Refrigerated Storage of Red Deer Epididymal Spermatozoa in the Epididymis, Diluted and with Vitamin C Supplementation

We have approached the problem of refrigerated storage of epididymal sperm samples from red deer by comparing three options: storing the genital (testicles within the scrotum), diluting the semen in extender or diluting the semen in extender supplemented with an anti-oxidant. Twenty-nine pairs of testes were collected. Spermatozoa from one of each of the pairs were immediately recovered, and diluted to 400 × 10⁶ sperm/ml in Tris-citrate-fructose with 20% egg yolk. Control group was stored as such, and Anti-oxidant group was supplemented with 0.8 m m vitamin C. The remaining epididymides and the diluted samples were stored at 5°C and spermatozoa were analysed at 0, 24, 96 and 192 h for: motility [computer-assisted semen analysis (CASA)], acrosomal integrity, sperm viability (eosine/nigrosine staining), normal tails and chromatin status [sperm chromatin structure assay (SCSA)]. In general, seminal quality decreased with storage time. Vitamin C supported progressive motility better at 24 h (median 42% vs 23% Control and 15% epididymis), reduced the incidence of tail abnormalities and protected chromatin. Storing the semen in the epididymis slowed down motility loss, but slightly increased the occurrence of tail abnormalities and viability was lower at 192 h. However, regarding chromatin status, sperm stored in the epididymis was protected similarly to those diluted in the medium supplemented with vitamin C. Although the differences between the three groups were small, there were some advantages in supplementing the extender with vitamin C. Besides, refrigerating the epididymis may be a good option when immediate processing is not available.     

In Vitro Sperm Penetration Speed and its Relationship with In Vivo Bull Fertility

The in vitro tests utilized to evaluate sperm quality represent an interesting and important approach to evaluate ejaculated fecundant capacity. In the present work, the oocyte-penetrating ability of sperm from two bulls with low and two with high in vivo fertility rate was investigated. Sperm-quality parameters, such as sperm concentration, total and progressive motility, acrosome and total sperm anomalies, proximal cytoplasmic drops and live : dead sperm ratio were assessed and batches of sperm homologues in these parameters were selected. In expt 1, a sperm : oocyte ratio of 3000 was used and the oocytes were fixed 5, 10, 15 and 18 h post-insemination (hpi). In expt 2, the sperm : oocyte ratio was reduced to 1000 and the eggs were fixed at 8 and 10 hpi. The results, analysed by chi-square test, showed significant differences (p <0.001) among bulls at 15 hpi in expt 1 and 8 hpi in expt 2; nevertheless, no accordance was found between sperm penetration rate and in vivo fertility. At 18 hpi, the low-fertility bulls exceeded the two high-fertility bulls, supporting previous reports that suggest an opposite correlation between in vivo and in vitro fertility rate at low doses of heparin. Furthermore, a more efficient zona binding ability of one high-fertility bull was pointed out in expt 2 after the reduction of sperm : oocyte ratio, as it reached the highest percentage of penetration when compared with all the others.     

Dimethylformamide Improves the In vitro Characteristics of Thawed Stallion Spermatozoa Reducing Sublethal Damage

