Comparison of density gradient and single layer centrifugation of stallion spermatozoa: Yield,motility and survival

Reasons for performing study: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4°C overnight, were also available for testing. Results: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18–20% when cold‐stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. Potential relevance: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.     

Modulation of seminal plasma content in extended semen improves the quality attributes of ram spermatozoa following liquid preservation at 3–5°C

Seminal plasma (SP) is known to induce motility and capacitation in spermatozoa curtailing their lifespan when preserved. Hence, this study was conducted to examine the effects of removal of SP from sperm surface prior to liquid preservation either by high dilution (1/15) or by washing and the poststorage treatment with SP (15% and 25%, v/v) on the quality attributes of liquid‐preserved ram semen. Over the period of storage, the rapid motility (66.0% and 71.1% vs. 58.3%), straightness (87.1% and 82.1% vs. 79.4%), average path velocity (152.3 and 152.0 µm/s vs. 133.3 µm/s) and the straight‐line velocity (131.3 and 127.8 µm/s vs. 108.5 µm/s) were significantly (p < 0.05) higher in both the high‐dilution and wash groups as compared to the control (1/3 dilution). The functional membrane integrity (82.3% vs. 77.2%) and noncapacitated sperm count (65.0% vs. 58.7%) were also significantly (p < 0.05) higher in the high‐dilution and wash groups, respectively, as compared to the control. The poststorage treatment of sperm with SP significantly (p < 0.05) increased the functional membrane integrity (70.1% vs. 53.8%) and most of the motility attributes as compared to the control (without SP). In conclusion, both the removal of SP prior to liquid preservation and poststorage treatment with SP significantly improved the quality attributes of ram spermatozoa.     

New strategies of boar sperm cryopreservation: Development of novel freezing and thawing methods with a focus on the roles of seminal plasma

Cryopreservation of boar spermatozoa offers an effective means of long‐term storage of important genetic material. Many researchers have investigated how to improve reproductive performance by artificial insemination (AI) using cryopreserved boar spermatozoa. Recently, we and other groups reported that high conception rates (70–80%) can be achieved by AI with frozen‐thawed boar spermatozoa using a modified temperature program during freezing, or a novel cryopreservation extender to improve sperm quality (including sperm survivability, motility, membrane status and fertilization ability) after thawing, or a novel sperm infusion method, deep intra uterine insemination. However, these techniques have not yet been used for commercial pig production. The variation in sperm freezability among boars or among ejaculations in an identical boar is one of the main reasons for this problem. In our previous study, it was revealed that some components of seminal plasma have a negative effect on the freezability of boar sperm. One of these factors is bacteria‐released endotoxin (lipopolysaccharide: LPS). LPS binds to Toll‐like receptor‐4 (TLR‐4) expressed on the sperm surface, resulting in induction of apoptosis. On the other hand, seminal plasma suppresses cryo‐capacitation induced by thawing stress. On the basis of these findings, we designed a novel protocol of AI using frozen‐thawed boar sperm.     

Upregulation of CRISP-3 and kallikrein in stallion seminal plasma is associated with poor tolerance of cooled storage

For unknown reasons, stallion fertility and sperm longevity during cooled storage of semen vary markedly between individuals. Spermatozoa from individual stallions react differently to the presence, or the removal, of seminal plasma (SP). The aim was to evaluate differences in protein content in stallion seminal plasma with either a positive or a negative effect on sperm chromatin integrity during storage. Stallion semen samples from different ejaculate fractions were stored at 5°C for 24 hr. Sperm survival was assessed after storage using a sperm chromatin structure assay. Protein expression in SP with either positive or negative effects on sperm survival during storage was studied using two-dimensional differential gel electrophoresis and liquid chromatography–mass spectrometry. Lower sperm chromatin integrity was associated with upregulation of the proteins kallikrein, CRISP-3 and HSP-1, while higher chromatin integrity was associated with upregulation of TIMP-2. In the sperm-rich fractions, kallikrein and CRISP-3 differed significantly between SP samples with differing effects on sperm chromatin integrity. In the sperm-poor fractions, TIMP-2 and HSP-1 differed significantly between the two SP groups. Differences in the seminal plasma proteome are associated with sperm longevity during cooled storage.     

