Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15 °C

Boar semen is occasionally transferred to different locations in liquid form at 15 °C for cryopreservation. However, the use of frozen boar semen is limited due to the high susceptibility of boar sperm to cold shock. The aim of this study was to help improve the quality of frozen boar semen by determining the changes in sperm membrane and ROS during the cryopreservation processes of 15 °C-stored boar semen. Semen was collected from ten Duroc boars and transferred to our laboratory in liquid form stored at 15 °C. After cooling to 5 °C and freezing-thawing, conventional sperm parameters (total motility, progressive motility, and normal morphology), plasma membrane integrity, acrosomal membrane status, and intracellular ROS were evaluated. Sperm function, as assessed by conventional parameters, was unaffected by cooling but was decreased by freezing-thawing (P<0.05). However, the cooling and freezing-thawing processes led to damages in the sperm plasma membrane, and the cooling process caused increase in mean PNA (peanut agglutinin)-fluorescence intensity in viable acrosome-intact sperm (P<0.05). In ROS evaluation, the cooling process decreased intracellular (·)O(2) and H(2)O(2) in viable sperm (P<0.05), while the freezing-thawing process increased intracellular H(2)O(2) (P<0.05) without change in intracellular (·)O(2) in viable sperm. Our results suggest that, in liquid boar semen stored at 15 °C, cooling may be primarily responsible for the destabilization of sperm membranes in viable sperm, while freezing-thawing may induce reductions in sperm function with increase in membrane damage and H(2)O(2).     

Effects of centrifugation, glycerol level, cooling to 5 degrees C, freezing rate and thawing rate on the post-thaw motility of equine sperm

Five experiments evaluated the effects of processing, freezing and thawing techniques on post-thaw motility of equine sperm. Post-thaw motility was similar for sperm frozen using two cooling rates. Inclusion of 4% glycerol extender was superior to 2 or 6%. Thawing in 75 degrees C water for 7 sec was superior to thawing in 37 degrees C water for 30 sec. The best procedure for concentrating sperm, based on sperm motility, was diluting semen to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium at 20 degrees C and centrifuging at 400 x g for 15 min. There was no difference in sperm motility between semen cooled slowly in extender with or without glycerol to 5 degrees C prior to freezing to -120 degrees C and semen cooled continuously from 20 degrees C to -120 degrees C. From these experiments, a new procedure for processing, freezing and thawing semen evolved. The new procedure involved dilution of semen to 50 x 10(6) sperm/ml in centrifugation medium and centrifugation at 400 x g for 15 min, resuspension of sperm in lactose-EDTA-egg yolk extender containing 4% glycerol, packaging in 0.5-ml polyvinyl chloride straws, freezing at 10 degrees C/min from 20 degrees C to -15 degrees C and 25 degrees C/min from -15 degrees C to -120 degrees C, storage at -196 degrees C, and thawing at 75 degrees C for 7 sec. Post-thaw motility of sperm averaged 34% for the new method as compared to 22% for the old method (P<0.01).     

Effects of exposing bovine sperm to bovine serum albumin,or freeze-thawing,on sperm-bound amidase activity

Isolation of a self-selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm-bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm-bound amidase (P < .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5°C or layered onto a solution of 6% BSA in extender at 37°C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5°C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm-bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P < .01). However, the amount of exposed sperm-bound amidase also was greater (P < .05) this was a deleterious change. Immediately after thawing, more (P < .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37°C there was no difference. After freezing and thawing of sperm, amidase activity was higher (P < .05) for the isolated sperm than for nonisolated cells. Thus, isolation of sperm using a 6% BSA gradient increased the proportion of progressively motile sperm, but decreased the percentage of sperm with an intact acrosome, based on measurements of amidase activity.     