A total of 42 ejaculates were used in the experiment; six ejaculates per stallion, obtained from seven Pure Spanish stallions (PRE), were split and frozen in freezing media with different concentrations and combinations of cryoprotectant (CPA): (i) Cáceres (skim milk based extender) containing 2.5% glycerol (2.5GL), (ii) Cáceres containing 1.5% glycerol and 1.5% dimethylformamide (1.5%GL–1.5%DMFA), (iii) Cáceres extender supplemented with 1.5% glycerol and 2.5% dimethylformamide (1.5%GL–2.5%DMFA) and (iv) Cáceres extender supplemented with 4% dimethylformamide (4%DMFA). After at least 4 weeks of storage in liquid nitrogen (LN), straws were thawed and semen analysed by computer‐assisted sperm analysis and flow cytometry (membrane lipid architecture (Merocyanine 540), integrity and sublethal damage (YoPro‐1) and mitochondrial membrane potential (JC‐1)). After thawing, better results were observed in samples frozen in 4%DMFA or in combinations of 1.5%GL–2.5%DMFA, in fact total motility increased by 16% in the 4%DMFA group compared to 2.5%GL (P < 0.05). Also, there was an increment in the percentage of progressive motile sperm in the 1.5%GL–2.5%DMFA group (9.8% 2.5GL vs 19% in the 1.5%GL–2.5%DMFA group p < 0.05); also, samples frozen in the 4%DMFA group had more intact (YoPro‐1 negative) sperm post‐thawing, 29.3% in 2.5%GL vs 36.7% in 4%DMFA group (p < 0.05). Membrane lipid architecture was not affected by any of the cryoprotectants tested, while samples frozen in 4%DFMA had a lower percentage of mitochondria with lower membrane potential. It is concluded that DMFA improves the outcome of cryopreservation of stallion spermatozoa mainly reducing sublethal cryodamage.     

In Vitro Fertilization and Development of OPU Derived Goat and Sheep Oocytes

The recent upgrade in IVP technology seen in cattle can be adapted to embryo production in small ruminants to overcome limitations exhibited by surgical procedures on preserving the reproductive potential of donors and the efficiency of embryo production. The aim of the present study was to assess the current procedures used in cattle for the production of IVP embryos in goats and sheep based on laparoscopic-aided ovum pick-up (LOPU) supplied oocytes. Sexually matured goat and sheep donors were treated during the breeding season with FSH and subjected to laparoscopic-guided follicular puncture under general anaesthesia. The collected cumulus-oocyte complexes were matured in medium 199 and fertilized by frozen-thawed spermatozoa using Talp medium supplemented with heparin and oestrus-sheep serum. Cleaved ova were either cultured in sheep in vitro fertilization medium plus amino acids or transferred to sheep oviducts. Blastocyst rate, hatching rate and development rate up to term were used as markers of embryo function. The results obtained for goat and sheep involving 30 and 35 donors respectively (10 and 9 LOPU sessions) were 81.2% and 85.2% of oocyte collection rate; 88.3% and 98.6% oocyte incubation rate; 85.6% and 76.0% fertilization rate; 82.4% and 93.4% of cleavage rate; 50.0% and 61.5% IVP blastocyst rate; 42.1% and 45.5% blastocyst rate in oviducts; 73.0% and 66.7% embryo survival up to term, respectively. The results are comparable to those obtained in small ruminants and in bovines suggesting that requirements for embryo production and development are similar.     

Autologous Whole Ram Seminal Plasma and its Vesicle-free Fraction Improve Motility Characteristics and Membrane Status but not In Vivo Fertility of Frozen–Thawed Ram Spermatozoa

Motility characteristics (assessed subjectively and with computer-assisted semen analysis) and membrane status (after staining with chlortetracycline) of washed and non-washed frozen-thawed ram spermatozoa were evaluated after incubation in buffer and buffer containing autologous whole seminal plasma or one of its two fractions: the pellet of membrane vesicles obtained by ultracentrifugation (and used at three times normal protein concentration) or the vesicle-free supernatant fraction. Whole seminal plasma and supernatant, but not membrane vesicles, improved the motility characteristics of spermatozoa after 3 and 6 h of post-thaw incubation compared with the control buffer. Resuspension and incubation with whole seminal plasma, supernatant or membrane vesicles lowered the proportion of acrosome-reacted frozen-thawed spermatozoa compared with the control buffer. Unwashed frozen-thawed semen from three rams, incubated with autologous whole seminal plasma or its fractions and inseminated using cervical or intrauterine artificial insemination, had no effect on pregnancy rates of ewes in synchronized oestrus. However, fertility was higher after laparoscopic than cervical insemination (44.9 vs 12.3%, p < 0.001). In conclusion, resuspension and incubation of frozen-thawed ram spermatozoa in autologous whole seminal plasma or its vesicle-free supernatant fraction improved their motility characteristics and, with membrane vesicles, membrane status, but these benefits were not reflected in improved fertility after cervical or intrauterine insemination.     

