Effect of liquid storage and cryopreservation of boar spermatozoa on acrosomal integrity and the penetration of zona-free hamster ova in vitro

The effect of liquid storage and cryopreservation of boar spermatozoa on sperm motility, acrosomal integrity, and the penetration of zona-free hamster (ZFH) ova was examined. The sperm penetration assay (SPA) provides valuable information on specific events of fertilization and is a potentially useful indicator of sperm fertility. Ejaculated semen from 4 boars was subjected to 3 treatments: fresh (FRE, no storage), liquid-stored (LIS, stored at 18°C for 3 days), and frozen (FRO, frozen by pellet method and stored at ?196°C for 3 days). A highly motile sperm population was isolated by the swim-up procedure (1 hr). FRE and LIS were incubated an additional 3 hr at 39°C in a Tris-buffered medium to elicit capacitation and the acrosome reaction. Sperm motility and acrosomal integrity were assessed before and after incubation. For the SPA, sperm and eggs were incubated at 39°C for 3 hr in Hams F-10 medium. Each egg was assessed for sperm penetration, sperm binding, and stage of development. Percentages of sperm motility and sperm with a normal apical ridge (NAR) prior to incubation were 78 and 78 (FRE), 75 and 69 (LIS), and 28 and 50 (FRO). After incubation, percentages of motility, NAR, and acrosome-reacted sperm were 34, 10, and 73 (FRE); 43, 24, and 51 (LIS); and 18, 13, and 59 (FRO). A somewhat higher (P < .05) percentage of ZFH ova was penetrated by FRE (45.8) than by LIS (42.0). Penetration of ZFH ova by FRO was markedly (P < .05) reduced (30.2). Sperm penetration was not significantly correlated with motility or acrosomal integrity before or after incubation, regardless of treatment. These data suggest that the SPA can be used in conjunction with conventional measures of semen analysis in assessing the potential fertilizing capacity of boar sperm and that liquid storage is superior to frozen storage with respect to preserving sperm fertility.     

Molitity and acrosomal changes in ionophore-treated bovine spermatozoa and their relationship with in vitro penetration of zone-free hamster oocytes

Frozen-thawed spermatozoa from Friesian bulls held at stud in Ireland were used to assess the effect of ionophore on motility, acrosome reaction and heterologous in vitro fertilization. Bovine spermatozoa penetrated zone-free hamster oocytes following treatment with calcium ionophore in the absence of bovine serum albumin (BSA) and in the presence of 10 mM caffeine. Sperm velocity was stimulated in concentrations of caffeine <2.5 mM following dilution with medium containing BSA. Sperm attachment to the plasmalemma showed no association with penetration rates of zona-free hamster oocytes. Penetrated oocytes in regimens with >0.1 mM ionophore did not progress through Meiosis II. Increasing concentrations of ionophore induced the acrosome reaction more rapidly, although this was associated with reduced motility. Hyperactive motility was observed in calcium ionophore-treated spermatozoa which were capable of penetrating zona-free hamster oocytes. Sperm velocity remained unchanged. whereas the track:vector ratio, a measurement of curvilinear movement, was reduced. This work may have important implications for the assessment of bovine fertility and cytogenetic analysis of bovine sperm.     

In vitro fertilization of bovine oocytes in protein-free culture system

We studied the possibility of fertilization of bovine oocyte-cumulus complexes, matured in vitro in a protein-free medium, in a protein-free culture system without preliminary capacitation of spermatozoa. The development of embryos to the morula-blastocyst and blastocyst stage was considered as a criterion of successful fertilization. It was shown that replacement of bovine serum albumin for polyvinyl alcohol or polyvinylpyrrolidone in Tyrode medium for fertilization did not affect significantly the development to the morula-blastocyst stage and the number of cells in blastocysts. It was also found that replacement bovine serum albumin for polyvinyl alcohol in all used media, Tyrode medium for washing of oocytes, medium for sperm preparation to fertilization, and Tyrode medium for fertilization, did not affect significantly the development to the morula-blastocyst and blastocyst stages, as well as on the number of cells in blastocysts. The results obtained suggest that in vitro fertilization of bovine oocyte-cumulus complexes is possible in a protein-free culture system without significant reduction in the capacity for in vitro development of the obtained embryos and number of cells in blastocysts.     

