The chemotactic properties of porcine seminal components toward neutrophils in vitro

Our objectives were to investigate the mechanisms of postbreeding inflammation in swine by examining the chemotactic properties of polymorphonuclear neutrophilic granulocytes (PMN) and of various populations of spermatozoa and seminal plasma. Epididymal spermatozoa from two boars obtained under sterile conditions, washed ejaculated spermatozoa from two boars, and pooled seminal plasma from eight boars of known fertility were examined for chemotaxis to PMN. The chemotaxis of blood-derived PMN in response to sperm and seminal plasma was evaluated and expressed as a percentage of a positive control (lipopolysaccharide-activated blood plasma). The mean chemotactic effect of washed sperm alone (4.4+/-0.04) and of epididymal sperm alone (3.4+/-0.06) was not different from that of the negative controls (3.1+/-0.05) of McCoy's medium with 10% heat-inactivated fetal calf serum. A marked chemotactic effect was detected when washed ejaculated and epididymal sperm were incubated with blood plasma, compared with blood plasma without spermatozoa (P < 0.001). Washed sperm in blood plasma (86.2+/-5.6) and epididymal sperm in blood plasma (83.9+/-7.7) were different from blood plasma alone (11.2+/-1.5), but no differences were detected between the two populations of sperm. This effect, however, was not completely inhibited by heat inactivation of the blood plasma. The chemotactic response of washed ejaculated and epididymal spermatozoa incubated in lipopolysaccharide-treated, heat-inactivated blood plasma were greater than that of the negative control (P < 0.05). Polymorphonuclear neutrophilic granulocyte migration toward seminal plasma was similar to the negative control (4.0+/-0.04 vs 3.1+/-0.05). It seems that porcine epididymal sperm and ejaculated sperm activate chemotactic components in porcine blood plasma and heat-inactivated blood plasma, suggesting that, at least partially, a heat-stable (noncomplement) blood plasma component may be involved in sperm-induced PMN chemotaxis. In contrast, porcine seminal plasma was not chemotactic to PMN. These results support the hypothesis that spermatozoa play an active role in initiating postbreeding endometritis.     

The positive effects of seminal plasma during the freezing process on cryosurvival of sperm with poor freezability in the rhesus macaque (Macaca mulatta)

The objective was to examine the effect of seminal plasma on cryopreservation of sperm from rhesus macaques. Sperm cryosurvival was evaluated by sperm motility and acrosomal integrity. Compared with slow cooling (-0.4 C/min) from 37 C (body temperature) to 4 C, rapid cooling (-16 C/min) caused cold shock in rhesus macaque sperm. The cryosurvival of sperm was decreased regardless of the presence or absence of seminal plasma (P<0.05). However, the presence of seminal plasma during cold shock at a rapid cooling rate improved sperm motility and acrosomal integrity in individual monkeys. Male-to-male variation in sperm cryosurvival was observed after cryopreservation (P<0.05), and the presence of seminal plasma during sperm cryopreservation improved sperm motility and acrosomal integrity in individual monkeys (P<0.05). Furthermore, by adding seminal plasma from monkeys with good sperm cryosurvival to sperm freezing extender, the frozen-thawed motility and acrosomal integrity of sperm from monkey with poor cryosurvival were improved (P<0.05). The present study indicated that seminal fluid is beneficial to sperm undergoing cold shock or cryopreservation in individual monkeys. The cryosurvival of sperm from rhesus macaques with poor sperm freezability could be improved by the presence of seminal plasma from males with good sperm cryosurvival. This finding provides a useful method for genetic preservation in this important species.     

