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1.
The objective of the present study was to investigate the effect of testicular tissue lysate (TTL) on developmental competence of germinal vesicle (GV) stage porcine oocytes. Two types of TTL were prepared through repeated freeze–thaw in liquid nitrogen, one from whole testicular tissue (wTTL) and other from either of four different sections of testes, namely just beneath the tunica albuginea (TA), from the transitional area between the seminiferous cord/tubules and the mediastinum testis (TR) and from the intermediate area (parenchymal tissue origin) and CE (cauda epididymis origin). The whole or section‐wise TTL treatments were given for 44 hr during in vitro maturation (IVM). Oocyte maturation was done in either of the two media, namely defined (high‐performance basic medium for porcine oocyte maturation, commercially available) and serum containing (TCM199). After maturation, oocytes were co‐incubated with fresh spermatozoa for 6 hr and then transferred to embryo culture media. Treatment of GV stage oocytes with wTTL (1 mg/ml) increased the cleavage and morula percentage rate (69.23 ± 6.23 and 48.15 ± 6.77, respectively) than that of their control (58.33 ± 8.08 and 32.54 ± 5.53, respectively) in defined media, and in serum‐containing media, cleavage and morula percentage rate were almost equal in both treatment (54.56 ± 7.79 and 34.70 ± 6.78, respectively) and control (59.52 ± 8.21 and 38.52 ± 6.54, respectively). However, effect of wTTL was not significant. In case of section‐wise TTL supplements, TR section significantly (p < .01) improved cleavage and morula rate (58.43 ± 7.98 and 36.14 ± 6.89, respectively) followed by TA. In conclusion, present study indicates that IVM, in vitro fertilization and in vitro culture of embryo are improved in the presence of TTL, particularly its TR section. Further study is expected to reveal the principal components of TTL which may prove useful for IVM.  相似文献   

2.
The objective of this study was to find out the impact of L‐carnitine (10 mM) on developmental regulation of preimplantation sheep embryos cultured in vitro when supplemented in maturation medium and post‐fertilization medium separately. Subsequent objective was to observe the L‐carnitine‐mediated alteration in expression of apoptotic genes (Bcl2, Bax, Casp3 and PCNA) in sheep oocytes and developing embryos produced in vitro. Oocytes matured with L‐carnitine showed significantly (p < .05) higher cleavage (67.23% vs 43.12%), morula (47.65% vs 28.58%) and blastocysts (32.12% vs 13.24%) percentage as compared to presumptive zygotes cultured with L‐carnitine during post‐fertilization period. So it is suggested to use L‐carnitine during maturation than post‐fertilization period. Antiapoptotic and proliferative effects of L‐carnitine were confirmed by inducing culture medium with actinomycin D (apoptotic agent) and TNFα (antiproliferative agent), respectively, with and without L‐carnitine. Oocytes and embryos cultured with actinomycin D and TNFα showed developmental arrest with significant (p < .05) decrease in morula and blastocysts percentage but s upplementation of L‐carnitine to actinomycin D and TNFα induced culture medium showed similar result as that of control . L‐carnitine supplementation during IVM significantly (p < .05) upregulated the expression of Bcl2 and PCNA genes in majority of the developmental stages. Although L‐carnitine upregulated the expression of Bax in initial developmental stages but downregulated at latter part, whereas the expression of Casp3 was upregulated upto 16‐cell stage but after that there was no difference in expression. Expression of GAPDH gene was not affected by L‐carnitine supplementation. In conclusion, L‐carnitine acted as an antiapoptotic and proliferative compound during embryo development and supplementation of L‐carnitine during IVM altered the expression of apoptotic genes in the developmental stages of embryos.  相似文献   

3.
The acquisition of equine oocyte developmental capacity is ensured by the follicular environment, such as granulosa cells, which could reflect the meiotic development potential of immature oocytes. This study evaluated the relationship between DNA fragmentation of granulosa cells, using the chromatin dispersion test, and equine oocyte meiotic development after in vitro maturation. Granulosa cells and cumulus–oocytes complexes (n = 50) were recovered from slaughterhouse‐derived ovaries. Oocytes were in vitro matured, stained and evaluated under fluorescence microscopy. Maturation rates were classified into outstanding, medium and poor levels of maturation using 25th and 75th percentiles as thresholds. For DNA assessment, each sample was processed with the Ovoselect® kit (Halotech DNA). High, low and total DNA fragmentation percentages were compared among levels of maturation rates by ANOVA, followed by Duncan test. Results were expressed as mean ± SE. Total and high DNA fragmentation rates of granulosa cells were significantly higher (p < 0.05) in follicles whose oocytes had reached outstanding maturation level than those originating from follicles whose oocytes had reached poor maturation level. In conclusion, the DNA fragmentation analysis of equine granulosa cells can be a valuable test to identify equine oocytes showing the best meiotic competence after in vitro maturation.  相似文献   