Enhancement of the acrosome reaction of hamster spermatozoa by the proteolytic enzymes,kallikrein, trypsin,and chymotrypsin

The involvement of a kallikrein−kinin system in the motility of mammalian spermatozoa has been suggested by several investigators. We found that incorporation of kallikrein (0.1–1.0) unit/ml) in the sperm incubation medium did not enhance the motility of hamster spermatozoa that were already active. However, this enzyme significantly increased the incidence of the acrosome reaction. Trypsin (1.8–18 units/ml) and chymotrypsin (0.34–3.4 units/ml) also increased the incidence of the acrosome reaction, and accelerated its onset. Kinins (bradykinin and kallidin) added to the medium in a wide concentration range (1 ng/ml to 1 mg/ml) had no marked effects on either the motility or the acrosome reaction. A kallikrein−kinin system is apparently not of primary importance at least for the acrosome reaction. The enhancement of the acrosome reaction by exogenous proteinases may be due in part to accelerated removal or alteration of the sperm surface coat (glycoprotein) by the enzyme peior to the acrosome reaction. Exogenous proteinases may also act synergistically with endogenous (acrosomal) proteinases (and other enzymes) in altering membrane proteins and dispersing the acrosome matrix during the course of teh acrosome reaction.     

In vitro fertilization of pig oocytes matured in vitro

The development of in vitro fertilization (IVF) techniques in pigs as well as in other species is of great importance because of the possible applications of this technology in different research fields. Methods of IVF vary in different incubation periods and temperatures, in the hormone concentrations used, and in the treatment of the sperm samples. It has been particularly difficult to succeed in the achievement of fertilization in the pig. In the present study we used FSH and LH concentrations of 2 IU/ml for oocyte maturation, an incubation temperature of 37°C, and dilution of spermatozoa for capacitation, and we achieved a high fertilization rate (50 to 75%) with no cases of polyspermy.     

Effect of the cumulus on in vitro fertilization of bovine matured oocytes

The effect of the cumulus on in vitro fertilization in bovines was examined. Follicular oocytes were cultured in medium 199 plus OCS and extra granulosa cells. Frozen-thawed bovine spermatozoa was separated by the swim-up technique, suspended in Talp medium and capacitated with heparin. Fresh sheep and goat semen was incubated for 4 h at room temperature, washed and spermatozoa were then suspended in Talp medium and capacitated by incubation at 38.5 °C and 5% CO₂ in air and heparin.

In experiment 1, cumulus-enclosed oocytes, denuded oocytes and denuded oocytes plus additional cumulus cells were incubated with a reduced concentration of bovine spermatozoa for 8 or 18 h. In Experiment 2, cumulus enclosed and denuded oocytes were incubated with bovine spermatozoa for 4, 6, 8 and 18 h using a sperm concentration adjusted to secure high fertilization rates. In Experiment 3, cumulus-enclosed and denuded bovine oocytes were incubated with either sheep or goat spermatozoa for 18 h. Fertilization rates were then calculated and compared statistically. The results showed that 1) the cumulus improved the fertilization rate only when cumulus cells were associated with the oocytes 2) the timing of sperm penetration was not modified by the cumulus and started at 4 h after sperm incubation and 3) the presence of the cumulus improved the heterologous fertilization rate only when sheep spermatozoa were used. The results suggest that the cumulus improves fertilization rate by providing a capacitation-inducing mechanism and by facilitating the interaction between capacitated spermatozoa and the zona pellucida surface.     

Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract

The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm². Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract.     

Sperm characteristics and in vitro fertilization ability of thawed spermatozoa from Black Manchega ram: Electroejaculation and postmortem collection