A comparative study on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen in boars

A comparative study was carried out on the chemical composition of plasma from the cauda epididymidis, semen fractions, and whole semen of boars. A total of 22 boars were used in this study. The boars, which ranged in age from 8 to 14 months, were of Swedish Landrace and Swedish Yorkshire breed. All boars used presented a normal semen picture. A dummy sow and an artificial vagina were employed for semen collection. The semen was collected as whole semen and as semen fractions in 10 nil volumes. The contents of the cauda epididymidis was removed post mortem.The following parameters were investigated: sperm concentration, dry weight of spermatozoa and of seminal plasma, osmotic pressure, sodium, potassium, chloride, inorganic phosphorus, calcium, magnesium, total protein, GOT, GPT, and alkaline phosphatase in seminal plasma. Paper electrophoresis was carried out on seminal plasma. Tlxe results of the analysis are summarized in Tables 1–6.The sperm concentration was approximately 3.2 mill./mm3 in the cauda epididymidis, 1 mill./mm³ in the sperm-richest fraction (II) and 0.25 mill./mm3 in whole semen. The dry weight (expressed in per cent dry matter) of spermatozoa was highest in the cauda epididymidis (25.47 %), showing a tendency to decreasing in semen fractions I—IV and was lowest in whole semen (15.29 %). The per cent dry weight in plasma was higher in the cauda epididymidis (4.56 %) than in semen fraction I (2.20 %). In semen fractions I—IV the per cent dry weight rose from 2.20 (U to 4.51 % and reached the level of approximately 3.80 % in the sperm-free fractions V—VII. The osmotic pressure was significantly higher in the cauda epidi-dymal plasma than in the whole seminal plasma or the seminal plasma fractions. The same phenomenon was observed in a boar where the cauda epididymal content was collected in vivo from a patent established fistula. There appears to be a connection between the per cent dry weight of spermatozoa and the osmotic pressure, which means that the per cent dry weight of the cauda epididymal spermatozoa decreases when mixed with the accessory gland secretions, which have a lower osmotic pressure. The fall in per cent dry weights is thought to be caused by an intake of water.The amount of sodium, chloride and magnesium was higher in ejaculated seminal plasma than in cauda epididymal plasma. The reverse was true for inorganic phosphorus and potassium. Moreover the sperm-free fractions contained more sodium, chlorides and magnesium than the sperm-containing fractions, while the concentration of potassium and inorganic phosphorus was comparatively higher in the sperm-containing fractions. A connection is apparent between sperm concentration and the potassium, inorganic phosphorus and magnesium levels. Statistical analysis of the values of chloride and magnesium revealed significant differences between individual boars for most of the semen fractions.The concentration of plasma proteins in the cauda epididymidis was approximately the same as in whole semen and in the semen fractions except for fraction I, which contained a relatively low concentration. As regards total protein there were significant differences between individual boars in most of the semen fractions as well.The paper electrophoretic pattern of epididymal plasma was different from that of semen plasma. Thus there were three or four distinct components in the cauda epididymidis numbered 1, 2, 3, and 4, and three distinct components in whole seminal plasma numbered 3, 4, and 5, while the sperm-richest semen fractions contained four components (2, 3, 4, and 5) and the others three components, namely 3, 4, and 5.The level of GOT was high in the cautlu cpiflidymill contents (99.1 i. u./ml) compared with that for whole seminal plasma (99.1 i.u/ml). In semen fractions there was a clear positive correlation between the level of GOT and the sperm concentration. The GPT concentration wis as a whole low and. in contrast to GOT. somewhat higher in the sperm-free fractions than in the sperm-containing fractions. The concentration of alkaline phosphatase was very high in cauda epididymal plasma (31,463 i. u./ml) as well as in the sperm-rich fractions (e.g. 7,096 i. u./ml in fraction II). Preliminary investigation has moreover revealed a very low alkaline phosphatase concentration in seminal plasma of vasectomized boars, which condition suggests thai the main origin for alkaline phosphatase in boars is the testis and epididymis.     