4.
Buffalo is considered short-day breeder in tropical and subtropical part of the world and seasonality and photoperiodism impart major influence on its fertility. However, its impact on in vitro embryo production (IVEP) remains elusive. Therefore, this study investigated the effect of seasonal variations and photoperiodism on morphological and molecular parameters of IVEP in buffalo. For this purpose, we conducted two different experiments on the oocytes obtained by aspirating follicles from abattoir derived ovaries. In Exp. I, retrospective analysis was performed for oocyte recovery, blastocyst and hatching rate, during four consecutive seasonal periods (i.e. January–March, April–June, July–September and October–December). In Exp. II, oocytes from peak breeding and non-breeding seasons were subjected to 24 hr in vitro maturation and evaluated for polar body extrusion to assess maturation rate. Results showed that embryo development was markedly low during second quarter (April–June) and maximum during fourth quarter (October–December) of the year; referred as non-breeding and breeding seasons, respectively. Comparative data analysis demonstrated that poor oocyte quality is major reason for lesser efficiency of embryo production during non-breeding season than peak breeding season as suggested by poor oocyte recovery (2.31 ± 0.10 vs. 3.65 ± 0.27) and maturation rate (33.32 ± 2.1 vs. 63.15 ± 7.31). Subsequently, comparative gene expression analysis of blastocysts during peak breeding season significantly upregulated pluripotency gene (OCT-4) and downregulated heat shock protein 90, as compared to non-breeding season. Therefore, it could be divulged from the present study that seasonal variations and photoperiodism have profound effect on oocyte quality and subsequent embryo development. It is recommended to find suitable additives for in vitro maturation that could mitigate seasonal effects.  相似文献   

5.
Glycerol‐based extenders are widely utilized for freezing equine semen, but media combining methylformamide may better preserve sperm motility and mitochondrial function. Semen is cryopreserved utilizing either a Styrofoam box filled with liquid nitrogen or an automatic freezer. The objective of this experiment was to compare the post‐thaw characteristics of the same ejaculates cryopreserved in a Styrofoam box or in an automatic freezer, utilizing a glycerol‐based extender (Gent) and an extender that combines methylformamide and glycerol (BotuCrio®). For that, one ejaculate from 30 stallions collected in two different centres was used. For data analysis, a mixed linear model with laboratory, medium and freezing method and respective interactions as fixed effects was used. Stallion was taken into account as a random effect. There was no influence (p > .05) of laboratory, while stallion effect was marked. Semen frozen in BotuCrio® in the automatic freezer had higher (p < .001) VCL than semen cryopreserved in Gent using the Styrofoam box. VCL was also higher (p = .068) for semen frozen in BotuCrio® in the Styrofoam box than for semen cryopreserved in Gent using the same method. The difference between percentage of sperm with intact plasma membrane frozen in Gent using the Styrofoam box (44.43% ± 2.44%) compared to spermatozoa cryopreserved in BotuCrio® using the same method (40.78% ± 2.42%) approached significance (p = .0507). The percentage of sperm with intact acrosome membrane was higher (p < .05) in semen frozen in BotuCrio® (79.08% ± 1.79%) than semen frozen in Gent (75.15% ± 1.80%). A higher (p = .0125) percentage (32.24% ± 2.18%) of semen extended in Gent and cryopreserved in the Styrofoam box had high mitochondrial membrane potential than semen frozen in BotuCrio® using the same method (26.02% ± 2.15%). Fertility studies are warranted to assess whether differences found have any effect on the fertility of inseminated mares.  相似文献   

