Evaluation of membrane integrity of frozen/thawed deer spermatozoa: short communication

A simultaneous live/dead and acrosome staining, originally described for domestic mammals, was successfully applied on red deer (Cervus elaphus) and fallow deer (Dama dama) spermatozoa collected from the cauda epididymidis and vas deferens of shot stags. The staining is simple enough for routine application. Seven classes of spermatozoa were distinguished in the smears of frozen/thawed semen samples. Morphology, including cytoplasmic droplets, was evaluated as well. Percentage of live cells with intact acrosomes and with no other morphological aberrations might be a practical index of semen quality.     

Effect of chelating and antioxidant agents on morphology and DNA methylation in freeze-drying rabbit (Oryctolagus cuniculus) spermatozoa

Freeze-drying (FD) has been exhaustively tried in several mammalian species as an alternative technique to sperm cryopreservation, but few studies have been done in rabbits (Oryctolagus cuniculus). The main objective of this study was to compare the protective effect of various antioxidants added to EDTA medium on structural and functional components of FD rabbit spermatozoa and on their status of global DNA methylation. FD media used were composed of basic FD medium (10 mM Tris-HCl buffer and 50 mM NaCl) supplemented with either 50 mM EDTA alone (EDTA) or added with 105 µM of rosmarinic acid (RA, EDTA-RA) or 10 µM of melatonin (MLT, EDTA-MLT). The effect of each medium on the preservation of FD spermatozoon structure was evaluated with light and scanning electron microscopy (SEM). Global DNA methylation was quantified in all FD sperm samples as well as in fresh spermatozoa. Morphologically, fracture points were evidenced in the neck, mid and principal piece of the spermatozoon tail. No differences in spermatozoon fracture points were evidenced among FD treatments: intact spermatozoa were the largest (p < .01) category, whereas the most frequent (p < .01) injury was the neck fracture, resulting in tailless heads. At SEM, the head of spermatozoa showed a well-conserved shape and intact membrane in all treatments. DNA methylation status was the same in all FD treatments. In conclusion, supplementation of EDTA, EDTA-RA and EDTA-MLT during FD preserved rabbit sperm morphological integrity and methylation status as well. Therefore, the difficulty of getting viable offspring using FD semen is likely unrelated to the impact of the lyophilization process on DNA methylation and morphology of lyophilized spermatozoa.     

Fertility of deep frozen boar spermatozoa: influence of thawing diluents and of boars.

2 insemination trials were conducted to assess the effect of different thawing diluents on deep frozen boar spermatozoa. The diluents used were boar seminal plasma, protein free seminal plasma, isotonic glucose solution, and OLEP (fructose, sodium pyruvate, CaC12, MgC12, KCL, NaC1, and NaHC03). The electrolyte levels and pH and osmotic pressure of the latter are similar to those of boar seminal plasma. 139 gilts were inseminated. The percentage of fertilized ova was similar for OLEP, protein free seminal plasma, and boar seminal plasma. However, thawing in isotonic glucose solution gave markedly poorer results. There were also differences in the fertility of spermatozoa from the different boars, though these were independent of the effects of the various thawing diluents. The possible effects of the thawing diluents are discussed. The need for methods of selecting boars with spermatozoa of good freezing quality is emphasized.     

Improved assessment of frozen/thawed mouse spermatozoa using fluorescence microscopy

Genetically modified (GM) animals are unique mutants with an enormous scientific potential. Cryopreservation of pre-implantation embryos or spermatozoa is a common approach for protecting these lines from being lost or to store them in a repository. A mutant line can be taken out of a breeding nucleus only if sufficient numbers of samples with an appropriate level of quality are cryopreserved. The quality of different donors within the same mouse line might be heterogeneous and the cryopreservation procedure might also be error-prone. However, only limited amounts of material are available for analysis. To improve the monitoring of frozen/thawed spermatozoa, commonly used in vitro fertilization (IVF) followed by embryo transfer were replaced with animal-free techniques. Major factors for assessing spermatozoa quality (i.e., density, viability, motility, and morphology) were evaluated by fluorescence microscopy. For this, a live/dead cell staining protocol requiring only small amounts of material was created. Membrane integrity was then examined as major parameter closely correlated with successful IVF. These complex analyses allow us to monitor frozen/thawed spermatozoa from GM mice using a relatively simple staining procedure. This approach leads to a reduction of animal experiments and contributes to the 3R principles (replacement, reduction and refinement of animal experiments).     