Incubation of Post-Thaw Epididymal Cat Spermatozoa with Seminal Plasma

In domestic cats, epididymal spermatozoa have lower initial motility and viability than ejaculated spermatozoa and it is possible that seminal plasma compounds are behind these effects. The aim of this study was to investigate whether co-incubation of post-thaw epididymal cat spermatozoa with seminal plasma was able to improve sperm quality. Epididymal cat spermatozoa from 11 cats were cryopreserved. After thawing, each sperm sample was divided into two aliquots, centrifuged and incubated with two different media; Tris buffer (control) or pooled seminal plasma (treatment). Sperm quality was observed at 0, 2, 4 and 6 h after incubation. The results demonstrated that all of the sperm parameters except acrosome integrity were lower in the treatment group compared to the control group (p < 0.05); the percentages of motility (46.4 ± 15.4 vs 40.0 ± 9.4), the scores of progressive motility (3.1 ± 0.4 vs 2.8 ± 0.5), the percentages of spermatozoa with intact plasma membrane (46.3 ± 9.7 vs 39.6 ± 8.9) and intact acrosome (36.5 ± 16.2 vs 32.9 ± 15.1), as well as at all time points. In conclusion, the seminal plasma seems less beneficial to the post-thaw epididymal cat spermatozoa than the Tris buffer.     

Alkaline and Acid Phosphatase Activity, pH and Osmotic Pressure of Boar Semen

Alkaline phosphatase activity was recorded in forty ejaculates of the sperm rich fraction of boar semen as 9,790 ± 5,250 Klein-Babson-Read units per 100 ml. of seminal plasma. Acid phosphatase activity in the same ejaculates was 681 ± 304 Babson-Read units per 100 ml. of seminal plasma. No alkaline phosphatase activity was detected in the seminal plasma of vasectomized boars.

The pH of the sperm rich fractions was 7.69 ± 0.33 and the osmotic pressure was 313.56 ± 7.98 milliosmols.

     

The effect of exogenous administration of oestrogens on the function of the epididymis and the accessory sex glands in the boar.

Three normal, sexually mature young boars of Swedish Yorkshire breed were treated with daily intramuscular injections of diethylstilboestrol and/or oestradiolbenzoate for a total period of 13–15 weeks. Whole ejaculates were collected on a dummy sow with an artificial vagina twice a week. The ejaculate was examined as regards semen volume, sperm concentration, sperm motility and sperm morphology. The seminal plasma was analysed for Na, K, Cl, Mg, inorganic phosphate, total protein and fructose. Testicles, epididymides and the accessory sex glands were obtained at slaughter. Material from epididymal segments A, B, D_b, F_a and F_b was collected and examined for sperm morphology. Spermatocrit, osmotic pressure and plasma concentrations of Na, K, Gl, total protein and GPC were assessed in material from epididymal segment F_a. The sex glands and the accessory sex glands were examined maeroscopically and microscopically.The semen volume, the sperm concentration and consequently the total sperm count per ejaculate showed a gradual increase during the course of the investigation. This is to be expected as the boars used were comparatively young at the beginning of the experiment. These values were all within normal limits for adolescent boars.Taking all the characteristics examined into consideration no conclusive evidence was found that an exogenic administration of oestrogens to intact boars has any influence on the function of the epididymis and the accessory sex glands.     

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.     

Assessment of Sperm Function Parameters and DNA Fragmentation in Ejaculated Alpaca Sperm (Lama Pacos) by Flow Cytometry