Effect of docosahexaenoic acid and alpha-tocopherol enrichment in chicken sperm on semen quality, sperm lipid composition and susceptibility to peroxidation

The aim of the present experiment was to study the effect of fish oil and Vitamin E rich diets on semen production, sperm functions and composition in broiler breeders. The following parameters were measured: semen volume and concentration, sperm motility and viability, sperm susceptibility to induced peroxidation, sperm lipid and alpha-tocopherol contents. Dietary n-3 PUFA were successfully transferred into spermatozoan phospholipid by fish oil feeding according to the following main features: (a) the C22:6n-3 and C22:5n - 3 contents were increased, but C22:4n-6 remained the peculiar and major polyunsaturate; (b) the content and proportion of total PUFA did not change; (c) the proportional increase of n-3 PUFA was compensated by the decrease of n-6 PUFA, an increase in the proportion of n-9 fatty acids was also found. The sperm content of alpha-tocopherol was doubled increasing the dietary availability of the vitamin to 300 mg/kg of feed. The specific n-3 PUFA and Vitamin E enrichment of chicken sperm affected cell functions. Significant interactions between the two treatments were also found for some parameters. The best sperm quality condition in control sperm (rich mainly in n-6 PUFA) was found supplying 200mg Vitamin E/kg of feed to the male breeders, and in contrast in n-3 rich sperm supplying 300 mg Vitamin E/kg.     

Development of a simple sperm cryopreservation model using a chemically defined medium and goat cauda epididymal spermatozoa

This investigation was carried out to develop a simple sperm cryopreservation model using a chemically defined synthetic medium (modified Ringer's solution) and mature goat cauda epididymal sperm as the model system. Rates of cooling, freezing, and maximum freezing temperature were manipulated with the help of a computer-controlled programmable biofreezer. Highly motile goat cauda sperm dispersed in a modified Ringer's solution was subjected to the freezing protocol: cooling 0.25 degrees C min(-1) to 5 degrees C, 5 degrees C min (-1) to -20 degrees C, 20 degrees C min(-1) to -100 degrees C, prior to plunging into liquid nitrogen. In the absence of any cryoprotective agent, all of the spermatozoa lost their motility. Addition of glycerol (0.22 to 0.87 M) caused a dose-dependent increase of sperm motility recovery. The highest recovery of forward and total motility was (32 and 35%, respectively) at 0.87 M. Further increase of the glycerol concentration caused a marked decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery (0-18%) of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells. Like glycerol, dimethyl sulfoxide (Me(2)SO) and ethylene glycol also showed a dose-dependent increase in motility recovery as well as a biphasic curve of cryoprotection. At optimal concentrations, dimethyl sulfoxide (1.00 M) and ethylene glycol (1.29 M) were effective in recovering sperm motility to the extent of 20 and 13%, respectively. Thus these reagents have markedly lower cryoprotection potential than glycerol.     

Effect of cold shock and cooling rate on calcium uptake of ram spermatozoa

Rapid cooling (cold shock) of washed ejaculated ram sperm irreversibly reduced motility and respiration and greatly increased uptake of ⁴⁵Ca²⁺. The effect was greater as the temperature of cooling was reduced from 15°C to 0°C, and a substantial increase in sperm calcium levels was even observed after slow cooling to temperatures below 10°C. The rise in calcium uptake on freezing sperm to −79°C was not as great as that on cold shocking sperm to 0°C.Inactivation of sperm by mild heat (50°C) had no significant effect on calcium uptake but subsequent cold shock increased the sperm calcium. Reverse immobilization of sperm by low concentrations of formaldehyde significantly reduced calcium uptake on cold shock. Addition of detergents to sperm immediately reduced motility, respiration and calcium uptake of control and cold-shocked sperm to zero.     

Artificial insemination using cryopreserved sperm in the silkworm,Bombyx mori

We report in this paper that female moths artificially inseminated with cryopreserved sperm (-196 degrees C) could oviposit eggs when the sperm was preserved for 356days, and that the fertilization rate and the number of eggs laid were almost equivalent to those obtained in normally mated moths. The optimal cooling rate for sperm freezing was 5-65 degrees C/min for maintaining a high fertility of sperm. The simple and reliable method of cryopreservation was to put the semen first in a deep freezer at -80 degrees C and thereafter put them in liquid nitrogen. When female moths of 'white 2' egg-color mutant strain were inseminated with a mixture of frozen-thawed sperm from males of normal-colored egg strain and non-frozen sperm from males of the 'white 2', female moths deposited a majority of 'white 2' eggs and a very small number of eggs of normal color. The result shows that there was a competitive fertilization of sperm between the two strains of the silkworm, and that sperm fertility was reduced to a considerable extent by freezing at -196 degrees C. These results may contribute not only to basic studies on fertilization in Lepidoptera but also to the development of long-term preservation procedure of genetic resources by using cryopreserved sperm of Bombyx mori.     