提高奶牛细管冻精精子活力的试验研究

为了提高奶牛细管冻精精子的活力,试验探索了稀释液种类、最佳熏蒸距离、最佳冷冻温度、最佳熏蒸时间、不同解冻温度及时间对精子活力的影响。结果表明:精子在由柠檬酸钠和果糖组成的稀释液中存活时间长,在三羟甲基氨基甲烷稀释液中冷冻后精子活力高于其他稀释液;牛细管冻精最佳熏蒸距离为2.5 cm,时间影响不显著(5～10 min均可);用50℃温水解冻15 s的精子活力比其他解冻温度和时间时的精子活力要好。     

Fertility of deep frozen boar spermatozoa at various intervals between insemination and induced ovulation: influence of boars and thawing diluents.

The fertilizing ability of deep frozen boar spermatozoa was assessed after artificial insemination at various intervals after human chorionic gonadotropin (HCG) induced ovulation in gilts. Boar seminal plasma and OLEP were used as thawing diluent agents. The fertilization rates were similar when insemination was performed 2 or 6 hours before the time of estimated ovulation (40 hours post-HCG administration). However, fertility rates markedly declined when insemination was performed earlier than 6 hours before expected ovulation. In inseminations performed 2 hours before ovulation, the fertility rate was higher with specimens treated with boar seminal plasma diluent than with OLEP. As the difference between the time of insemination and that of ovulation increased, the difference in fertilizing ability of spermatozoa from the 2 boars used also increased. The results show that spermatozoa which have a low resistance to freezing-thawing also have a diminished fertile life in the female genital tract.     

Hysteroscopic insemination of low numbers of flow sorted fresh and frozen/thawed stallion spermatozoa

The objective of this experiment was to determine the effects of flow cytometric sorting and freezing on stallion sperm fertility. A 2 x 2 factorial design was used to delineate effects of flow sorting and freezing spermatozoa. Oestrus was synchronised (July-August) in 41 mares by administering 10 ml altrenogest (2.2 mg/ml) per os for 10 consecutive days, followed by 250 microg cloprostenol i.m. on Day 11. Ovulation was induced by administering 3,000 iu hCG i.v. either 6 h (fresh spermatozoa) or 30 h (frozen/thawed spermatozoa) prior to insemination. Mares were assigned randomly to one of 4 sperm treatment groups. Semen was collected from 2 stallions with an artificial vagina and processed for each treatment. Treatment 1 (n = 10 mare cycles) consisted of fresh, nonsorted spermatozoa and Treatment 2 (n = 16 mare cycles) of fresh, flow sorted spermatozoa. Spermatozoa to be sorted were stained with Hoechst 33342 and sorted into X- and Y-chromosome-bearing populations based on DNA content using an SX MoFlo sperm sorter. Treatment 3 (n = 16 mare cycles) consisted of frozen/thawed nonsorted spermatozoa (frozen at 33.5 x 106 sperm/ml in 0.25 ml straws) and Treatment 4 (n = 15 mare cycles) of flow sorted frozen/thawed spermatozoa (frozen at 64.4 x 10(6) sperm/ml). Concentrations of sperm in both cryopreserved treatments were adjusted, based on predetermined average post-thaw motilities, so that each insemination contained approximately 5 x 10(6) motile spermatozoa. Hysteroscopic insemination of 5 x 10(6) motile spermatozoa in a volume of 230 microd was used for all treatments. Pregnancy was determined ultrasonographically 16 days postovulation. No differences were found (P>0.1) in the pregnancy rates for mares inseminated with fresh nonsorted (4/10 = 40.0%), fresh flow sorted (6/16 = 37.5%), frozen/thawed nonsorted (6/16 = 37.5%) and flow sorted frozen/thawed spermatozoa (2/15 = 133%). Pregnancy rates tended (P = 0.12) to be lower following insemination of frozen/thawed flow sorted spermatozoa. Further studies are needed with a larger number of mares to determine if fertility of flow sorted frozen/thawed spermatozoa can be improved.     