Flow cytometry has been shown to be an accurate and highly reproducible tool for the analysis of sperm function. The main objective of this study was to assess sperm function parameters in ejaculated alpaca sperm by flow cytometry. Semen samples were collected from six alpaca males and processed for flow cytometric analysis of sperm viability and plasma membrane integrity using SYBR‐14?PI staining; acrosomal membrane integrity using FITC‐conjugated Pisum Sativum Agglutinin?PI labelling; mitochondrial membrane potential (Δψm) by staining with JC‐1 and DNA Fragmentation Index (DFI) by TUNEL. The results indicate that the mean value for sperm viability was 57 ± 8 %. Spermatozoa with intact acrosome membrane was 87.9 ± 5%, and viable sperm with intact acrosomal membrane was 46.8 ± 9%, high mitochondrial membrane potential (Δψm) was detected in 66.32 ± 9.51% of spermatozoa and mean DFI value was 0.91 ± 0.9%. The DFI was inversely correlated with high Δψm (p = 0.04; r = ?0.41) and with plasma membrane integrity (p = 0.01; r = ?0.47). To our knowledge, this is the first report of the assessment on the same sample of several parameters of sperm function in ejaculated alpaca sperm by flow cytometry.     

Influence of Thawing Diluents on Vitality,Acrosome Morphology,Ultrastructure and Enzyme Release of Deep Frozen Roar Spermatozoa*

The present investigation was performed to study the effect of freezing and thawing on boar spermatozoa. Thirty-one ejaculates from four boars were investigated after thawing in three different thawing diluents (seminal plasma, OLEP, isotonic glucose solution).From each ejaculate one sample of 1 × 10⁹ spermatozoa was thawed in each of the thawing diluents. Each sample was examined in a thermoresistance test in which motility was stimulated with caffeine 30 min. and 3 hrs. after thawing. Furthermore, acrosome morphology and ASAT release from the spermatozoa were investigated for each sample. One ejaculate from the two most frequently used boars was examined by electron microscopy after thawing in each of the thawing diluents.Differences in the aspects studied appeared between isotonic glucose solution and the other two thawing diluents in the thermoresistance test, in the response to caffeine stimulation 3 hrs. after thawing and in the amount of ASAT released from the spermatozoa. The influence on the acrosome morphology varied between the thawing diluents, but the acrosomal alterations did not seem to be connected with the damage reflected by the thermoresistance test and by the measurement of extracellular ASAT activity.The ultrastructural investigation showed that all spermatozoa examined had some degree of ultrastructural alteration as compared with freshly ejaculated boar spermatozoa treated in the same way. This alteration could not be related to any of the thawing diluents.Of the various laboratory tests the thermoresistance test and the measurement of ASAT release are suggested to be sensitive indicators of sperm damage during freezing and thawing. These tests might be useful indicators of variations in sensitivity of spermatozoa to the freezing-thawing procedure.     

长白公猪血清和精浆中元素含量对精液品质的影响

本试验旨在研究不同精液品质特征长白公猪血清和精浆中元素含量的差异,分析血清和精浆中元素含量对精液品质的影响。基于107头长白公猪的1402次精液品质记录,将公猪群按照精液可利用率划分为3组:低利用率组(利用率<60%,n=21)、中等利用率组(60%≤利用率≤80%,n=27)和高利用率组(利用率>80%,n=59)。采集每头长白公猪的血清和精浆样品,采用电感耦合等离子体质谱法检测血清和精浆中营养元素和毒性元素含量。结果表明:1)低利用率组长白公猪的有效精子数和精子活力显著低于中等利用率和高利用率组(P<0.01),精子畸形率显著高于中等利用率和高利用率组(P<0.01)。2)不同利用率组间长白公猪的血清和精浆元素含量无显著差异(P>0.05);但在血清和精浆元素含量与精液品质参数相关性分析中发现,精浆铅元素含量与精子活力呈显著负相关(P<0.05),与精子畸形率呈显著正相关(P<0.05)。3)对精浆铅含量分组进一步分析发现,精浆铅含量为0μg/L时长白公猪的精子畸形率显著低于精浆铅含量>10.0μg/L时(P<0.05),精子畸形率降低约6.11%。总的来说,长白公猪精浆中毒性元素铅的存在会通过损害精子活力和形态,影响公猪精液品质。     

Seasonal changes in semen characteristics, composition of seminal plasma and frequency of acrosome reaction induced by calcium and calcium ionophore A23187 in Large White boars