The aim of this study was to assess two models of sperm collection on the quality and fertility of thawed spermatozoa from Black Manchega rams, a threatened breed. Sperm samples were collected by electroejaculation and postmortem from each male. Samples were diluted with Biladyl and frozen. Motility (subjective and objective by means of computer-assisted semen analysis), membrane integrity, and acrosomal status (microscopy) were assessed on fresh and thawed semen; plasmalemma integrity, mitochondrial membrane potential, DNA integrity, and acrosomal status were evaluated by flow cytometry on thawed semen. Thawed spermatozoa were used in a heterologous in vitro fertilization test. After thawing, the proportion of live spermatozoa with intact membrane (YO-PRO-1−/PI−) was higher for postmortem samples (P < 0.001), although the ratio of YO-PRO-1− spermatozoa within the PI− population was higher for ejaculated samples (P = 0.007). Likewise, the proportion of live spermatozoa having high mitochondrial membrane potential (MitoTracker+) and intact acrosomes (PNA−) was higher for postmortem samples (P < 0.001 and P < 0.001, respectively). Considering only live spermatozoa, the ratio of MitoTracker+/PNA− cells was higher for electroejaculated samples (P = 0.026 and P = 0.003). Both electroejaculated and postmortem samples fertilized oocytes. Nevertheless, electroejaculated samples yielded a higher percentage of hybrid embryos (P = 0.041). In conclusion, although postmortem spermatozoa had better sperm quality after thawing, electroejaculated spermatozoa showed higher ratios for sperm quality when only the live population was considered. Electroejaculated and postmortem samples might be used for germplasm banking of this threatened breed, but the fertility of postmortem spermatozoa might be lower.     

The zymogen form of acrosin in testicular,epididymal, and ejaculated spermatozoa from ram

The sperm-specific proteinase acrosin (EC 3.4.21.10) is found in spermatozoa as a zymogen. We have looked for different forms of this zymogen in testicular, epididymal, and ejaculated spermatozoa from ram and have compared total sperm extracts made immediately after cell disruption with extracts made later from isolated sperm heads. We have concluded that the autoactivatable zymogen form, known generally as proacrosin, is the only form of acrosin within intact mature ram spermatozoa; no other zymogen form was detected, although lower levels of proacrosin were found in some samples of testicular spermatozoa. From studies of the activation process, it appears that ram proacrosin is truly autoactivatable; no evidence could be found for the involvement of any auxiliary enzyme. Estimations of the molecular weight of proacrosin using gel chromatography (60,000) and SDS-polyacrylamide gel electrophoresis (51,300) indicated that the zymogen is monomeric. Comparison with the molecular weight of ram acrosin (44,000 or 40,000, using the two respective methods) indicated that a single acrosin molecule is derived from each zymogen molecule. The sperm acrosin inhibitor (molecular weight 11,000 or 8,000) was present in testicular spermatozoa as well as in ejaculated spermatozoa; there was no evidence that it was produced as a result of zymogen activation.     

Production,characterization, and use of ionophore-induced,calcium-dependent acrosome reaction in ram spermatozoa

A system has been developed for inducing a calcium-dependent acrosome reaction in ram spermatozoa in vitro using the calcium ionophore A23187. The resultant reaction is accompanied by release of the acrosomal enzymes hyaluronidase and acrosin, but there is no release of the cytoplasmic enzyme glucose 6-phosphate isomerase. In any given cell, the visible acrosome reaction apparently takes place rapidly, but there is a variable delay before the reaction occurs. Under optimum conditions, about 90% of treated spermatozoa show an acrosome reaction within one hour. Preincubation of the spermatozoa with the proteinase inhibitors p-amino-benzamidine or p-nitrophenylguanidinobenzoate allows two stages of the reaction to be distinguished ultrastructurally, a membrane fusion stage followed by a dispersal of the acrosomal matrix. In the presence of the inhibitors, the first stage is delayed but is completed within 1 hour, whereas the second remains largely incomplete. In the presence of calcium, ionophore concentrations which induce an acrosome reaction abolish sperm motility rapidly and completely. However, by adding serum albumin shortly after addition of ionophore, motility can be preserved while the acrosome reaction occurs as usual; the motility pattern observed under these conditions is of the “whip-lash” or “activated” type. Although the motile ionophore-treated spermatozoa were unsuccessful at penetrating normal mature sheep oocytes in vitro, they were able to penetrate zona-free oocytes, after which swelling and decondensation of the sperm head took place.     

猪卵巢体外保存温度对卵母细胞染色质构型和成熟能力的影响

Most porcine oocytes used in studies on embryo biotechnology and the in vitro production of embryos are currently obtained from the ovaries of slaughtered gilts. The duration and temperature during ovary transportation and handling might, therefore, affect the recovery of culturable COCs, chromatin configuration and developmental competence of oocytes. The effects of ovary storage temperature on chromatin configuration and in vitro maturation of porcine oocytes were examined in this study. Ovaries collected from a slaughterhouse were stored in vitro for 8 h under different temperatures. The results showed that more culturable COCs were isolated from the ovares stored at 39℃ than that from ovaries stored at 31℃ or 20℃ and before storage. Thirty-one centidegree was the best storage temperature in terms of cumulus expansion, nuclear maturation and morphology of the first polar body after in vitro maturation culture. The ability of cumulus expansion was completely lost in COCs derived from ovaries stored at 39℃ for 8 hours. Ovary storage （at both 31℃ and at 20℃ ） increased the proportion of oocytes with the GVc configuration in which chromatin condensed into a single big clump at the nucleolus and the functional significance of this configuration needs further investigations [ Acta Zoologica Sinica 51 （5）： 919 923, 2005].     

