms between standard oocyte mitochondria and the typical mitochondria with yolk-crystal inclusions. The observation of such intermediate forms is consistent with proposals that the yolk-crystal inclusions arise within a limited portion of the oocyte chondriome by a complex process of mitochondrial differentiation.