Sperm DNA integrity in Landrace and Duroc boar semen and its relationship to litter size

The sperm chromatin structure assay is a method for assessment of sperm DNA fragmentation, a parameter reported to be negatively related to field fertility in several mammal species. This method calculates a DNA fragmentation index (DFI) whose high values indicate abnormal chromatin structure. In this study, running from March 2010 until June 2017, the aim was to assess sperm DFI in stored liquid extended semen from two different pig breeds, Norwegian Landrace (NL; n = 693) and Norwegian Duroc (ND; n = 655), and to evaluate the influence on total number of piglets born (TNB). There was a significantly higher median DFI (p < 0.0001) in ejaculates from the 478 ND boars compared to the 452 NL boars. Data from 19,496 NL litters and 3,877 ND litters of the same boars were retrieved. For either breed, sow herd (p < 0.0001), parity (p < 0.05) and DFI (p < 0.05) showed significant effects on TNB. The DFI was negatively correlated to TNB in both breeds. The boars with the 5% lowest TNB had a least square means DFI of 3.05% and 2.24% in NL and ND, respectively, compared to 1.67% and 1.23% for the boars with the 5% highest TNB (p < 0.01). The DFI and the motility of the same semen samples were negatively correlated (p < 0.0001), and the high and low TNB groups showed significant differences in motility. However, this difference could not be used for practical prediction of TNB group (92.1% vs. 89.7%; p = 0.0038 and 92.3% vs. 89.5%; p = 0.018; NL and ND, respectively). In conclusion, our results indicate that sperm DNA integrity in semen with good motility and morphology may be an additional prediction parameter for fertility in pigs.     

Semen analysis of boars under intertropical conditions reveals the relevance of proximal and distal cytoplasm droplets for sperm functional integrity

This work aimed to establish whether the temperature humidity index (THI) under different intertropical zones affects the retention of cytoplasmic droplets (CDs), sperm function and DNA integrity in boars. With this purpose, two separate studies were devised. In the first one, 49 boars from six farms were collected every 45 days (230 ejaculates). THI were measured daily, and sperm parameters were evaluated. Boars were classified into three groups based on the incidence of ejaculates having more than 25% spermatozoa with CDs: persistent (at least three consecutive ejaculates), moderate (less than three ejaculates) and absent (no ejaculate having ≥25% spermatozoa with CDs). Farms were classified based on THI through cluster analysis into two groups. In the second study, 32 liquid-stored semen samples were classified based on three cluster analysis: low and high incidence of proximal (PCDs), distal (DCDs) CDs and a combination of PCD and DCDs. high THI farms presented significantly (p < .05) higher proportions of boars with moderate and persistent incidence of CD than those with low THI. In study 2, the presence of PCDs was negatively correlated with sperm DNA integrity (r = −0.691; p < .01). However, differences between groups were more apparent when ejaculates were classified based on both PCDs and DCDs than when PCDs or DCDs were considered separately. In conclusion, classification of boars according to the severity and persistence of CDs in boars allows understanding more clearly the dynamics of CD retention and the effects of ambient temperature and relative humidity. Additionally, the joint analysis of both PCDs and DCDs is necessary in routine sperm quality analyses.     