Effect of dietary selenium deficiency on the in vitro fertilizing ability of mice spermatozoa

Selenium is an essential micronutrient for mammals, being integral part of antioxidant system. The aim of the study was to evaluate the effect of selenium deficiency on in vitro fertilization (IVF) capacity of spermatozoa and on oxidative stress in these cells. Male C57BL/6N mice were maintained on selenium-deficient or selenium-sufficient diets (0.02 or 0.2 ppm of selenium as selenomethionine, respectively) for 4 months. Liver glutathione peroxidase activity measurements were used to confirm selenium deficiency. Sperm quality and IVF capability among both groups were evaluated. To assess oxidative damage, lipid peroxidation as malondialdehyde production was determined in spermatozoa as well as the testes. Ultrastructural analyses of spermatozoa nuclei using transmission electron microscopy were also performed. The percentage of eggs fertilized with sperm from selenium-deficient mice was significantly decreased by approximately 67%. This reduced fertilization capacity was accompanied by increased levels of lipid peroxidation in both the testes and sperm, indicating that selenium deficiency induced oxidative stress. Consistent with this finding, spermatozoa from selenium-deficient animals exhibited altered chromatin condensation. Deficiency in dietary selenium decreases the reproductive potential of male mice and is associated with oxidative damage in spermatozoa.     

Effect of antioxidants on preservation of motility,viability and acrosomal integrity of equine spermatozoa during storage at 5 degrees C

Preservation of liquid semen at 5 degrees C is an important technique in the breeding management of horses. Oxidative damage to spermatozoa during storage is a potential cause of the decline in motility and fertility during hypothermic storage of liquid semen. The objective of this study was to evaluate the use of water-soluble and lipid-soluble antioxidants to improve the maintenance of motility of equine spermatozoa at 5 degrees C during storage for 72 to 96 h. In Experiment 1, the effect of addition of catalase on the maintenance of motility, viability and acrosomal integrity was determined. Semen was collected, and these treatments were applied: catalase (0, 100 or 200 U/mL) in nonfat, dried skim milk extender (NFDSM; with or without seminal plasma) or 10% seminal plasma + NFDSM. Motility was determined by computerized semen analysis (CASA) at 0, 24, 48 and 72 h. Viability and acrosomal integrity were determined at 72 h of storage. There was no significant treatment effect on the maintenance of sperm motility during 72 h storage. In Experiment 2, the effect of adding lipid-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted to a final concentration of 25 x 10(6) sperm/mL in NFDSM containing butylated hydroxytoluene (BHT; 2.0, 1.0, or 0.5 mM), Vitamin E (4.0, 2.0, 1.0 mM), or Tempo (2.0, 1.0, or 0.5 mM). Although the addition of BHT significantly reduced (P < 0.05) progressive motility during storage compared to the control, there were no positive treatment effects of either Vitamin E or Tempo on maintenance of motility. In Experiment 3, the effect of adding water-soluble antioxidants on maintenance of motility was evaluated. Semen was diluted in NFDSM containing these treatments: Trolox (2.0 mM), Tempo (1.0 mM), Vitamin C (0.45 mg/mL), BSA (3% w/v), combinations of these antioxidants, or control. Adding these water-soluble antioxidants did not significantly improve the maintenance of motility during cooled storage at 5 degrees C. In conclusion, adding the enzyme scavenger, catalase, or a variety of lipid- and water-soluble antioxidants did not significantly improve the maintenance of motility during liquid semen storage at 5 degrees C.     