Response of Thawed Epidi dymal Red Deer Spermatozoa to Increasing Concentrations of Hydrogen Peroxide,and Importance of Individual Male Variability

Oxidative stress represents a challenge during sperm manipulation. We have tested the effect of increasing hydrogen peroxide (H₂O₂) levels on red deer spermatozoa after cryopreservation, and the role of male‐to‐male variation in that response. In a first experiment, eight thawed samples were submitted to 0, 25, 50, 100 and 200 μm H₂O₂ for 2 h at 37°C. Intracellular reactive oxygen species (H₂DCFDA‐CM) increased with H₂O₂ concentration, but we only detected a decrease in sperm function (motility by CASA and chromatin damage by sperm chromatin structure assay) with 200 μm . Lipoperoxidation assessed by the thiobarbituric acid reactive substance (TBARS) method increased slightly with 50 μm H₂O₂ and above. In a second experiment, samples from seven males were submitted to 0 and 200 μm H₂O₂ for 2 h, triplicating the experiment within each male. Males differed at thawing and regarding their response to incubation and H₂O₂ presence. We found that the kinematic parameters reflected male‐to‐male variability, whereas the response of the different males was similar for lipid peroxidation and viability. A multiparametric analysis showed that males grouped differently if samples were assessed after thawing, after incubation without H₂O₂ or after incubation with H₂O₂. Red deer spermatozoa are relatively resilient to H₂O₂ after thawing, but it seems to be a great male‐to‐male variability regarding the response to oxidative stress. The acknowledgement of this individual variability might improve the development of optimized sperm work protocols.     

Effects of Egg Yolk and Cooling Rate on the Survival of Refrigerated Red Deer (Cervus elaphus hispanicus) Epididymal Spermatozoa

Egg yolk is a common component to sperm refrigeration for most of the deer species, the role of which is to protect sperm membranes against cold shock. In addition, there have been many studies of conservation of ejaculated semen from stags, but few have been reported for epididymal spermatozoa. This work was designed to investigate the combined effects of cooling rates (slow: 0.23 degrees C/min vs rapid: 4.2 degrees C/min) from room temperature to 5 degrees C, and egg-yolk concentration (0, 5 or 20%) in the extender on the survival of Iberian red deer epididymal spermatozoa refrigerated at 5 degrees C. Heterospermic sperm samples were diluted to a final sperm concentration approximately 400x10(6) sperm/ml with a Tris-citrate-fructose (TCF)-egg-yolk diluent. Sperm quality was in vitro judged by microscopic assessments of individual sperm motility [sperm motility index (SMI)], and of plasma membrane (hypo-osmotic swelling test) and acrosome (NAR) integrities. Our results first showed that the presence of egg yolk in the extender significantly improves (p=0.01) the viability and sperm motility after sperm dilution. In addition, acrosome and plasma membrane integrities post-refrigeration did not differ significantly between cooling procedures; however, the SMI differed significantly between cooling procedures (slow: 46.6% vs rapid: 50.0%; p=0.01). Our results also showed that sperm quality was significantly (p<0.01) affected by the combined effects of egg-yolk concentration and cooling procedure, being rapid cooling with 20% of egg yolk the most suitable combination for epididymal sperm refrigeration. In conclusion, egg-yolk improved red deer epididymal spermatozoa characteristics after dilution. Rapid cooling protocol using TCF with 20% egg-yolk significantly improved sperm motility of red deer epididymal spermatozoa after cooling.     