烟草雌、雄配子DNA 含量变化

采用显微分光光度法测定了烟草( Nicotiana tabacum) 精细胞和卵细胞的DNA 含量。烟草是二胞花粉, 花粉萌发后生殖细胞在花粉管中分裂形成精细胞。授粉后45 h 花粉管到达子房, 在花粉管内的精细胞DNA 含量为1C。当花粉管在退化助细胞中破裂, 释放出的两个精细胞开始合成DNA。在与卵细胞融合前,两个精细胞DNA 含量接近2C。随着精细胞的到达及合成DNA, 卵细胞也开始合成DNA, 融合前的卵细胞DNA 含量也接近2C。精、卵细胞融合后, 合子DNA 含量为4C。烟草雌、雄配子是在细胞周期的G2 期发生融合, 属于G2 型。     

Effect of temperature on the fluidity of boar sperm membranes

Fluidity was used to assess changes in molecular organization of boar spermatozoa plasma membranes from (1) the head and (2) the rest of the sperm body and acrosome as a consequence of temperature. The initial fluidity of the head membranes at 25 degrees C was less than that of the sperm body membranes (P less than 0.05). When held at 25 degrees C, the fluidity of the head membranes decreased for 105 +/- 8 min and then stabilized for the remainder of the 160-min incubation. Calcium (10 mM) caused a significantly greater decrease in fluidity. The fluidity of the sperm body membranes increased slightly over time in the absence of Ca2+, but decreased significantly with Ca2+. Cooling from 25 to 5 degrees C and subsequent heating to 40 degrees C (0.4 degrees C/min) caused marked alterations in the fluidity of each membrane. Cooling the head membranes prevented the fluidity increase seen at 25 degrees C, while reheating caused a dramatic decrease in fluidity. Fluidity of the head membranes was now unaffected by Ca2+. Lipid phase transitions, indicated by sharp break points in data curves, were detected at the onset of reheating (7 +/- 3 C) and at 23 +/- 4 degrees C during reheating. Fluidity of the sperm body membranes decreased slightly and in a linear fashion with Ca2+. Without Ca2+, the sperm body membranes showed an additional lipid phase shift at 31 +/- 5 degrees C, which led to a rapid fall in fluidity. These results suggest that the fluidity, and therefore the molecular structure, of sperm head and body membranes differ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryopreservation of tench, Tinca tinca, sperm: Sperm motility and hatching success of embryos

The aim of the present study was to elaborate cryopreservation methods for ex situ conservation of tench. Success of cryopreservation was tested during two series of experiments. The first set of experiments studied the effects of two types of cryoprotectants (DMSO and a combination of DMSO with propanediol at ratio 1:1) at concentrations of 8 and 10% and three different equilibration times in two different immobilization solutions (IS) (Kurokura 180 and Kurokura) before freezing (0.0, 2.0 and 4.0h after T(0)). The K4 cooling programme was used to freeze 1ml of cryoextended sperm using 1.8ml cryotubes. Main monitored parameter was hatching rate after using of cryopreserved sperm. The second set of experiments studied the volume effect of 0.5, 1 and 5ml straws and compared these with 1.8ml cryotubes as well as the effect of the cooling programme (K4 and L1). Following the results of the first study, a combination of DMSO and propanediol (ratio 1:1) at concentration of 10% was added to extended sperm in Kurokura 180 IS. Main monitored parameter was hatching rate after using cryopreserved sperm, supplementary parameters were sperm velocity and motility percentage assessed at 10s post-activation. Sperm was collected directly into IS and stored at 4 degrees C for 2.5h. Thereafter were sperm samples pooled, equlibred in IS (first set of experiments) or directly mixed with cryoprotectants (DMSO or a mixture of DMSO with propanediol at ratio 1:1) and transferred to 1.8ml cryotubes or straws (0.5, 1 and 5ml). Then the cryotubes/straws were directly transferred to pre-programmed PLANER Kryo 10 series III and cooled using two different cooling programmes including a slow cooling programme (a) named K4 (from +4 to -9 degrees C at a rate of 4 degrees Cmin(-1) and then from -9 to -80 degrees C at a rate of 11 degrees Cmin(-1)) and a rapid cooling programme (b) named L1 (directly from +4 to -80 degrees C at a rate of 20 degrees Cmin(-1)). Both slow (K4) and rapid (L1) cooled samples were held 6min at -80 degrees C. Finally, samples were transferred into liquid N(2). The frozen spermatozoa were thawed in a water bath (40 degrees C) according to the frozen volume and checked for fertilization and hatching rates. Percentage of sperm motility and sperm velocity were measured using video recorded frames. ANOVA showed a significant influence of frozen and fresh sperm in all treatments. The hatching rates of 33.8% were obtained when sperm was equilibrated for 0h before freezing in IS of Kurokura 180 and frozen with a 10% of mixture 1:1 of DMSO and propanediol into straws of 5ml and cooled using program L1. The velocity of frozen-thawed spermatozoa ranged from 31 to 46microms(-1) and in post-thawed sperm was not significantly different according to frozen sperm volume, but a higher velocity was obtained when sperm was fast frozen using programme L1. A large volume of frozen sperm could reveal the best procedure for freezing, but also for simulating methods of artificial propagation for future practical use of frozen tench sperm at a large scale.     