不同脱水处理对桔梗花粉离体萌发及贮藏性能的影响

对桔梗花粉离体萌发条件进行了筛选,并采用花粉离体萌发法测定了不同干燥方式和贮藏时间处理对桔梗花粉生活力的影响;同时采用人工授粉的方法研究了不同处理后桔梗花粉授粉结实能力的变化。结果表明：（1）30℃培养1.5h是桔梗花粉离体萌发和花粉管生长的最佳培养条件。（2）不同干燥方式、不同干燥时间处理后桔梗花粉萌发率和花粉管长度均有所下降,但下降幅度不同,其中以干燥箱45℃烘干处理1.5h~2h下降最少,其花粉耐藏性也有明显提高。（3）不同干燥方式处理桔梗花粉后其授粉结籽率的变化明显不同,阴置2h处理的花粉结籽率最低,而干燥器内放置6h、日晒2h和烘干1.5h处理后花粉结籽率略有变化;干燥处理后随贮藏时间的增加,其授粉结籽率均呈下降趋势,但45℃烘干1.5h处理的花粉结籽率下降速度最慢,其授粉结籽能力可保持5d左右。     

Sperm binding, in vitro fertilization, and in vitro embryonic development of bovine oocytes fertilized with spermatozoa incubated with norepinephrine

The final stages of sperm maturation, fertilization, and early embryonic development occur within the oviduct and are essential for successful reproduction in mammals. Norepinephrine was previously identified in native bovine oviductal fluid and its in vitro effects on bull sperm capacitation and the acrosome reaction have been determined. It was unknown how physiological concentrations of norepinephrine influence sperm binding, fertilization, and embryo development. Therefore, the objective of this study was to determine if pre-incubating bovine spermatozoa with physiological concentrations of norepinephrine prior to insemination of bovine oocytes would improve sperm-oocyte binding, fertilization, and embryonic development in vitro. Norepinephrine, in concentrations representing those measured in bovine oviductal fluid, was used to treat bovine spermatozoa prior to insemination. Spermatozoa incubated in norepinephrine were used to inseminate bovine oocytes matured in vitro, and oocytes were evaluated for sperm binding and fertilization. Additional experiments were conducted to evaluate how early in the co-incubation period oocytes were fertilized by spermatozoa pre-incubated with norepinephrine, and to test the developmental competence of those oocytes fertilized with norepinephrine-treated sperm. Sperm binding to the zona pellucida was reduced by pre-incubation with norepinephrine. Rates of fertilization and embryo development did not increase as a result of pre-incubating spermatozoa with norepinephrine, but as early as 4h after insemination, spermatozoa treated with 20 ng/ml norepinephrine fertilized more oocytes than spermatozoa incubated in medium alone. Interestingly, this concentration of norepinephrine was found to capacitate spermatozoa in previous studies. These data suggest that oocytes fertilized by spermatozoa incubated in 20 ng/ml norepinephrine fertilize earlier in vitro than sperm pre-incubated in medium alone, and provide additional support for the role of norepinephrine in sperm capacitation and the acrosome reaction.     

Effect of follicle cells on the acrosome reaction, fertilization, and developmental competence of bovine oocytes matured in vitro

The role of follicle cells in the acrosome reaction of frozen-thawed bovine spermatozoa, in vitro fertilization, cleavage, and development in vitro was investigated. Cumulus-oocyte complexes were cocultured and matured in vitro with additional granulosa cells for 24 hr. Immediately before in vitro insemination, the oocytes were divided into three types with different follicle cells: denuded and corona- and cumulus-enclosed oocytes. The proportion of live, acrosome-reacted spermatozoa significantly increased at 3 and 6 hr after insemination in all types of oocytes. However, the mean proportion of live, acrosome-reacted spermatozoa that inseminated cumulus-enclosed oocytes at 6 hr after insemination was significantly higher than that of spermatozoa inseminating denuded oocytes (18.3% and 13.3%, respectively). The frequency of in vitro fertilization was significantly higher for cumulus-enclosed oocytes (65.4%) than for denuded and corona-enclosed oocytes (30.8% and 39.4%, respectively). Cumulus-enclosed oocytes when cocultured with oviduct epithelial cells also had significantly higher rates of cleavage (two- to eight-cell, 59.8%; eight-cell, 22.4%) and blastocyst formation (7.7%) than denuded and corona-enclosed oocytes. No eight-cell embryos or more advanced stages of embryonic development were observed in either denuded or corona-enclosed oocytes without the coculture. The present results indicate that cumulus cells at fertilization play an important role in inducing the acrosome reaction and promoting a high fertilization rate, cleavage, and development into blastocysts in vitro.     