The Effects of Cryopreservation on the Morphometric Dimensions of Iberian Red Deer (Cervus elaphus hispanicus) Epididymal Sperm Heads

Computer-automated sperm-head morphometry was used in this study to determine the effects of cryopreservation on red deer sperm-head morphometry. Epididymal sperm samples were collected from 40 mature stags and were divided. One portion was diluted at room temperature in a Tris-citrate egg yolk medium, containing 6% glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for length, width, area, perimeter and shape factor (length/width), for a minimum of 135 spermatozoa were determined for each slide by means of the Sperm-Class Analyser (SCA). Firstly, our results show that cryopreservation substantially reduced (p < 0.001) sperm motility and plasma membrane and acrosome integrities. In addition, sperm heads were significantly smaller in cryopreserved spermatozoa than in the companion extended samples for area (32.05 microm2 vs 32.56 microm2; p < 0.05), length (8.46 microm vs 8.53 microm; p < 0.0001) and shape factor (1.833 vs 1.849; p < 0.0001) for all stags. These differences were found within 29 of 40 stags (75%) for at least three of the morphometric parameters. The individual variability (CV) of sperm head measurements from extended samples was negatively correlated (p < 0.005) with the per cent of change in sperm head measurements after cryopreservation for area (r = -0.465), width (r = -0.483) and perimeter (r = -0.375). Thus, the lower the sperm head variability in the extended samples, the greater the sperm change as a consequence of the cryopreservation. These results suggest that the variability (heterogeneity) in sperm head dimensions of individual stags may be a good indicator of sperm freezability.     

Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex‐sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex‐sorted sperm frozen at low sperm concentrations (20 × 10⁶ sperm/ml; S20 group). Non‐sorted spermatozoa frozen at 1000 × 10⁶ (C1000 group) and 20 × 10⁶ (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post‐thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non‐sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post‐thaw motility of sex‐sorted spermatozoa frozen at low concentrations.     

Optimizing the Conditions for In Vitro Maturation and Artificial Activation of Sika Deer (Cervus nippon hortulorum) Oocytes

With the goal of establishing experimental protocols for cloning sika deer, various conditions for in vitro maturation (IVM) and artificial activation of sika deer oocytes were examined. In vitro maturation was evaluated in seven different culture media. The highest rate of oocyte maturation was 75.4% in 10 μg/ml follicle‐stimulating hormone (FSH), 1 μg/ml LH, 0.2 mm cysteamine and 50 ng/ml epidermal growth factor (EGF) after 24 h of IVM. The efficiency after 24 h of IVM did not differ significantly (p > 0.05) from that observed after 20 h. Cysteamine (0.2 mm ) significantly increased the maturation rates after 20 h (from 59.1% to 67.2%, p < 0.05) and after 24 h (from 63.2% to 71.6%, p < 0.05) of IVM. The IVM rates of oocytes collected during the oestrous season (75.4%) and the anoestrous season (23.3%) were significantly different at 24 h. The 20 μg/ml FSH, 2 μg/ml LH, 0.4 mm cysteamine and 100 ng/ml EGF significantly increased the maturation rates (from 23.3% to 54.2%, p < 0.01) at 24 h during the anoestrous season. For the activation experiments, the most effective method was chemical activation [ionomycin + 6‐dimethylaminopurine (6‐DMAP)], which promoted the development of sika deer oocytes to the blastocyst stage (32.4%). Our results indicate that in vitro matured sika deer oocytes are good candidates for parthenogenetic activation and that chemical treatment is needed for relatively efficient activation of the oocytes. These optimized conditions for IVM and parthenogenetic activation may be useful for efforts to restore populations of the endangered sika deer using the somatic cell nuclear transfer technique.     

塔里木马鹿茸间充质干细胞体外培养及细胞特性的研究

鹿茸间充质干细胞是一类新发现的干细胞。为获得鹿茸间充质干细胞的生物学特征,建立鹿茸间充质干细胞体外培养模式,通过采用改良组织块细胞培养法对生长30 d的塔里木马鹿茸间充质层细胞体外分离培养,观察细胞形态特征,MTT比色法检测细胞生长特点。结果表明:间充质层样品组织块于1 d完全贴壁,贴壁后培养2 d即可观察到组织块边缘迁出少量细胞,贴壁后培养3 d,大量的间充质干细胞从组织块中迁出,并不断增殖,在组织块贴壁后5 d细胞生长融合。传代培养的2~6代细胞在培养到96 h细胞增殖达到高峰。原代培养细胞呈梭形,细胞核呈椭圆形,细胞呈菊花状排列生长;传代细胞形态呈梭形、不规则形,且排列无规则。本研究结果将为鹿茸间充质干细胞的后续研究奠定基础。     