Studies on boar semen. III. Sperm concentration and seminal plasma total solids followed in Danish AI boars through a 10-year-period

Semen samples from 1531 young boars eligible for AI service were examined for normality. Sperm concentration (s.c.) was determined by the hemocytometer technique in 1348 of the samples. Seminal plasma total solids (t.s.) were determined on all samples in order to control whether the semen samples originated from complete ejaculates.The hemocytometer counts showed an arithmetic mean of 370 × 10⁶/ml with a standard deviation of 188×10⁶/ml. No correlation was found either between s.c. and age or between s.c. and season of the year.The seminal plasma t.s. showed an arithmetic mean of 4.6% with a standard deviation of 1.35%. No correlation was found between t.s. and s.c. or between t.s. and the age of the boars. Neither was there any association between t.s. and the season of the year.Values of t.s. below 1.6% combined with aspermia were regarded as the result of incomplete ejaculations and the following retest from such cases gave ejaculates showing normal values concerning s.c. and t.s. A drop in t.s. combined with an admixture of pathological cells to the ejaculate may indicate an inflammatory condition in the vesicular glands.     

三种因素对雄蜂精子数量和活动率影响的研究

本文较详细地研究了雄蜂体重、性成熟程度和天气情况对雄蜂精子总数和精子活动率的影响,结果表明:雄蜂总精子数与雄蜂的体重呈正相关。性成熟很充分的雄蜂其精子总数相对较多,但精子活动率却低于性成熟一般的雄蜂。虽然在不同的天气里雄蜂精子总数差异不大,但精子活动率有着比较明显的差异。本实验旨在采集到高质量的雄蜂精液,为蜜蜂人工授精技术的发展提供理论依据。     

高温对荷斯坦种公牛精液品质及精清生化指标的影响研究

以荷斯坦种公牛为试验牛研究了高温对其精液品质和精清生化指标的影响。结果表明:(1)高温可造成种公牛精液品质显著下降。使原精活力、精子密度、活精子百分数和顶体完整率分别比春季下降3·13%(P<0·05)、34·8%(P<0·01)、15·0%(P<0·01)和17·8%(P<0·01),精子畸形率比春季上升25·5%(P<0·01)。(2)夏季荷斯坦种公牛精清睾酮含量仅为4·31pg/mL,极显著低于非高温季节(P<0·01)。(3)高温环境使得荷斯坦种公牛精清钾、钙、镁处于最低水平(P<0·01),精清碱性磷酸酶(ALP)、酸性磷酸酶(ACP)处于最高水平(P<0·01),使精清钠由春季至夏季呈显著下降趋势。     

Retracted: Influence of Glutamine Supplementation on Motility and Fertilization Success of Frozen–Thawed Persian Sturgeon (Acipenser persicus) Sperm

Amino acids have an important biological role for the prevention of cell damage during cryopreservation. The aim of this study was to investigate the effects of glutamine on post‐thaw sperm motility and fertilization success in the Persian sturgeon (Acipenser persicus). Sperm collected from six fish was cryopreserved in extenders containing different glutamine concentrations (2.5, 5 and 10 mm ). Sperm samples diluted at the ratio of 1 : 1 using the extenders were subjected to cryopreservation. After dilution, the sperm suspensions were sucked into 250‐μl straws; the straws were placed on the tray, frozen in nitrogen vapour and plunged into liquid nitrogen. Then, sperm were thawed in a water bath at 40°C for 5 s and used for analysis. Our results revealed that an increase in the concentration of glutamine caused a significant increase in the motility percentage, curvilinear velocity (VCL) and also fertilization success in the Persian sturgeon (p < 0.05). Comparing all concentrations of glutamine, the best concentration for sperm motility and fertilization rate was 10 mm . In addition, higher post‐thaw motility percentage, VCL, and fertilization and hatching rates were obtained with the extender at the concentration of 10 mm (p < 0.05). The findings of this study showed that glutamine was of greater benefit to Persian sturgeon sperm motility during frozen–thawed process.     

Measurement of carbonic anhydrase isozyme VI (CA‐VI) in swine sera,colostrums, saliva,bile, seminal plasma and tissues