Osmotic tolerance of avian spermatozoa: influence of time, temperature, cryoprotectant and membrane ion pump function on sperm viability

Potential factors influencing sperm survival under hypertonic conditions were evaluated in the Sandhill crane (Grus canadensis) and turkey (Meleagridis gallopavo). Sperm osmotolerance (300-3000 mOsm/kg) was evaluated after: (1) equilibration times of 2, 10, 45 and 60 min at 4 °C versus 21 °C; (2) pre-equilibrating with dimethylacetamide (DMA) or dimethylsulfoxide (Me₂SO) at either 4 °C or 21 °C; and (3) inhibition of the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ionic pumps. Sperm viability was assessed using the eosin-nigrosin live/dead stain. Species-specific differences occurred in response to hypertonic conditions with crane sperm remaining viable under extreme hypertonicity (3000 mOsm/kg), whereas turkey sperm viability was compromised with only slightly hypertonic (500 mOsm/kg) conditions. The timing of spermolysis under hypertonic conditions was also species-specific, with a shorter interval for turkey (2 min) than crane (10 min) sperm. Turkey sperm osmotolerance was slightly improved by lowering the incubation temperature from 21 to 4 °C. Pre-equilibrating sperm with DMA reduced the incidence of hypertonic spermolysis only in the crane, at both room and refrigeration temperature. Inhibiting the Na⁺/K⁺ and the Na⁺/H⁺ antiporter membrane ion pumps did not impair resistance of crane and turkey spermatozoa to hypertonic stress; pump inhibition actually increased turkey sperm survival compared to control sperm. Results demonstrate marked species specificity in osmotolerance between crane and turkey sperm, as well as in the way temperature and time of exposure affect sperm survival under hypertonic conditions. Differences are independent of the role of osmotic pumps in these species.     

Calcium influx into mouse spermatozoa activated by solubilized mouse zona pellucida,monitored with the calcium fluorescent indicator,fluo-3. inhibition of the influx by three inhibitors of the zona pellucida induced acrosome reaction: Tyrphostin A48, pertussis toxin,and 3-quinuclidinyl benzilate