Production of Diabetic Offspring Using Cryopreserved Epididymal Sperm by In Vitro Fertilization and Intrafallopian Insemination Techniques in Transgenic Pigs

Somatic cell nuclear transfer (SCNT) is a useful technique for creating pig strains that model human diseases. However, production of numerous cloned disease model pigs by SCNT for large-scale experiments is impractical due to its complexity and inefficiency. In the present study, we aimed to establish an efficient procedure for proliferating the diabetes model pig carrying the mutant human hepatocyte nuclear factor-1α gene. A founder diabetes transgenic cloned pig was generated by SCNT and treated with insulin to allow for normal growth to maturity, at which point epididymal sperm could be collected for cryopreservation. In vitro fertilization and intrafallopian insemination using the cryopreserved epididymal sperm resulted in diabetes model transgenic offspring. These results suggest that artificial reproductive technology using cryopreserved epididymal sperm could be a practical option for proliferation of genetically modified disease model pigs.     

Evaluation of Cheetah and Leopard Spermatozoa Developmental Capability after Interspecific ICSI with Domestic Cat Oocytes

The ICSI procedure is potentially of great value for felids, and it has not been extensively studied in these species. The objectives of this work were to determine the best conditions for ICSI in the domestic cat (DC) to generate interspecific embryos by injecting cheetah (Ch) and leopard (Leo) spermatozoa. Firstly, DC oocytes were matured with insulin–transferrin–selenium (ITS) or without it (MM) and cultured using atmospheric (21%) or low (5%) oxygen tension after ICSI. The group ITS‐5%O₂ showed the highest blastocyst rate (p < 0.05), 20.9% vs 8.7%, 7% and 6.5%, for MM‐21%O₂, MM‐5%O₂ and ITS‐21%O₂, respectively. The best conditions were used to generate the interspecific embryos, together with ionomycin activation (Io) after ICSI. Interspecific embryos resulted in high rates of blastocysts that were not positively affected by Io activation: 32.6% vs 21% for Ch and Ch‐Io, 9.8% vs 21% for Leo and Leo‐Io, and 20% vs 17.4% for DC and DC‐Io. We also evaluated DNA‐fragmented nuclei of experiment 1 and 2 blastocysts, using TUNEL assay. The fragmented nucleus proportion was higher in the ITS‐5%O₂ group, 67.6%. Surprisingly, interspecific blastocysts showed the lowest fragmented nucleus proportion: 27% and 29.9% for Ch and Leo, respectively. We concluded that ITS and 5%O₂ improve blastocyst formation in DC, although with a concomitant increase in DNA fragmentation. Most importantly, cheetah and leopard spermatozoa were able to generate blastocysts without artificial activation, which suggests that developmental capacity of wild felid spermatozoa can be evaluated by interspecific ICSI. This technique should be used to assist wild felid reproduction.     

Production of Lambs After the Transfer of Fresh and Cryopreserved in vitro Produced Embryos from Prepubertal Lamb Oocytes and Unsorted and Sex-sorted Frozen-thawed Spermatozoa