6.
The present experiment was aimed to compare the effect of different protein supplementation sources, foetal calf serum (FCS), oestrous dromedary serum (EDS) and BSA, in experiment 1, and the effect of different concentrations of epidermal growth factor (EGF), in experiment 2, on in vitro nuclear maturation of the dromedary oocytes. Cumulus oocyte complexes (COCs) were harvested from the ovaries collected from a local slaughterhouse by aspirating the visible follicles in PBS supplemented with 5% FCS. Pooled COCs were randomly distributed to 4‐well culture plates containing 500 μl of the maturation medium and cultured at 38.5°C in an atmosphere of 5% CO2 in air for 32–36 h. The basic maturation medium consisted of TCM‐199 supplemented with 0.1 mg/ml L‐glutamine, 0.8 mg/ml sodium bicarbonate, 0.25 mg/ml pyruvate, 50 μg/ml gentamicin, 10 μg/ml bFSH, 10 μg/ml bLH and 1 μg/ml estradiol. In experiment 1, this medium was supplemented with 10% FCS, 10% EDS or 0.4% BSA, whereas in experiment 2, it was supplemented with 0.4% BSA and 0, 10, 20 or 50 ng/ml of EGF. The oocytes were fixed, stained with 1% aceto‐orcein stain and their nuclear status was evaluated. Oocytes were classified as germinal vesicle, diakinesis, metaphase‐I, anaphase‐I (A‐I), metaphase‐II (M‐II) and those with degenerated, fragmented, scattered, activated or without visible chromatin as others. There was no difference (p > 0.05) observed in the proportion of oocytes reaching M‐II stage between the media supplemented with FCS (71.5 ± 4.8), EDS (72.8 ± 2.9) and BSA (72.7 ± 6.2). In experiment 2, a higher proportion (p < 0.05) of oocytes reached M‐II stage when the medium was supplemented with 20 ng/ml of EGF (81.4 ± 3.2) when compared with the media supplemented with 10 ng/ml (66.9 ± 4.1) and control (67.2 ± 7.1) groups. It may be concluded that the maturation media for dromedary camel oocytes can be supplemented with any of the three protein sources, i.e. FCS, EDS and BSA without any significant differences on the maturation rates. Also, a supplementation of 20 ng/ml of EGF in the maturation medium seems to be optimal and improves the nuclear maturation of dromedary camel oocytes.  相似文献   

7.
In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low‐molecular thiol compounds can be added to culture media. Beta‐mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n = 293, BME 0.1 n = 270, BME 0.7 n = 285) were stained with Hoechst 33342 and examined for nuclear stage after 26 hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n = 83, BME 0.1 n = 65, BME 0.7 n = 80). Cleavage rates were determined after 60 hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1 mM and 55.1% BME 0.7 mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7 mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.  相似文献   

8.
We report the cryopreservation of oocytes from Ban miniature pigs which are endemic in Vietnam. Immature cumulus‐oocyte complexes were collected from antral follicles of 7–8 mo old female cyclic Ban pigs and vitrified in micro‐drops. Oocyte morphology, lipid content, post‐warming survival, nuclear maturation, and embryo development were compared to those of oocytes from commercially slaughtered Landrace × Large white hybrid pigs. The size of oocytes in the two breeds was similar. However, significantly lower amounts of intracellular lipid were detected in Ban oocytes. There was no difference (p > 0.05) between Ban and Landrace × Large white oocytes in percentages of post‐warming survival (93.1 ± 3.4% vs. 70.7 ± 16.7%, respectively) and nuclear maturation after in vitro maturation (80.4 ± 5.1% vs. 90.0 ± 1.3% respectively). Similarly, cleavage (30.8 ± 7.8% vs. 10.3 ± 6.1%, respectively) and blastocyst development rates (9.4 ± 5.0% vs. 0.79 ± 0.79, respectively) were not different (p > 0.05) between vitrified Ban and Landrace × Large white oocytes after in vitro fertilization and embryo culture. In conclusion, high survival and maturation rates were achieved after vitrification of immature Ban oocytes and their cryo‐tolerance was similar to that of Landrace × Large white oocytes, despite the difference in lipid content. We succeeded to generate reasonable rates of blastocysts from vitrified Ban oocytes by in vitro fertilization.  相似文献   

9.
The study was designed to evaluate AndroMed® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 106 spermatozoa/ml) in tris‐citric egg yolk or AndroMed® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed® extender (45.5% vs. 49%). It is concluded that AndroMed® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.  相似文献   