褪黑激素对獭兔日增重及其毛皮皮尺的影响

选取体重为1.4kg左右日龄相近的健康獭兔60只,随机分为3组,每组20只,公母各半。分别皮下埋植褪黑激素0mg、10mg、20mg,研究褪黑激素对獭兔日增重及其毛皮皮尺的影响。结果表明:褪黑激素对獭兔的日增重增加4.1%(P>0.05)、毛皮长度以及毛皮厚度均无显著(P>0.05)影响;但埋植10mg组的獭兔毛皮宽度要显著(P<0.05)大于埋植20mg组。     

Mobility of Japanese quail spermatozoa and its relationship to egg fertility

Sperm mobility (SM) appears to be primary determinant of fertility in chicken and turkey. The aims of this study were to extend the concept to the Japanese quail by developing an assay to quantify SM, explaining the basis of SM using motility properties measured by CASA, and exploring the relationship between SM and egg fertility. The study was carried out in three stages: i) males (n = 20) and females (n = 20) were mated individually; ii) ejaculates were collected from 20 males, and SM was measured; iii) males (n = 20) and females (n = 20) were mated individually. In Stages I and III, data were collected for egg fertility, Sperm_OPVL and Holes_IPVL. In Stage II, SM assay was developed and assay conditions were defined: effect of sperm numbers on absorbance in Accudenz solution; effect of Accudenz concentration on sperm motility and mobility; effect of quail proctodeal gland foam extract and incubation temperature on SM at 37 and 41°C. The recorded absorbance of sperm movement was dependent on sperm numbers in the sperm suspension overlaying the Accudenz (p < .001). At 41°C, SM, progressively motile sperm, VCL, VSL and VAP were negatively affected by Accudenz concentration (p < .05). The effect of foam on SM and motility depended on an interaction between the concentration of foam extract and incubating temperature. Males were categorized into low, average and high SM phenotypes. These categories differed significantly (p < .001), but sperm motility and SM were not related to egg fertility. In conclusion, SM assay can be used to identify mobility phenotypes, but the poor relationship between SM and egg fertility indicates a need for further studies on interaction between the concentration of foam extract, incubating temperature, and in vivo sperm movement and egg fertilization success.     

褪黑激素对长毛兔产毛性能及兔毛品质的影响

试验旨在研究褪黑激素(Melatonin,MT)对长毛兔夏季产毛的影响。选择1.5岁左右的皖系长毛兔60只,其中公兔40只,母兔20只,随机分为4组,分别皮下埋植0、25、40、55 mg的褪黑激素,经过70 d的养毛期后测定各组产毛量及粗毛和细毛的长度变化差异,分析褪黑激素对长毛兔产毛性能的影响。结果显示:公兔40 mg埋植组产毛量显著高于埋植前产毛量(P<0.05),55 mg埋植组产毛量显著高于对照组产毛量(P<0.05);母兔对照组试验后剪毛量显著低于试验前剪毛量(331.0 g vs 284.8 g,P<0.05),而3个褪黑激素埋植组试验后产毛量下降幅度并不显著(P>0.05)。皮下埋植40 mg或55 mg褪黑激素能够增加皖系长毛兔公兔或母兔的粗毛长度,但对细毛长度和兔毛细度均无显著影响(P>0.05)。说明长毛兔皮下埋植40 mg和55 mg褪黑激素能够提高公兔夏季产毛量且抑制母兔夏季产毛量的降低,同时增加公、母兔粗毛长度。研究结果可为褪黑激素的合理利用提供参考,并为揭示其对促进长毛兔夏季兔毛生长的作用机制奠定基础。     

The effect of liposomes composed of phosphatidylserine and cholesterol on fertility rates using frozen thawed equine spermatozoa

The objective of this study was to determine if the addition of liposomes composed of phosphatidylserine (PS) and cholesterol (CH) to equine sperm would improve pregnancy rates after sperm were cryopreserved. Ejaculates from four stallions, collected every other day during May and June were split, treated with PSCH liposomes or HBS (Hepes Buffered Saline) and cryopreserved. Fifty-two mares were bred over eighty estrous cycles with the frozen-thawed semen. The one cycle pregnancy rates of the mares inseminated with semen treated with liposomes (45%) were similar to mares inseminated with control semen (48%; p>0.05). Thus, at least at the levels used, PSCH liposomes added to equine sperm did not improve pregnancy rates of mares inseminated with cryopreserved semen.     