Swine secretory carbonic anhydrase VI (CA‐VI) was purified from swine saliva and an antibody to CA‐VI was generated. A specific and sensitive enzyme‐linked immunosorbent assay (ELISA) has been developed for the measurement of swine CA‐VI. The assay can detect as little as 5 ng/mL of swine CA‐VI. Typical standard curves were determined for a range of CA‐VI solutions (7.8 to 500 ng/mL). The coefficients of variation for these solutions were less than 5%. When 500, 250 or 100 ng/mL of swine CA‐VI was added to swine sera, the recoveries were 102.0%, 109.7% and 100.2%, respectively. The concentrations of CA‐VI in the saliva (26.2 ± 30.4 µg/mL), sera (3.3 ± 4.9 ng/mL), bile (153.0 ± 114.0 ng/mL), seminal plasma (124.0 ± 39.0 ng/mL) and parotid gland (441.3 ± 90.0 µg/g wet tissue), submaxillary gland (88.1 ± 124.4 µg/g wet tissue), sublingual gland (58.6 ± 24.6 µg/g wet tissue) and gallbladder (2.4 ± 1.3 µg/1g wet tissue) were determined by ELISA. The concentration of CA‐VI in colostrum was 163.3 ± 101.4 ng/mL and did not decrease within 10 days following parturition. An immunohistochemical reaction to anti‐CA‐VI antiserum was observed in the columnar epithelial cells lining the gallbladder. These data suggest that secretory CA‐VI plays various roles in pH regulation and the maintenance of ion and fluid balance.     

A comparative study of the effects of intramuscular administration of gonadorelin,mating and intrauterine infusion of either raw seminal plasma or seminal plasma purified β‐NGF on luteal development in llamas

The aim of this study was to compare the effect of the intramuscular administration of 50 μg of gonadorelin acetate versus natural mating, intrauterine infusion (i.u.) of a physiological relevant dose of either raw llama seminal plasma (SP) or purified beta‐nerve growth factor from seminal origin (spβ‐NGF) on ovulation rate and corpus luteum (CL) development and function in llamas. Females with a follicle (≥8 mm) were assigned to groups: (i) i.m. administration of 50 μg of gonadorelin acetate (GnRH; positive control; n = 4); (ii) single mating (mating; n = 6); (iii) i.u. infusion of 4 ml of llama SP (SP; n = 4); or (iv) i.u. infusion of 10 mg of spβ‐NGF contained in 4 ml of PBS (phosphate‐buffered saline) (spβ‐NGF; n = 6). Ovaries were examined by power Doppler ultrasonography at 0, 1, 3, 6, 12 and 24 hr after treatment to determine preovulatory follicle vascularization area (VA), and additionally every 12 hr until Day 2 (Day of treatment = Day 0) to determine ovulation. Afterwards, ovaries were examined every other day until Day 8 to evaluate CL diameter and VA. Blood samples were collected on Days 0, 2, 4, 6 and 8 to determine plasma progesterone (P4) concentration. Ovulation rate did not differ (p = .7) among groups, but treatment affected (p < .0001) preovulatory follicle VA. Neither treatment administration nor treatment by time interaction affected (p ≥ .4) CL diameter, VA and plasma P4 concentration. Mating tended (p = .08) to increase CL VA when compared to the seminal plasma group by Day 8. Intrauterine administration of seminal plasma or spβ‐NGF does not increase CL size and function when compared to i.m. GnRH treatment, suggesting that the administration route of spβ‐NGF influences its luteotrophic effect in llamas.     

Effects of dietary curcumin supplementation on seminal quality indices and fertility rate in broiler breeder roosters

1. Decreased semen quality is an underlying contributor to age-related subfertility in broiler breeder roosters. This study investigated the effects of dietary curcumin (derived from turmeric) supplementation as an antioxidant source on semen quality and fertility in broiler breeder roosters.

2. Twenty-eight Ross 308 roosters were randomly allotted to four groups with seven birds in each and were fed a standard diet supplemented with different levels of curcumin at 0 (C₀), 10 (C₁₀), 20 (C₂₀) and 30 (C₃₀) mg/bird per day from 48 through to 61 weeks of age. Body weight and semen quality traits were evaluated on a weekly basis and seminal concentrations of malondialdehyde (MDA) as a measure of antioxidation status were quantified at one-week intervals during the first 11 weeks of the trial (48–59 weeks of age). Semen samples from last 2 weeks (60 and 61 weeks of age) were used to artificially inseminate to assess the sperm-egg penetration (SP) in perivitelline membrane and fertility rates.