Cumulus-oocyte complexes from hormone-stimulated 3-4-week-old (n=43) and 6-7-week-old (n=12) prepubertal lambs were matured in vitro and incubated with unsorted, or X- or Y-spermatozoa separated with a high-speed cell sorter (SX MoFlo)frozen-thawed. Presumptive zygotes were then cultured to the blastocyst stage, and transferred to recipients fresh or after cryopreservation (frozen). Oocyte cleavage was higher (p <0.05) with unsorted (515/926, 55.6%) than X- or Y-spermatozoa (261/672, 38.8% and 229/651, 35.2%, respectively) and blastocyst formation (% zygotes) by Day 9 of in vitro culture was lower (p <0.05) for X- (102/261, 39.1%) than unsorted spermatozoa (249/515, 48.3%), but did not differ between Y-spermatozoa (103/229, 45.0%) and unsorted spermatozoa, or between X- and Y-spermatozoa (p >0.05). For fresh embryos, survival to term was 50.0% (3/6) for unsorted, 0.0% (0/6) for X- and 16.7% (1/6) for Y-spermatozoa-derived embryos (p >0.05), and for frozen embryos was 4.0% (2/50) for unsorted, 9.1% (2/22) for X- and 2.9% (1/34) Y-spermatozoa-derived embryos (p >0.05). Of the two lambs born from X-spermatozoa-derived embryos, one was female (50%), and from the two Y-spermatozoa-derived lambs, both were male (100%), demonstrating that lambs can be produced after the transfer of fresh and cryopreserved IVP embryos derived from prepubertal lamb oocytes and frozen-thawed sex-sorted sperm.     

Ultrastructural Characteristics of Non-matured and In Vitro Matured Oocytes Collected from Pre-pubertal and Adult Domestic Cat Ovaries

The present study describes the ultrastructural characteristics of cat oocytes before maturation and after 12- and 24-h in vitro maturation (IVM). Oocytes were recovered from pre-pubertal and adult queen ovaries after ovariohysterectomy and a proportion were stored in glutaraldehyde at 4°C until examination by transmission electronic microscopy (TEM). Those selected for maturation were cultured before TEM in DMEM for 12 and 24 h at 38°C in a humidified environment of 5% O₂, 5% CO₂ and 90% N₂. Specimens were divided into six groups: non-matured oocytes from pre-pubertal queens (PP0), non-matured oocytes from adult queens (A0), 12-h in vitro matured oocytes from pre-pubertal queens (PP12), 12-h in vitro matured oocytes from adult queens (A12), 24-h in vitro matured oocytes from pre-pubertal queens (PP24) and 24-h in vitro matured oocytes from adult queens (A24). Across the treatment groups, it was possible to observe differences in the thickness of the perivitelline space, the penetration of cumulus cell projections forming a junctional complex, distribution and density of small vesicles, lipid droplets, microvilli, mitochondria and cortical granules and variable degrees of development of Golgi complexes. These findings demonstrated that ultrastructural analysis of oocytes matured in vitro is a valuable tool to evaluate oocyte cytoplasmic maturation and that this IVM protocol was efficient in inducing gradual morphological changes necessary for cytoplasmic maturation of pre-pubertal and adult cat oocytes.     

Expression of the Orexigenic Peptide Ghrelin and the Type 1a Growth Hormone Secretagogue Receptor in Sheep Oocytes and Pre-implantation Embryos Produced In Vitro

Recent studies have implicated the peripheral actions of ghrelin in reproductive tissues. Here, we provide evidence that both ghrelin and its receptor GHSR-1a (the type 1a growth hormone secretagogue receptor) are expressed at both mRNA and protein levels in sheep oocytes and pre-implantation embryos produced in vitro . Real-time RT-PCR experiments confirmed that ghrelin mRNA levels varied depending on developmental stage, with the highest expression in metaphase II (MII) oocytes, higher expression at the 2-cell stage, and minimal expression in germinal vesicle (GV) oocytes, 4- and 8-cell stages, and in the blastocyst. The levels of GHSR-1a mRNA decreased from GV to MII, increased immediately at the 2-cell stage and then remained stable until the blastocyst stage. Ghrelin protein was detected mainly in the cytoplasm close to the plasma membrane in both inner cell mass and trophectoderm cells, while GHSR-1a protein was most abundant in the plasma membrane. In conclusion, the presence of the ghrelin signalling system within the sheep oocytes and pre-implantation embryos opens up the possibility of a potential regulatory role of this novel molecule in reproductive function.     