Conservation science in a terrorist age: the impact of airport security screening on the viability and DNA integrity of frozen felid spermatozoa.

In response to growing terrorism concerns, the Transportation Security Administration now requires that all checked baggage at U.S. airports be scanned through a cabinet x-ray system, which may increase risk of radiation damage to transported biologic samples and other sensitive genetic material. The objective of this study was to investigate the effect of these new airport security regulations on the viability and DNA integrity of frozen felid spermatozoa. Semen was collected from two domestic cats (Felis silvestris catus) and one fishing cat (Prionailurus viverrinus), cryopreserved in plastic freezing straws, and transferred into liquid nitrogen dry shippers for security screening. Treatment groups included frozen samples from each male scanned once or three times using a Transportation Security Administration-operated cabinet x-ray system, in addition to non-scanned samples (i.e., negative control) and samples previously scanned three times and exposed to five additional high-intensity x-ray bursts (i.e., positive control). Dosimeters placed in empty dry shippers were used to quantify radiation exposure. Following treatment, straws were thawed and spermatozoa analyzed for post-thaw motility (percentage motile and rate of progressive movement), acrosome status, and DNA integrity using single-cell gel electrophoresis (i.e., the comet assay). Dosimeter measurements determined that each airport screening procedure produced approximately 16 mrem of radiation exposure. Our results indicated that all levels of radiation exposure adversely affected (P < 0.05) post-thaw sperm motility, but the percentage of acrosome-intact spermatozoa did not differ (P > 0.05) among treatment groups. Results also showed that the amount of double-stranded DNA damage was greater (P < 0.05) in sperm samples from both cat species scanned three times compared to samples scanned once or negative controls. Findings suggest that new airport security measures may cause radiation-induced damage to frozen spermatozoa and other valuable biologic samples transported on passenger aircraft and that alternative modes of sample transportation should be used whenever possible.     

外源褪黑素处理方式对荷斯坦奶牛体内褪黑素富集与代谢的影响

为研究正常情况下经产荷斯坦奶牛24 h内血液中褪黑素浓度的变化情况,探索不同外源褪黑素处理方式对荷斯坦奶牛血液褪黑素浓度的影响,本研究采用颈静脉注射、颈部皮下注射、颈部皮下埋植和口腔灌服外源褪黑素等方式处理经产荷斯坦奶牛,观察血液中褪黑素浓度的变化情况并分析褪黑素在体内的分泌规律及代谢速率。结果显示,在24 h内,经产荷斯坦奶牛血液中褪黑素浓度变化明显,04：00浓度最高,为8.30 ng/mL,16：00浓度最低,为2.08 ng/mL。皮下埋植和口腔灌服外源褪黑素以后,血液中褪黑素浓度升高幅度较小;皮下注射46.4、4.64和0.464 mg褪黑素后,血液中褪黑素浓度迅速升高,1 h后达到峰值,分别为561.94、487.03、92.89 ng/mL,极显著高于注射前（P<0.01）;静脉注射46.4、4.64和0.464 mg褪黑素后,血液中褪黑素浓度迅速升高,0.5 h之后达到峰值,分别为767.68、639.19、110.56 ng/mL,极显著高于注射前（P<0.01）,高水平褪黑素可稳定持续48 h。本研究证明了荷斯坦奶牛体内褪黑素分泌有明显的昼夜节律性,静脉或皮下注射外源褪黑素可以迅速提高血液中褪黑素的浓度,且血液中褪黑素的代谢速度与褪黑素的浓度高度相关,随着血液中褪黑素浓度的增加,褪黑素的代谢速度明显加快。     

Effects of Different Melatonin Injection Ways on the Enrichment and Metabolism of Blood Melatonin in Holstein Cows