3. Except for body weight and ejaculate volume, other characteristics, including semen concentration, total sperm production, progressive motility and plasma membrane integrity were linearly improved by the increasing levels of curcumin supplementation (P < 0.01). However, dietary curcumin levels were linearly and quadratically associated with decreased seminal concentration of MDA (P < 0.01 and P < 0.03), percentage of abnormal sperm (P < 0.01 and P < 0.07) and increased plasma membrane functionality (P < 0.01 and P < 0.04), respectively. The SP holes in perivitelline membrane were increased in a linear and quadratic manner in response to increasing levels of curcumin (P < 0.01). Moreover, fertility rate was linearly improved (P < 0.01) as the dosage of curcumin increased, and resulted in 8, 12 and 14% improvements in the birds fed C₁₀, C₂₀ and C₃₀, compared to C₀, respectively.

4. In conclusion, the results showed that increasing levels of dietary supplementation of curcumin was associated with beneficial effects on semen quality indices and fertility rate in aged broiler breeder roosters.     

Effect of relaxin on the motility, acrosome reaction and utilization of glucose of fresh and frozen-thawed bovine spermatozoa

The study was conducted to investigate the effect of relaxin on motility, acrosome reaction (AR), viability and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa. Both semen samples were washed twice through centrifugation (5 min at 600 g), and preincubated for 1 h at 39°C for swim up. The swim‐up separated spermatozoa were resuspended in a sperm Tyrode's albumin lactate pyruvate (Sp‐TALP) medium containing 0 (control) and 40 ng/mL porcine relaxin and incubated for 0–6 h. Sperm motility was determined on the basis of movement quality examined by a phase contrast microscope. Sperm viability and AR were evaluated by using the triple staining technique. The incorporation and oxidation of ¹⁴C‐glucose was assessed by a liquid scintillation counter. Motility was improved (P < 0.05) in both fresh and frozen‐thawed spermatozoa by the addition of relaxin to the Sp‐TALP medium, whereas relaxin showed no significant effect on viability in either fresh or frozen‐thawed spermatozoa. The percentage of AR increased (P < 0.05) when fresh or frozen‐thawed spermatozoa were incubated with relaxin. In contrast, the incorporation and oxidation of ¹⁴C‐glucose increased (P < 0.05) in both kinds of spermatozoa incubated with relaxin. Thus the results demonstrated that the addition of relaxin to the Sp‐TALP medium increased the motility, AR and utilization of glucose in fresh and frozen‐thawed bovine spermatozoa.     

肉鸡血浆VLDL浓度的选择效应—肉仔鸡体重和血浆VLDL

以父线代肉种鸡为材料,用比浊法测定血浆ＶＬＤＬ浓度,分别以１６周龄,６周龄血浆ＶＬＤＬ浓度为选择指标建立零世代,一世代肉鸡高脂系和低脂系地肉鸡体重和血浆ＶＬＤＬ浓度的选择反应。结果：１．零世代,一世代肉鸡血浆ＶＬＤＬ浓度变异很大,变异系数分别为４３．６％－７４．６％和３８．７％－７９．９％,二个世代选择后,血浆ＶＬＤＬ浓度变异系数在高,低脂质中分别为３７％－５２％和３１％－４６％,表明对血浆ＶＬＤ     

Retracted: Determination of some blood and seminal plasma ions in the beluga,Huso huso (Linnaeus, 1758)

Blood and seminal plasma ionic parameters are essential for monitoring health status, detecting illnesses, fish stock conservation and development of artificial propagation methods via extender improvement. In this study, comparison of blood and seminal plasma ionic parameters in beluga, Huso huso (30–45 kg, 1–2 m, n = 10), was made. The results obtained show that Na⁺ (82.54 ± 5.46), Cl^‐ (15.95 ± 0.72) and K⁺ (3.57 ± 0.15) were predominant ions in the seminal plasma (as mm ). Blood ionic values (as mm ) were determined for Na⁺ (110.2 ± 1.26), K⁺ (3.77 ± 0.081), Cl^? (60.12 ± 1.5), Ca²⁺ (2.05 ± 0.35) and Mg²⁺ (1.9 ± 0.16). Results of the comparison between ionic parameters of seminal and blood plasma indicated that the concentrations of all parameters of blood plasma with the exception of K⁺ were significantly (p < 0.05) higher than those of seminal plasma.     