Phosphofructokinase and Malate Dehydrogenase Participate in the In Vitro Maturation of Porcine Oocytes

Oocyte maturation depends on the metabolic activity of cumulus–oocyte complex (COC) that performs nutritive and regulatory functions during this process. In this work, the enzymes [phosphofructokinase (PFK) and malate dehydrogenase (MDH)] were tested to elucidate the metabolic profile of porcine COCs during the in vitro maturation (IVM). Enzymatic activity was expressed in U/COC and U/mg protein (specific activity) as mean ± SEM. In vitro maturation was performed with 2‐oxoglutarate (5, 10 and 20 mm ) or hydroxymalonate (30, 60 and 100 mm ) inhibitors of PFK and MDH, respectively. The PFK and MDH activities (U) remained constant during maturation. For PFK, the U were (2.48 ± 0.23) 10^?5 and (2.54 ± 0.32) 10^?5, and for MDH, the U were (4.72 ± 0.42) 10^?5 and (4.38 ± 0.25) 10^?5 for immature and in vitro matured COCs, respectively. The specific activities were significantly lower after IVM, for PFK (4.29 ± 0.48) 10^?3 and (0.94 ± 0.12) 10^?3, and for MDH (9.08 ± 0.93) 10^?3 and (1.89 ± 0.10) 10^?3 for immature and in vitro matured COCs, respectively. In vitro maturation percentages and enzymatic activity diminished with 20 mm 2‐oxoglutarate or 60 mm hydroxymalonate (p < 0.05). Viability was not affected by any concentration of the inhibitors evaluated. The U remained unchanged during IVM; however, the increase in the total protein content per COC provoked a decrease in the specific activity of both enzymes. Phosphofructokinase and MDH necessary for oocyte IVM would be already present in the immature oocyte. The presence of inhibitors of these enzymes impairs the meiotic maturation. Therefore, the participation of these enzymes in the energy metabolism of the porcine oocyte during IVM is confirmed in this study.     

Comparison of the TBARS Assay and BODIPY C11 Probes for Assessing Lipid Peroxidation in Red Deer Spermatozoa

Several methods are used to measure lipid peroxidation (LPO) in spermatozoa. The objective of this study was comparing the thiobarbituric acid reactive species (TBARS) method and the BODIPY 581/591 C₁₁ (B581) and BODIPY 665/676 C₁₁ (B665) fluorescent probes to measure induced peroxidative damage in thawed epididymal spermatozoa from Iberian red deer. Samples from three males were thawed, pooled, diluted in PBS, incubated at room temperature and assessed at 0, 3, 6 and 24 h under different experimental conditions: Control, hydrogen peroxide (H₂O₂) 0.1 mm or 1 mm , or tert‐butyl hydroperoxide (TBH) 0.1 mm or 1 mm . LPO was assessed by the TBARS assay [malondialdehyde (MDA) detection] and by the fluorescence probes B581 and B665 (microplate fluorimeter and flow cytometry). Increasing MDA levels were only detectable at 1 mm of TBH or H₂O₂. Both fluorescence probes, measured with fluorometer, detected significant increases of LPO with time in all treatments, except Control. Flow cytometry allowed for higher sensitivity, with both probes showing a significant linear relationship of increasing LPO with time for all oxidizing treatments (p < 0.001). All methods showed a good agreement, except TBARS, and flow cytometry showed the highest repeatability. Our results show that both B581 and B665 might be used for LPO analysis in Iberian red deer epididymal spermatozoa, together with fluorometry or flow cytometry. Yet, the TBARS method offered comparatively limited sensitivity, and further research must determine the source of that limitation.     

Evaluation of the Lipid Content in Bovine Oocytes and Embryos with Nile Red: a Practical Approach

In this study, the fluorescent lipid dye Nile Red, was used to demonstrate that the lipid content of immature bovine oocytes is correlated with the morphological appearance of the ooplasm. Oocytes with a uniform dark cytoplasm contained significantly more intracellular lipids in lipid droplets compared with oocytes with a granulated or pale cytoplasm (p < 0.05). Furthermore, this lipid-analysing technique was applied for the first time on single bovine in vitro embryos, showing a significant increase of the lipid content in lipid droplets after culture in the presence of serum (p < 0.05).