In order to exploring the change pattern of melatonin in blood under the normal conditions of the multiparous Holstein cows within 24 h,the change of melatonin in blood of Holstein cows after the intravenous injection,subcutaneous implantation in neck,neck subcutaneous injection and drench were studied and the melatonin secretion rule and its metabolic rate were analyzed. The results showed that melatonin secretion changes obviously in the blood within 24 h,the concentration peak appeared at 04:00 (8.30 ng/mL),lowest at 16:00 (2.08 ng/mL). After subcutaneous implantation and drench,the concentration of melatonin in the blood rose relatively flat compared to other groups. Subcutaneous injection of 46.4, 4.64 and 0.464 mg melatonin,the concentration in the blood rose rapidly, the peak appeared at 1 h after the injection,which were 561.94,487.03,92.89 ng/mL,respectively,extremely significantly higher than pre-injection (P<0.01).Intravenous injection of 46.4,4.64 and 0.464 mg melatonin,the concentration in the blood also rose rapidly,the peak appeared at 0.5 h after the injection,which were 767.68,639.19,110.56 ng/mL,respectively,extremely higher than pre-injection (P<0.01).This study proved that the secretion of melatonin in the Holstein cows had obvious circadian rhythm,intravenous or subcutaneous injection of melatonin could improve the concentration of melatonin rapidly and there was a high correlation between the melatonin metabolic rate and the melatonin concentration in the blood,with the increase of the concentration of melatonin,the metabolism velocity was accelerated obviously.     

Identification of sperm morphometric subpopulations in cooled‐stored canine sperm and its relation with sperm DNA integrity

The aims of this study were to (i) identify different morphometric subpopulations in cooled‐stored canine sperm and their patterns of distribution during cool‐storage for up to 240 hr and (ii) determine whether or not morphometric sperm subpopulations (sP) are related to sperm DNA integrity. For that purpose, morphometric parameters were analysed by computer‐assisted sperm analysis (CASA) and sperm DNA fragmentation (sDFi) using the sperm Halomax test. Four morphometric sperm heads subpopulations were identified: sP1 (large and rounded), sP2 (large and elongated), sP3 (small and rounded) and sP4 (small and elongated). sP1 was the most predominant subpopulation for up to 72 hr and thereafter sP3 increased progressively. sDFi increased after 48 hr of cool‐storage. Although sP3 showed a positive correlation with sDFi, and both increased over time, it could not be ensured that only the sperm with fragmented DNA are accumulated in sP3. In conclusion, sP3 and DNA fragmentation increased progressively during cool‐storage, becoming possible indicators of sperm damage. However, it cannot be concluded that sP3 only contains sperm with fragmented DNA.     

Comparative evaluation of DNA integrity using sperm chromatin structure assay and Sperm‐Ovis‐Halomax during in vitro capacitation of cryopreserved ram spermatozoa

This study aimed to compare the ability of sperm chromatin structure assay (SCSA^®) and Sperm‐Ovis‐Halomax^® to detect DNA fragmentation in frozen‐thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF‐ESS) at multiple time points (0–240 min). Incubation in SOF‐ESS had no significant effects on SCSA^® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm‐Ovis‐Halomax^® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA^® and Sperm‐Ovis‐Halomax^® may quantify different types of DNA damage in ram spermatozoa under these experimental conditions.     

Relationship between ATP content and motility in bovine spermatozoa with reference to the effects of the bull and the A.I. centre.

Deep-frozen semen from 28 bulls belonging to 6 different A.I. centres was studied after thawing and the ATP content in the spermatozoa was assayed using a bioluminescence technique. The sperm motility was subjectively estimated under a phase contrast microscope and the sperm concentration of each ejaculate was calculated in a haemocytometer. The overall mean ATP content was 16.6 nmoles ATP/spermatozoa x 10(8). There was a significant variation in ATP content between A.I. centres. Significant differences between bulls in ATP content were found as well as a significant correlation between ATP concentration and the number of motile spermatozoa. This may indicate that ATP assessment may be useful as an additional, objective laboratory test.