The fluorescent calcium indicator, fluo-3, was loaded as the membrane permeant tetraacetoxymethyl (AM) ester into cauda epididymal mouse sperm at 25°C for 20 min in the absence of bovine serum albumin (BSA) and presence of the dispersant, Pluronic F-127. Excess indicator was removed by two centrifugation washes at 100g for 10 min, a procedure that did not impair sperm motility. Upon resuspension in medium containing 20 mg/ml BSA to promote capacitation, the sperm cells exhibited readily detectable fluorescence uniformly distributed in the cytoplasm. Cell fluorescence was stable over the time of the experiments and was responsive to changes in intracellular calcium concentration, [Ca²⁺]_j. Initial [Ca²⁺]_j was 231 ± 58 nM (±SE, n = 43). Addition of heat-solubilized mouse zonae pellucidae to capacitated sperm increased [Ca²⁺]_j by 106 ± 19 nM (±SE, n = 18), the higher steady-state concentration being reached after 30 min. Subsequent addition of the non-fluorescent calcium ionophore Br-A23187 resulted in a further increase of 114 ± 18 nM (± SE, n = 18), the higher steady-state concentration being reached after 6 min. The increase in [Ca²⁺]_j induced by solubilized zonae pellucidae was largely blocked by 3-quinuclidinyl benzilate (QNB) an antagonist of muscarinic receptors that was earlier shown to block the zona pellucida induced acrosome reaction in mouse sperm (Florman and Storey, 1982: Dev Biol 91:121–130). This [Ca²⁺]_j increase was completely blocked by the tyrosine kinase inhibitor, tyrphostin A48, and by the inactivator of G₁ proteins, pertussis toxin. At the concentrations at which they blocked the zona pellucida-induced increase in [Ca²⁺]_j all three inhibitors also blocked the zona pellucidainduced acrosome reaction. These results indicate that [Ca²⁺]_j increase in is an early, if not the initial, reaction in the sequence leading to zona pellucida induced acrosomal exocytosis in mouse sperm. The observation that the three inhibitors, each having a different mode of action, all block the zona pellucida induced [Ca²⁺]_j suggests that the sperm plasma membrane receptors mediating the zona pellucida induced acrosome reaction may function as a complex, whose formation is activated by zona pellucida ligand binding. © 1994 Wiley-Liss, Inc.     

Sperm biology and control of reproduction in sturgeon: (II) sperm morphology, acrosome reaction, motility and cryopreservation

In the present review, sperm morphology, acrosome reaction, motility, short-term storage and cryopreservation are summarized and discussed in sturgeon (Chondrostei, Acipenseriformes). The elongated head of spermatozoon comprises an acrosome with 8?C12 posterolateral projections. Usually three endonuclear canals are observed in the nucleus. Proximal and distal centrioles and 3?C6 mitochondria are located in the midpiece region. The flagellum consists of an axoneme with a typical ??9?+?2?? structure of microtubules and presents a ribon-like structure due to two lateral membranous fins. Egg water, Ca²⁺ and Mg²⁺ can trigger acrosome reaction. Trypsin- and chymotrypsin-like activities are reported in sturgeon sperm. These physiological properties of sturgeon sperm are identified as serine activity with 33?kDa molecular mass and can be inhibited by their respective inhibitors. The K⁺ prevents sperm activation in seminal plasma, and hypo-osmolality or decrease of extracellular K⁺ triggers sperm activation. Extracellular Ca²⁺ is involved in flagellar beating pattern and sperm velocity. After activation, sperm motility, velocity, and flagellar beating frequency, wavelength and amplitude decrease, while number of waves and curvature increase. Sturgeon sperm can be stored for several days at 4?°C; however it is better to add K⁺ into the immobilizing medium because it prevents sperm activation during incubation. Regarding sperm cryopreservation, methanol is a better cryoprotectant than DMSO. Either short-term storage or cryopreservation of sperm generates damage to spermatozoa that lead to reduction of sperm motility performance. Some studies suggest using an activation medium containing Ca²⁺ for enhancing sperm motility performance of incubated or frozen-thawed sperm.     

Fresh and frozen-thawed sperm quality, nuclear DNA integrity, invitro fertility, embryo development, and live-born offspring of N-ethyl-N-nitrosourea (ENU) mice