This study attempted to explain the mechanisms regulating boar fertility by examining seasonal changes in semen characteristics, the composition of seminal plasma and responsiveness of sperm acrosomes to Ca(2+) and the Ca(2+) ionophore A23187 (Ca(2+)/A23187). Sperm-rich and sperm-poor fractions were separately collected from 3 mature fertile Large White boars once a month over a one-year period. During the period of study, ambient temperature and relative humidity were recorded for within the stall in which the boars were kept and the semen characteristics, composition of the seminal plasma of sperm-rich fractions, and occurrence of the acrosome reaction in response to Ca(2+) (3 mM)/A23187 (0.3 microM) were examined. The highest mean maximum and minimum ambient temperatures were recorded in August-September, whereas the lowest mean maximum and minimum ambient temperatures were recorded in December and January, respectively. There was a moderate peak in relative humidity from July to October. The lowest percentages of motile spermatozoa and of spermatozoa with intact acrosomes and highest percentage of spermatozoa with abnormal morphology and strongest agglutination were seen in August-September. The total protein and albumin concentrations were lowest in August-September. Testosterone levels increased gradually as day length decreased after the summer solstice (June) and peaked in October-November. The percentage of acrosome reactions in response to Ca(2+)/A23187 was highest with the quickest response in August-September, as shown by the shortest time required for 50% of relative acrosome reactions. The farrowing rates were lowest in these same 2 months. These results suggest that seasonal infertility in Large White boars may be due, at least in part, to a combination of low motility, abnormal morphology including acrosomal abnormality, and early occurrence of the acrosome reaction in response to stimulus, possibly resulting from a decrease in acrosomal stabilizing proteins in the seminal plasma during summer. These changes may be modulated by heat/humidity stress and/or photoperiod-regulated testosterone.     

Comparison of two methods of seminal plasma removal on buffalo (Bubalus bubalis) sperm cryopreservation

Cryopreservation causes damage to spermatozoa, and methods minimizing this damage are therefore needed. Although much discussed, seminal plasma removal has become an alternative to improve sperm quality and viability after freezing and has been applied to different species in attempt to obtain good results. The objective of this study was to evaluate semen quality in buffaloes submitted to two methods for seminal plasma removal (filtration and centrifugation). Semen samples were collected from seven Murrah buffalo bulls (Bubalus bubalis) once a week for 8 weeks. Each ejaculate was divided into three groups: control (presence of seminal plasma), centrifugation and filtration. Sperm kinetics was evaluated with the computer‐assisted sperm analysis (CASA) system. Plasmalemma and acrosomal membrane integrity, mitochondrial membrane potential and reactive oxygen species (ROS) were measured by flow cytometry, and lipid peroxidation was evaluated by the thiobarbituric acid reactive substances (TBARS) assay. Seminal plasma removal did not improve sperm kinetics compared to the control group. Centrifugation increased the number of cells with damaged acrosomal membranes (0.77 ± 0.05) and filtration caused greater plasmalemma and acrosomal membrane damage (22.18 ± 1.07). No difference in the mitochondrial membrane potential was observed between groups. In contrast, ROS production was higher in the centrifugation group compared to the control and filtration groups, although no differences in TBARS formation were detected. In conclusion, seminal plasma removal did not improve the quality of thawed buffalo semen compared to control in terms of sperm kinetics, membrane integrity, mitochondrial membrane potential or lipid peroxidation.     