10.
Straws of sex‐sorted sperm are usually packaged at a low concentration (e.g., ~2.1 × 106 sperm/ml) and cost significantly more than unsorted conventional semen from the same sire. In order to maximize the efficiency of using sex‐sorted sperm under in vitro fertilization conditions, the selection of an appropriate sperm separation technique is essential. In this study, the effect of using different silane‐coated silica colloid dilutions and layering configurations during centrifugation of sex‐sorted sperm was examined over an extended period of incubation time. Sperm recovery and viability after centrifugation using the colloid separation technique were measured along with several sperm motility parameters using CASA. For this purpose, frozen and thawed sex‐sorted sperm samples were centrifuged using mini‐volume single‐layer (40%, 60% and 80%) and mini‐volume two‐layer (45%/90%, 40%/80% and 30%/60%) separation configurations using PureSperm®. A single layer of 40% PureSperm® recovered significantly more sex‐sorted sperm (78.07% ± 2.28%) followed by a single layer of 80% PureSperm® (68.43% ± 2.33%). The lowest sperm recovery was obtained using a two‐layer PureSperm® dilution of 45%/90% (47.57% ± 2.33%). Single‐layer centrifugation recovered more sorted sperm (68.67% ± 1.74%) than two layer (53.74% ± 1.74%) (p < .0001). A single layer of 80% PureSperm® exhibited the highest sorted sperm viability (72.01% ± 2.90%) after centrifugation (p < .05). The mini‐volume single layer of 80% PureSperm® was determined to be an effective alternative to a two‐layer centrifugation configuration for sex‐sorted sperm selection. In addition, single‐layer colloid dilution of 80% performed either as well as or significantly outperformed the other treatments, as well as the control, with regard to motility (MOT) for all time periods of analysis.  相似文献   

11.
The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed® and SL‐2; Bioxcell®) and a liposome‐based extender (LS; OptiXcell®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.  相似文献   

12.
Spontaneous nuclear maturation of mammalian oocytes can occur when physically removed from the ovarian follicle during in vitro oocyte maturation (IVM), largely because of a decrease in cyclic adenosine monophosphate (cAMP) concentration. Modulation of oocyte cAMP during IVM by using phosphodiesterase inhibitors has been shown to maintain elevated oocyte cAMP concentrations and control meiotic resumption of bovine and ovine oocytes. This study determined the effect of inclusion of isobutyl-1-methylxanthine (IBMX) during collection and the first 12 hours of incubation of equine oocytes on cAMP concentration and glucose metabolism of cumulus–oocyte complexes (COCs). Abattoir-derived COCs were collected in aspiration medium with (Asp-IBMX) or without (Asp) IBMX. Cumulus–oocyte complexes were then incubated for 12 hours in IVM medium with (Mat-IBMX) or without (Mat) IBMX, followed by additional 24 hours in Mat medium. The cAMP concentration, glucose consumption, lactate production, and metaphase II rates of the COCs were assessed. Cumulus–oocyte complexes aspirated into Asp-IBMX (62.2 ± 2.6 fmol per COC) medium had higher cAMP concentration than Asp (31.8 ± 2.8 fmol per COC) control group (P < .05). Likewise, at 12 hours of IVM, Mat-IBMX group (33.2 ± 2.1 fmol per COC) had higher cAMP concentration than the Mat group (7.68 ± 0.5 fmol per COC; P < .05). Glucose consumption and lactate production were lower during the first 12 hours of incubation in COCs cultured in Mat-IBMX (P < .05). Isobutyl-1-methylxanthine prevented the rapid drop in cAMP concentration and altered metabolism of glucose by the COC. Preventing the sudden drop in cAMP prevents the premature nuclear maturation of in vitro–matured oocytes causing poor developmental competence.  相似文献   