Effects of beta-endorphin and Naloxone on in vitro maturation of bovine oocytes

Bovine cumulus-oocyte complexes (COCs) and mural granulosa cells express the mRNA coding for the micro-opioid receptor. The addition of beta-endorphin (beta-end) to oocytes cultured in hormonally-supplemented in vitro maturation (IVM) medium had no effect on the rates of oocytes reaching the metaphase II (MII) stage, but significantly decreased the maturation rate (P < 0.05) and arrested oocytes at metaphase I (MI) after culture in hormone-free medium (P < 0.001). Naloxone (Nx) reverted this inhibitory effect of beta-end. Moreover, Nx "per se" showed a dose-dependent dual effect. When added at high concentration (10 x (-3) M), it significantly reduced the rate of oocytes in MII (P < 0.001), thus increasing the rate of oocytes arrested in MI. However, Nx added at low concentration (10 x (-8) M) significantly increased oocyte maturation (P < 0.001). High concentration of Nx induced an increase in both intracellular calcium concentration ([Ca(2+)](i)) and in the activity of the mitogen-activated protein kinase (MAPK) also called extracellular-regulated kinase (ERK) in cumulus cells of bovine COCs. Blocking the rise in [Ca(2+)](i) with the calcium chelator acetoxymethylester-derived form of bis (o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM) reversed the Nx-dependent inhibition of meiotic maturation observed at high Nx concentrations. Whereas blocking ERK with the MAPK/ERK kinase (MEK) inhibitor, PD98059, had no effect on this process. Therefore, we concluded that the mocro-opioid receptor, by inducing [Ca(2+)](i) increase, participates in the cumulus-oocyte coupled signaling associated with oocyte maturation.     

Effects of long-term chilled storage of red deer epididymides on DNA integrity and motility of thawed spermatozoa

We have carried out a study on the influence of prolonged cold storage (5 degrees C) of Iberian red deer epididymides on post-thaw sperm motility and DNA integrity. Twenty-nine pairs of testes, with attached epididymides, were collected during November and December. Spermatozoa from one of each of the pairs were immediately recovered, evaluated and frozen (control). The remaining epididymides were cooled to 5 degrees C and stored for 24, 96 and 192 h (experimental groups), after which spermatozoa were collected and frozen. Samples were evaluated before freezing, after thawing, and after a 2-h period of incubation at 37 degrees C. Motility was evaluated by means of a CASA system and chromatin stability was assessed following the Sperm Chromatin Structure Assay (SCSA). Our results showed that, during the first 96 h, the motility (total and progressive) did not significantly decline when assessed after cryopreservation, although there was a significant decline when epididymides had been stored for 192 h at 5 degrees C (P<0.001). The present study demonstrates that motility and DNA status of thawed spermatozoa collected from refrigerated epididymes, at least 96 h post-mortem, were good enough to consider their eventual use. Most importantly, sperm DNA integrity after thawing was apparently not affected by storage time, even after 192 h.     

Temporal distinctions in the synthesis and accumulation of proteins by oocytes and cumulus cells during maturation in vitro of bovine oocytes

Successful in vitro maturation (IVM) of bovine oocytes requires continual and/or episodic protein synthesis by cumulus-oocyte complexes. This study was designed to expose time-dependent changes in protein synthesis and accumulation by bovine oocytes and cumulus cells during routine IVM. Silver staining after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated little if any change in protein species present or their relative contents in oocytes during IVM; one notable exception, however, was the gradual accumulation of a 39-kDa polypeptide between 4–24 hr of maturation culture. Cumulus cells, on the other hand, exhibited no qualitative differences during the period examined, but total protein content did increase during IVM. Metabolic labeling with [³⁵S]-methionine, however, demonstrated changes in protein synthesis, both quantitative and qualitative, by both cell types. Oocytes exhibited a steady or slightly increasing rate of synthesis during the first 12 hr of IVM; thereafter, protein synthesis declined to about 10% of the initial rate by 40 hr in culture. In contrast, protein synthesis in cumulus cells was relatively constant during the first 24 hr. Of greater interest is the demonstration that the synthesis of at least seven oocyte-specific and five cumulus-specific proteins was stage-dependent during maturation. These results indicate that maturation of bovine oocytes is associated with the synthesis of several distinct and temporally expressed proteins which may play roles in the highly ordered sequence of events that culminates in oocyte maturation. © 1996 Wiley-Liss, Inc.