Artificial insemination of frozen epididymal sperm in beagle dogs

Freeze-storage of epididymal sperm is an important technique for the preservation of gametes in animals, including those becoming extinct. We froze canine sperm recovered from the cauda epididymis and investigated the fertility. The qualities of sperm from the cauda epididymis before freezing were: mean sperm motility, 89.4 +/- 1.6 (SE) %; sperm viability, 89.1 +/- 1.1%; and these were significantly higher than those of sperm from the caput-corpus epididymis (P<0.01, P<0.05). The number of sperm recovered from both cauda epididymides varied among animals: 6.3-122.3 x 10(7), mean 61.5 +/- 10.0 x 10(7). Freezing was used only for sperm recovered from the cauda epididymis. The sperm motility and viability after thawing were 19.5 +/- 2.5% and 53.1 +/- 3.3%, respectively. These were slightly lower than those of frozen-thawed ejaculated sperm, but the differences were not significant. When 2 x 10(8), 3 x 10(8), or 4 x 10 (8) sperm were inseminated in the unilateral uterus, only one animal inseminated with 3 x 10(8) sperm was fertilized (1/16, 6.3%). When 1 x 10(8) sperm were inseminated in the bilateral uterine tubes, one of six animals (16.7%) was fertilized. Therefore, although the qualities of epididymal sperm after thawing were similar to those of ejaculated sperm, the conception rate obtained with frozen-thawed epididymal sperm was low in beagle dogs. It is necessary to investigate the differences in damage between epididymal sperm after thawing and ejaculated sperm and to develop a method for improving the conception rate.     

Preservation of Epididymal Stallion Sperm in Liquid and Frozen States: Effects of Seminal Plasma on Sperm Function and Fertility

Three separate experiments were conducted to improve preservation of stallion epididymal sperm. In the first one, two different cooling extenders (Kenney and Gent) were compared. Sperm viability and motility patterns were assessed in 10 different epididymal sperm samples after 0 hours, 24 hours, 48 hours, 72 hours, and 96 hours of preservation at 4°C. No significant differences were observed in any of the evaluated parameters either between extenders or throughout the storage period. The second set of experiments was designed to determine whether supplementing thawing medium (INRA Freeze) with seminal plasma had any impact on the quality of frozen-thawed epididymal sperm. Ten epididymal frozen-thawed sperm samples coming from separate stallions were used and different functional parameters (sperm membrane integrity and lipid disorder, motility, intracellular Ca²⁺ levels, and intracellular concentrations of peroxides and superoxides) were evaluated after incubation with or without 50% seminal plasma. Supplementing thawing medium with seminal plasma had no impact on sperm function and survival. The third experiment was an in vivo study. Twenty-five mares were inseminated with epididymal frozen-thawed sperm and seminal plasma, and 21 were bred with epididymal frozen-thawed sperm only. Pregnancy rates obtained for mares artificially inseminated with epididymal frozen-thawed sperm and seminal plasma were significantly (P < .05) higher than those observed when seminal plasma was not infused (64% vs. 19%). Taken together, our data indicate that the quality of epididymal stallion sperm can be maintained at 4°C for up to 96 hours. In addition, not only does supplementing frozen-thawed epididymal sperm with seminal plasma have any damaging effect on their quality but it may also improve pregnancy rates after artificial insemination.     

Effects of Seminal Fluid Fractions on Plasma and Acrosome Membrane Integrity and Mitochondrial Membrane Potential Determined by Flow Cytometry in Chilled Canine Spermatozoa