13.
The morphological and morphometric characteristics of the ovary are fundamental properties for in vitro oocyte maturation. Nuclear maturation, including first polar body (1PB) extrusion, cytoplasmic maturation and cumulus cell (CC) expansion are the criteria for in vitro maturation (IVM) of oocyte. This study was designed to determine the effect of morphological and morphometric features of the ovary on CC expansion and 1PB extrusion during IVM of oocyte in the adult female dromedary camel. The weight, volume and three dimensions of ovaries from slaughtered dromedary camels and oocytes inside zona diameter and zona pellucida thickness were measured. The follicles were classified in regard to the size and oocytes according to their ooplasm appearance and CC compactness. Aspirated cumulus oocyte complexes (COCs) were incubated for 48 hr (with a 6‐hr interval) in Hams‐F10, and CC expansion and 1PB extrusion were assessed. Significant differences were seen in the shape, weight, volume and three dimensions of the ovaries between ≤4‐year‐old and >4‐year‐old dromedary camel (p < .5). Approximately, 95.82% of follicles were 2–4 mm in diameter. The mean (±SD) of inside zona diameter of the oocyte and zona pellucida thickness was 132.22 ± 13.8 and 14.64 ± 2.24 μm, respectively, in >4‐year‐old dromedary camel. The CC expansion and 1PB extrusion were seen in 86% and 21.88% of COCs, respectively. Age and sexual conditions of dromedary camel influence the morphological and morphometric characteristics of the ovary. Most COCs retrieved from 2–6 mm follicles are cultivable. The most slaughterhouse‐derived COCs retrieved from 2–6 mm follicles of non‐pregnant dromedary camels are excellent and good and yielding a most favourable diameter to achieve the developmental competence for IVM in an optimal time of 24–30 hr; the optimal time for CC expansion is 24–30 hr in this species. However, the CC expansion is a prerequisite process, but not sufficient for IVM.  相似文献   

14.
The purpose of the present study was to assess the effect of crocin supplementation during oocyte maturation on the antioxidant defence and anti‐apoptotic ability and subsequent developmental competence of porcine oocytes. Oocytes were cultured in media containing 0, 300, 400 or 500 µg/ml of crocin. Upon maturation, the maturation rates, reactive oxygen species (ROS) and glutathione (GSH) levels, mRNA expression of genes (SOD, CAT, GPx, Bcl‐2, BAX and Caspase3), expression of cleaved caspase3 and subsequent embryo cleavage rates were measured. Results indicated that the maturation rate of the 400 µg/ml group was 86.80% (p < 0.01). The ROS concentration of the 500 µg/ml group was the lowest (p < 0.01). The GSH concentration of the 400 µg/ml group was the highest (p < 0.01). The SOD, CAT and GPx mRNA expression levels were the highest in the 300, 400 and 500 µg/ml groups, respectively, with the expression levels of all genes being significantly higher than that of the control group (p < 0.01). The Bcl‐2/BAX mRNA expression ratio in 400 and 500 µg/ml groups significantly higher than other groups and significantly decreased caspase3 expression level (p < 0.01). The expression level of cleaved caspase3 in the 500 µg/ml treatment group was the lowest, significantly lower than that of the control group (p < 0.01). The cleavage rate of the 400 µg/ml group was 62.50% (p < 0.01). These experimental results show that the supplementation of in vitro culture medium with 400 µg/ml of crocin significantly enhanced the antioxidant defence and anti‐apoptotic ability and subsequent cleavage rate of porcine embryo.  相似文献   

15.
The goal of this study was to confirm the vasopressor and cardiac effects of POTENAY® INJETÁVEL (POT), a mephentermine‐based product, given to cattle with induced vascular/cardiac depression. Ten healthy Holstein cattle (206 ± 13 kg) followed a randomized‐complete‐block design (RCBD) utilizing crossover study design. Each animal randomly received (1 ml/25 kg, IM) of either POT (n = 10) or volume‐matched placebo control (0.9%NaCl, CP, n = 10). A subset of animals (n = 5) received POT first (day 0) while the remaining (n = 5) received CP; after a six‐day washout period, cattle received the opposite compound. Animals were anesthetized and catheterized for systemic/left ventricular hemodynamic monitoring. Myocardial dysfunction/hypotension was induced by increasing the end‐tidal isoflurane concentration until arterial blood pressure was 20% lower than at baseline and remained stable. Once the animal was determined to be hypotensive and hemodynamically stable, steady‐state hypotensive baseline data (BL2) were acquired, and treatment with either POT or CP was given. Data were acquired post‐treatment at every 15 min for 90 min. POT improved cardiac output (+68 L/min, ±14%, p < 0.05), MAP (+14 mmHg, ±4%, p < 0.05), HR (+22 bpm, ±8%, p < 0.05), and peak rates of ventricular pressure change during both systole (dP/dtmax: +37 mmHg/s ±13%, p < 0.05) and diastole (dP/dtmin: +31 mmHg/s, ±7%, p < 0.05). No improvements were noted following placebo‐control administration. Results indicate that POT improves cardiac performance and systemic hemodynamics in cattle with induced cardiovascular depression when given as single intramuscular injection.  相似文献   