Effect of nicotinic acid on the plasma membrane function and polyunsaturated fatty acids composition during cryopreservation in boar sperm

This study was conducted to evaluate the effect of nicotinic acid on plasma membrane integrity and fatty acid composition in frozen–thawed boar sperm. Boar semen was cryopreserved using freezing extender containing nicotinic acid (NA), then plasma membrane integrity, osmotic equilibration, lipid peroxidation and fatty acid were analysed. The plasma membrane integrity of frozen–thawed sperm was significantly higher in the 10 mM NA than in the 0 and 20 mM NA treatment groups (p < 0.05). Additionally, the osmotic equilibration ability was not different in treatment groups, but lipid peroxidation was significantly decreased in the 10 mM NA treatment group (p < 0.05). The saturated fatty acids were significantly decreased in the 10 mM NA treatment group, and C18:1n‐9, C18:2n‐6, C20:4n‐6, C22:5n‐6 and C22:6n‐3, and total polyunsaturated fatty acids (PUFAs) were significantly increased in the 10 mM NA treatment groups (p < 0.05). In summary, 10 mM NA improved plasma membrane integrity, inhibited lipid peroxidation and increased PUFAs in frozen–thawed boar sperm. These results suggest that NA may be useful to protect the plasma membrane and inhibit the loss of PUFAs for sperm cryopreservation in pigs.     

Effects of exogenous lactoferrin on characteristics and functions of bovine epididymal,ejaculated and frozen-thawed sperm

The purpose of this study was to investigate effects of addition of lactoferrin on characteristics and functions of bovine epididymal, ejaculated, and frozen-thawed sperm. The addition of lactoferrin was significantly (p < .05) effective on increasing values of progressive motility, straightness, and linearity in caput epididymal sperm and values of motility in cauda epididymal sperm. When ejaculated sperm were incubated in capacitation medium, percentages of motile and progressively motile sperm decreased largely within the first period of 30 min, followed by only minor changes. However, the addition of lactoferrin significantly lessened the early decreases of these parameters and additionally promoted capacitation-dependent changes of chlortetracycline staining patterns (from F pattern to B pattern). In other experiments, when ejaculated sperm were exposed to oxidative stress with 100-µM H₂O₂, the addition of lactoferrin partially protected them from dysfunction of flagellar movement and loss of progressive movement. In final experiments with frozen-thawed samples incubated in the capacitation medium, the addition of lactoferrin effectively survived dying sperm and suppressed occurrence of sperm agglutination. These results may suggest biological and biotechnological potentials of lactoferrin for modulation of bovine sperm viability, motility, capacitation state, and preservation in vitro.     

Effect of Foetal Bovine Serum on sperm motility,acrosome reaction and spermatic interaction to zona pellucida in alpacas (Vicugna pacos)

The use of foetal bovine serum (FBS) in cell culture media is quite common. However, little is known about the effect of FBS on sperm. The severe difficulties in alpaca reproduction demand the search of new methods for in vitro reproductive management. In the present study, we use for the first time FBS as a supplement in the culture medium for sperm in alpaca, and the effect of FBS on motility, acrosome reaction and sperm binding to the zona pellucida in this species was evaluated. A concentration of 10% v/v FBS was used. The sperm motility with FBS at the first hour was 32.8% (vs. control = 30.0%), whereas at the second hour sperm motility with FBS was 30.2% (vs. control = 28.8%). The acrosome reaction reached an average of 44.0% for treatment with FBS (vs. control = 30.1%). The sperm‐zona pellucida binding assay showed that the samples incubated with FBS had an average of 2.7 bound sperm (vs. control = 1.7). Only a significant difference was observed for sperm motility at the first hour and for the acrosome reaction. It is concluded that FBS favours the capacitation of sperm in alpaca.     

Effects of sulfanilamide on boar sperm quality,bacterial composition,and fertility during liquid storage at 17°C

Sulfanilamide (SA) is an effective broad‐spectrum antibacterial agent in human and veterinary medicine. The purpose of this study was to evaluate the effects of SA on boar sperm quality during liquid storage at 17°C and determine the optimal concentration of SA and its effects on bacterial growth, microbial composition, and maternal fertility. Boar ejaculates were diluted with a basic extender, containing different concentrations of SA, and stored in a 17°C incubator for 6 days. The sperm motility, plasma membrane integrity, and acrosome integrity were measured daily. The results showed that when the concentration of SA was 0.02 g/L, the sperm quality parameters were significantly higher than those of all other treatment groups (p < .05). We also monitored the bacterial growth and compared the differences in the microbial species between the 0.02 g/L SA group and the control by 16S rDNA sequencing. The results revealed that some bacteria, such as Staphylococcus and Pseudomonas, were considerably lower in the 0.02 g/L SA group than in the control group (p < .05). In addition, preserved semen was used for artificial insemination, and results showed that 0.02 g/L SA group had a higher litter size, and its pregnancy rate was 92.5%.