Efficient collection, freezing, reliable archiving of sperm, and re-derivation of mutant mice are essential components for large-scale mutagenesis programs in the mouse. Induced mutations (i.e. transgenes, targeted mutations, chemically induced mutations) in mice may cause inherited or temporary sterility, increase abnormal sperm values, or decrease fertility. One purpose of this study was to compare the effect(s) on fresh and frozen-thawed sperm quality, spermatozoa DNA integrity, unassisted in vitro fertility (IVF) rate, in vitro embryo development rate to blastocysts, and live-born offspring rates in non-ENU (control) animals and the F1-generation of N-ethyl-N-nitrosourea (ENU)-treated male mice (765 mg/kg C57BL6/J or 600 mg/kg 129S1/SvImJ total dose). The second purpose was to determine the effect(s) of parental oocyte donor strain on in vitro fertilization, in vitro embryo development to blastocysts, and live-born offspring rates using sperm and unassisted IVF to re-derive animals from non-ENU control and ENU mice. Sperm assessment parameters included progressive motility, concentration, plasma membrane integrity, membrane function integrity, acrosome integrity, and DNA integrity. There were no significant differences in fresh sperm assessment parameters, DNA integrity, unassisted in vitro fertility rate, in vitro embryo development rate to blastocysts, and live-born offspring rates between non-ENU and C3B6F1/J or B6129S1F1/J ENU mice. In addition, there were no significant differences in frozen-thawed sperm assessment parameters and DNA integrity rates for non-ENU control and ENU C3B6F1/J or B6129SF1/J mice. In vitro fertilization and in vitro embryo development to blastocysts were effected from strain genetic variability (P < 0.05). However, the cryopreservation process caused an increase of DNA fragmentation in non-ENU control and ENU C3B6F1/J or B6129S1F1/J hybrid mice compared to fresh control sperm (P < 0.01). Unlike the combinations of hybrid sperm and hybrid oocyte, increasing frozen-thawed sperm DNA fragmentation decreased the embryo development rate to blastocyst compared to fresh sperm when C57BL6, C3H, or 129S inbred mice were used as oocyte donors (P < 0.05).     

Effects of UV irradiation and hydrogen peroxide on DNA fragmentation, motility and fertilizing ability of rainbow trout (Oncorhynchus mykiss) spermatozoa

Preservation of DNA integrity is essential for protection of sperm quality. This study examined, with the use of comet assay, DNA fragmentation of rainbow trout (Oncorhynchus mykiss) spermatozoa subjected to UV irradiation (2,075 microW/cm(2), 0-15 min) or oxidative stress induced by hydrogen peroxide (0-20mM). Sperm motility and fertilizing ability were also measured. A dramatic increase in DNA fragmentation was recorded after 5 min UV irradiation but no significant changes in sperm motility were observed at this time. Longer irradiation resulted in a decrease in motility parameters and further increase of DNA fragmentation. UV irradiation caused a clear decrease in the percentage of eyed embryos and most of the embryos did not hatch. When highly diluted sperm suspensions (50,000-fold) were exposed to 0.1mM H(2)O(2) evident increase in DNA fragmentation was observed. On the other hand, when more concentrated sperm suspensions (diluted only 40-fold) were employed (in order to conduct motility and fertilization measurements at the same time) 1-20mM H(2)O(2) caused only moderate increase in DNA fragmentation and dose-dependent decline in sperm motility and fertilizing ability. This suggests that toxic effects of H(2)O(2) were primarily related to inhibition of sperm motility. Our results demonstrate that comet assay can be used for monitoring the effectiveness of fish sperm DNA inactivation by UV irradiation. Therefore, the comet assay together with sperm motility analysis can be applied in optimization works of gynogenetic procedures in fish. Lack of effectiveness of H(2)O(2) in inducing major DNA fragmentation suggests presence of mechanisms of antioxidative defense in rainbow trout spermatozoa.     

Effect of two cooling protocols on the post-thaw characteristics of Iberian ibex sperms

The rate at which lethal intracellular ice forms during sperm cryopreservation is highly dependent on the cooling protocol. The present work compares two cooling protocols for use with Iberian ibex (Capra pyrenaica) sperm by assessing the effects on the motility, viability, and size of frozen-thawed sperm cells. Ejaculates, obtained from six adult ibex males via transrectal, ultrasound-guided massage of the accessory sex glands plus electroejaculation if necessary, were cooled via either 1) Protocol 1 (decelerating cooling), involving cooling in liquid nitrogen vapor from 5 °C to −35 °C (40 °C/min), from −35 °C to −65 °C (17 °C/min), and then from −65 °C to −85 °C (3 °C/min); or 2) Protocol 2 (accelerating cooling) involving cooling in a biological freezer from 5 °C to −5 °C (4 °C/min), from −5 °C to −110 °C (25 °C/min), and then from −110 °C to −140 °C (35 °C/min). Compared to fresh ejaculates, sperm quality at thawing was found to be reduced by both protocols (p < .05), but especially by Protocol 1. Sperm head size was also significantly reduced by both protocols, although the Protocol 1 sperm heads were also significantly smaller than those of Protocol 2 sperms heads (p < .05). In fresh sperm samples, clustering analyses revealed two subpopulations of sperms with different morphometric characteristics, SP1 with larger cells, and SP2 with smaller cells. Both cooling protocols caused reduction in the proportion of SP1 cells, and an increase in the proportion of SP2 cells. In conclusion, the decelerating cooling protocol (Protocol 1) caused greater cryodamage to the sperm cells than the accelerating protocol (Protocol 2).     