16.
The present study was conducted to investigate the effects of the activator factor of the WNT pathway, chir98014, leading to the in vitro sheep oocyte maturation medium, on the cumulus cell development, different nuclear maturation stages and the following process of embryonic development. Experiments included (a) addition of different concentrations (0, 0.1, 0.5, 1 µm) of chir98014 to the maturation medium and evaluation of the cumulus cell expansion, (b) addition of different concentrations of chir98014 to the maturation medium and investigation of different nuclear maturation stages, (c) addition of different concentrations of chir98014 to the maturation medium and examination of the subsequent embryonic maturation process and (d) addition of different concentrations of chir98014 to the embryonic development culture medium (the first 48 hr) and investigation of the subsequent embryonic development process. The extracted data were analysed using the SPSS software, considering the significance level of p < .05 and making the mean comparisons. The results showed that the addition of the 0.1 µM concentration of chir98014 to the maturation medium had no significant effects on the oocyte maturation and embryo development post-fertilization but it enhanced the Cumulus-oocyte complexes (COCs) expansion. In the fourth experiment, the low concentration of chir98014 in the embryo culture media improved the embryo development process, whereas the high one had a detrimental effect on it, as compared to the control group. Thus, the presence of the lower concentrations of this compound in the embryonic culture medium had favourable effects on the development of embryos.  相似文献   

17.
The effect of porcine or ovine FSH on the maturation rate of porcine oocytes and on the time course of meiotic progression was studied. Groups of 20 grade‐A cumulus oocyte complexes, aspirated from slaughterhouse cycling‐gilt ovaries, were cultured in vitro in 400 μl of Modified Parker's Medium supplemented with oestrous cow serum and porcine FSH (Folltropin®‐V, 0.50 mg/ml) or ovine FSH (OvagenTM, 0.44 iu/ml), in four‐well dishes under mineral oil, at 38.5°C, 5% CO2 in humidified air. At the end of each 3‐h interval, from 3 to 42 h of culture, the nuclear status of oocytes was assessed microscopically (1000×), after fixation (methanol/acetic acid: 3/1) and orcein (2%) staining. Oocytes were classified as (i) immature (IMM), i.e. oocytes at germinal vesicle stage, germinal vesicle break down and prophase I, (ii) metaphase I (MI) and (iii) metaphase II (MII), i.e. oocytes at anaphase I, telophase I and metaphase II. Data were analysed using regression analysis, chi‐square and t‐test. Nuclear status was assessed in 1610 oocytes (porcine FSH: 787, ovine FSH: 823). Most of the oocytes were at MI from 24 to 33 h (porcine FSH 60.27%, ovine FSH 42.80%, p < 0.001) and at MII from 36 to 42 h (porcine FSH 80.38%, ovine FSH 67.45%, p < 0.01) of culture. Significantly higher maturation rate was observed in porcine FSH than in ovine FSH treated oocytes (86.69 ± 12.97%, 71.34 ± 9.86%, mean ± SD, p < 0.05), after 42 h of culture. In conclusion, under the specific culture conditions, porcine FSH seems to support pig oocyte maturation better than ovine FSH.  相似文献   

18.
旨在探讨体外成熟（IVM）液中添加血小板活化因子（PAF）对牦牛卵母细胞发育能力及基因表达影响。本试验将牦牛1 025个卵丘-卵母细胞复合体（COCs）分为4组,分别在含不同浓度PAF（0、10-8、10-7和10-6mol·L-1）的IVM液中成熟后,与奶牛精子体外受精（IVF）和体外培养（IVC）,比较分析各组卵母细胞成熟率、卵裂率和囊胚率以及COCs和胚胎中抗凋亡（BCL-2）、促凋亡（BAX）、表皮生长因子（EGF）及其受体（EGFR）、转录因子（OCT-4和NANOG）和c-fos等基因的表达量差异。结果表明,IVM液中PAF浓度为10-7mol·L-1组的卵母细胞成熟率（（92.16±0.19）%）、卵裂率（（77.20±0.85）%）和囊胚率（（46.71±0.68）%）显著高于其他组（P<0.05）。qPCR结果显示,在该组成熟的COCs中BCL-2、EGF、EGFR、c-fos表达量显著上调（P<0.05）,而促凋亡基因BAX表达量显著下调（P<0.05）,囊胚中EGF、EGFR、OCT-4基因表达量显著上调（P<0.05）。各组间2-、4-、8-细胞、桑椹胚和囊胚中BCL-2、BAX和NANOG表达量差异不显著,但桑椹胚和囊胚中BCL-2表达量均明显下调,而BAX表达量均明显上调。综上所述,IVM液中适宜浓度PAF（10-7mol·L-1）可以促进牦牛卵母细胞成熟和胚胎发育,调控细胞凋亡和增殖相关基因的表达。  相似文献   