Depending on the mammal species, the use of seminal plasma during semen processing for cryopreservation has been found to have both beneficial and detrimental effects. This study was designed to determine the effects of the second (SF) and third [prostatic fluid, (PF)] ejaculate fractions on plasma membrane and acrosome integrity, mitochondrial membrane potential (ΔΨm), phosphatidylserine (PS) translocation and sperm motility in chilled canine spermatozoa by flow cytometry. After pooling the second sperm‐rich fraction of ejaculates from six dogs, samples for each assay were preserved at 5°C for 72 h in egg yolk‐TRIS extender (EYT) alone (control) or supplemented with seminal fluid from the second (EYT‐SF) or third (EYT‐PF) ejaculated fractions. After cold storage, groups EYT‐SF and EYT‐PF showed significantly higher percentages of sperm cells with an intact acrosome [68.8 ± 1.4%, 69.6 ± 2.6% (p < 0.01)] and intact plasma membrane [48.1 ± 2.8%, 50.4 ± 8.2% (p < 0.001)] than that observed in EYT [51.7 ± 3.2% and 33.3 ± 4.1% respectively]. Only in EYT‐SF was PS translocation significantly reduced compared to EYT‐PF and EYT [3.9 ± 0.4%, 10.2 ± 2.2% and 9.0 ± 1.5%, respectively (p < 0.001)]. However, significantly diminished sperm motility was observed in EYT‐SF and EYT‐PF compared to EYT [36.8 ± 2.1%, 35.5 ± 2.3% and 78.4 ± 4.7% (p < 0.001)]. No significant differences were detected in ΔΨm (p > 0.05). In conclusion, supplementing semen extenders with seminal fluid from the second or third fractions of the ejaculate supplementation helps to preserve the integrity of the plasma and acrosome membranes along with the mitochondrial membrane potential but seems to compromise the motility of canine spermatozoa chilled for 72 h.     

Response of Boar Sperm to the Treatment with Cholesterol‐Loaded Cyclodextrins Added Prior to Cryopreservation

Cryopreserved boar sperm is not used extensively for artificial insemination, owing to the poor fertility rates of the sperm after freezing and thawing. The sperm membrane is damaged as the cells are cooled from body temperature to 5°C (cold shock), as well as during the freeze–thaw process. Increasing the cholesterol content of boar sperm membranes could help them survive cryopreservation, similar to sperm from other species that are cold shock sensitive. The aim of this study was to determine the optimal cholesterol‐loaded cyclodextrin (CLC) concentration to use for boar sperm cryopreservation, and the influence of CLCs on the cryosurvival of sperm from boars classified as good or poor freezers. Treating boar sperm with 1 mg of CLC/120 × 10⁶ sperm slightly improved (p < 0.05) the percentage of viable sperm after freezing–thawing. On the other hand, sperm, from both good and poor freezers, responded similarly to CLC treatment. Nevertheless, additional studies will be needed to study the effect of this treatment on other parameters of sperm quality.     

Seminal changes in boars after heat stress

The object of the present study was to investigate the influence of elevated ambient temperature on sperm production, sperm morphology and composition of seminal plasma in boars. A total of 8 boars were used, 4 of them were exposed to 35°C, in a climate room, during 100 h and 4 served as controls and were kept at 20°C during 100 h in the climate room.Ejaculate volume and total sperm count per ejaculate remained unaltered. An obvious decrease in sperm motility was seen in all heat exposed boars 15–21 days after the exposure. The most consistent increase in sperm abnormalities were proximal cytoplasmic droplets and abnormal sperm heads. The highest levels were found during the 4th week after exposure. All the sperm characteristics assessed had returned to normal levels at the end of the experimental period, which means 7–8 weeks after the end of exposure.Only minor and inconsistent alterations were found in the seminal plasma components analysed and these changes were observed both in control and experimental boars.     

Assisted reproduction for felid species conservation—Sperm competences at risk

Cryobanking of gametes in combination with artificial insemination is an essential option to support conservation programmes for endangered and threatened species. About two-thirds of the felid species are classified as ‘near threatened’, ‘vulnerable’ or ‘endangered’ ( www.cites.org ), and mostly, epididymal sperm are collected from euthanized or castrated male felids and cryopreserved. However, epididymal compared with ejaculated and cryopreserved compared with fresh sperm have a limited potential to fertilize if vaginal non-surgical insemination is applied in feline species. Missing or highly diluted seminal fluid in epididymal and cryopreserved sperm, as well as a potential interference of extender ingredients with the natural interactive properties of sperm in the female genital tract is discussed as potential drawback which hampers a proper sperm transit and fertilization besides the limited longevity of cryopreserved feline sperm. Individual components in seminal fluid as well as cryoextenders may adversely alter sperm properties and have a different impact on fertility and preservation success. The identification and investigation of beneficial as well as detrimental components is a precondition to deduce options for improving the process of cryopreservation in felids, particularly, if only epididymal sperm are available.     