Rapid evolution of sperm length in response to increased temperature in an ectothermic fish

The Intergovernmental Panel on Climate Change predicts an average global temperature increase of 1.8–4.0 °C by 2100. Tropical ectotherms are expected to be particularly sensitive to this temperature increase because they live close to their thermal limits. We investigated the phenotypic plasticity and evolutionary response of sperm traits in guppies (Poecilia reticulata) to increased temperatures after 6, 18, and 24 months. Guppies with evolution temperatures of 25 °C (control) or 28 °C were reared in either 25 or 28 °C in a 2 × 2 common garden design. The plastic response to increased temperature was a decreased sperm length, velocity, and path linearity. The evolutionary response was a subsequent increase in sperm length, resulting in complete compensation after just 6 months (at most four generations) in 28 °C water. Sperm velocity and linearity showed no sign of evolution even after 24 months. This study provides evidence that some reproductive traits can respond via rapid evolution to the temperature increase associated with climate change.     

Qualitative and quantitative decline in spermatogenesis of the follicle-stimulating hormone receptor knockout (FORKO) mouse

Sertoli cells express functional receptors for FSH, one of the two pituitary hormones that regulate spermatogenesis in mammals. We recently produced genetic mutant (FORKO) mice that lack FSH receptor, in order to examine the effects on testicular function and fertility. Mutant males exhibited weight loss of testis, epididymis, and seminal vesicle as well as low levels of testosterone. Except for reduced seminiferous tubular diameter, no gross changes were apparent upon histological examination. Analysis of testicular germ cells by flow cytometry revealed a significant increase in the percentage of 2C cells (spermatogonia and non-germ cells) and a significant decrease in the percentage of HC cells (elongated spermatids) of FORKO males. The absolute number of homogenization-resistant elongated spermatids was also significantly reduced in the mutant males. A 2-fold increase in c-kit-positive 2C cells was recorded in the mutant males. Elongated spermatids of FORKO males showed a dramatic increase in propidium iodide binding suggesting reduced nuclear compaction. The increase in size of the sperm head in mutants, as well as susceptibility to dithiothreitol-induced decondensation, suggests the inadequate condensation of sperm chromatin. Sperm chromatin structure assay, a technique that reflects DNA stability, revealed that sperm from FORKO males are susceptible to acid denaturation, indicating the poor quality of sperm. These data allow us to conclude that genetic disruption of FSH receptor signaling in the rodent induces major changes that might contribute to reduced fertility.     

Protective action of vitamins on the spermatogenesis in lead-treated Swiss mice

The protective action of vitamins C and E against lead acetate-induced reduced sperm count and sperm abnormalities in Swiss mice has been studied. Intraperitoneal injection of lead acetate (10mg/kg body weight) in the present study stimulates lipid peroxidation in the testicular tissue, indicated by a significant increase in malondialdehyde content in the experimental mice group. This is associated with an increased generation of noxious reactive oxygen species (ROS). Significantly reduced sperm count associated with increased sperm abnormality percentage in the lead-injected mice group compared to controls substantially proves the ongoing damaging effects of lead-induced ROS on developing germ cells. However, intraperitoneal administration of vitamin C (Vit C) at a concentration equivalent to the human therapeutic dose (10 mg/kg body weight) was able to minimize significantly the testicular malondialdehyde content with a concomitant increase in sperm count and significant decrease in the percentage of abnormal sperm population. Vitamin E (Vit E) (100 mg/kg body weight) treatment of a batch of lead-injected mice had a similar effect as Vit C but with a comparatively lower efficacy. On the other hand, coadministration of both vitamins (Vit C + Vit E) at the above mentioned doses to lead-treated mice led to the most significant decline in malondialdehyde content along with elevated sperm count and reduction in the percentage of abnormal sperm population. The protective action and the synergistic action of both vitamins (C and E) against lead-induced genotoxicity are discussed.