19.
The objective of this study was to determine the influence of powdered coconut water (ACP‐318®) diluted in high glucose (11.0 mm ) TCM199 in the achievement of nuclear in vitro maturation (IVM) of canine oocytes. Cumulus oocyte complexes (COCs) (n = 632) were randomly allocated into three experimental groups named as group 1 (control group), group 2 (5% powdered coconut water) and group 3 (10% powdered coconut water). The percentage of meiotic resumption (MR) (GVBD to MII) was 39.1% (81/207), 50.2% (108/215) and 46.6% (98/210) for groups 1, 2 and 3 respectively (p < 0.05). There were no differences in MR rates among groups 2 and 3. The medium with ACP‐318® slightly enhanced the nuclear maturation of canine oocytes when a comparison was established with rates of maturation exhibited by oocytes in the experimental group 1 without ACP‐318® (p < 0.05). The results suggest that oocytes’ nuclear morphology integrity and meiosis achievement were positively influenced when exposed to high glucose TCM199 supplemented with 5% powdered coconut water. Further investigation must be performed for a better understanding of powdered coconut water influence in cellular events during IVM of dog oocytes.  相似文献   

20.
The objective of this research was to improve the techniques in processing chilled and frozen‐thawed horse semen. In a preliminary experiment (Exp. I), different techniques for sperm selection and preparation [Swim‐up, Glass wool (GW) filtration, Glass wool Sephadex (GWS) filtration; Percoll] were tested for their suitability for equine spermatozoa and results were compared with the routine procedure by dilution (Exp. I). In the main experiment (Exp. II), two sperm preparation techniques (GWS, Leucosorb®) refering to the results of Exp. I and a previous study of our group (Pferdcheilkunde 1996 12, 773) were selected for processing complete ejaculates either for cooled‐storage or cryopreservation. In a third experiment (Exp. III), pregnancy rates from inseminations with semen processed according to the techniques tested in Exp. II were compared with those obtained with semen processed according to routine procedures. In Exp. I (six stallions, six ejaculates/stallion), between 48 and 92% of spermatozoa were lost following the different sperm selection procedures (p < 0.05). Preparation of sperm increased percentage of progressively motile spermatozoa (pms) [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] and decreased percentage of sperm head abnormalities [Swim‐up, GW, GWS vs dilution, Percoll (p < 0.05)] probably by not improving the quality of individual cells, but by elimination of spermatozoa of inferior quality. In Exp. II (eight stallions, three ejaculates/stallion) Leucosorb® and GWS procedures allowed the filtration of large volumes (extended ejaculates) for routine laboratory practice. GWS and Leucosorb® filtration resulted in increased motility, membrane integrity and sperm viability after storage of spermatozoa until 48 h at +5°C when compared with control (diluted) and centrifuged semen (p < 0.05). Significantly more spermatozoa were recovered after centrifugation (87.8 ± 15.4%) compared with GWS (63.5 ± 18.6%) and Leucosorb® filtration (53.6 ± 22.3%). GWS or Leucosorb® procedure resulted in successful cryopreservation of stallion semen without centrifugation for removal of seminal plasma. The per cycle conception rate of inseminated mares using 200 × 106 pms transferred within 8 h after collection of semen was not affected by GWS filtration or Leucosorb® separation when compared with centrifugation (n.s.; Exp. III). In conclusion, GWS and Leucosorb® filtration results in the improvement of semen quality and should be considered as a method for stallion semen processing. Additional studies are needed for the evaluation of potentially higher fertilizing ability of stallion spermatozoa separated by techniques for sperm selection.  相似文献   

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