骨桥蛋白在长白公猪生殖细胞中的表达及其与繁殖性能的相关性

本试验旨在研究骨桥蛋白（osteopontin,OPN）在长白公猪生殖细胞中的表达和定位,并探究其与公猪繁殖性能的相关性。试验采集了长白公猪精液和不同阶段（3日龄、3月龄、6月龄和12月龄）的睾丸组织,通过蛋白印迹的方法检测OPN蛋白在精液和不同月龄睾丸中的表达,通过免疫组化的方法对OPN蛋白在公猪睾丸细胞中进行定位;同时,根据配种胎次≥ 20胎,3次配种公猪为同一头的标准,筛选并采集17头长白种公猪精液,统计相对应的1 388头母猪的生产成绩,计算得到公猪繁殖性能指标（包括窝产总仔数、窝产活仔数、分娩率和繁殖力）。低温离心精液分离得到精子和精浆,丙酮法提取精浆蛋白,Lysis buffer方法提取精子蛋白,最后运用BCA和ELISA的方法检测精子和精浆中OPN蛋白的含量,分析OPN蛋白与公猪繁殖性能的相关性。蛋白印迹结果显示,OPN在精子、精浆和各月龄阶段的长白公猪睾丸中均以两种形式表达（67.4和33.7 ku）,且67.4 ku的形式在3月龄公猪睾丸中表达量最高;免疫组化的结果显示,OPN在长白公猪的初级精母细胞、次级精母细胞和精子细胞中表达,在精母细胞、支持细胞和间质细胞中无表达;BCA和ELISA结果显示,精子中的OPN蛋白含量是精浆中的7倍（P<0.05）,精液中的OPN蛋白与公猪窝产活仔数显著正相关（P<0.05）。本试验结果表明,OPN在各阶段的长白公猪睾丸中都有表达,且在精子和精浆中也有表达,这可能与公猪的繁殖性能有关,从而为后期OPN蛋白在公猪受精力的研究奠定基础。     

GPx6在大白猪附睾细胞中的表达及其与繁殖性能的相关性

本试验旨在研究谷胱甘肽过氧化物酶6（glutathione peroxidase 6,GPx6）在大白公猪附睾细胞中的表达和定位,并探究其与公猪繁殖性能的相关性。选取15月龄的公猪和母猪各3头,取睾丸、附睾、前列腺、尿道球腺、精囊腺、卵巢和输卵管,提取蛋白,通过蛋白免疫印迹和免疫组化（IHC）检测组织和细胞中GPx6蛋白的表达和定位;根据评定大白公猪受精能力的数学模型,按照公猪配种胎次≥20胎、3次配种公猪为同一头的标准,筛选并采集符合要求的20头公猪精液,同时,统计相对应的1 279头母猪的生产成绩,计算得到公猪繁殖性能指标（包括窝产总仔数、窝产活仔数、分娩率和繁殖力）。提取精子蛋白和精清蛋白,通过BCA和ELISA检测精子和精清中GPx6蛋白的含量。使用SPSS软件的独立样本t检验及单因素方差分析（one-way ANOVA）,进行差异显著性分析,用双变量Pearson进行相关性分析,P<0.05表示差异或相关显著。结果表明,GPx6蛋白在附睾中高表达,IHC结果显示,GPx6蛋白在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达,在肌样细胞中不表达;精清蛋白中GPx6的含量是精子蛋白的7倍,精液中GPx6蛋白的含量与窝产活仔数、窝产总仔数呈负相关关系。结果提示,GPx6在附睾的顶细胞、基底细胞、晕细胞、主细胞和精子中表达,且其在精液中的含量会影响公猪的繁殖性能,这为GPx6对公猪受精能力影响的研究奠定了理论和试验基础。