Effectiveness of in vitro Methods for Predicting in vivo Fertilizing Capacity of Boar Spermatozoa Cryopresemed with 2% or 4% Glycerol1

Contents: Thirteen ejaculates from each of two boars that were greatly different in post-thaw sperm quality were frozen in either 2 or 4% glycerol (final concentration/split-sample). Frozen-thawed sperm were evaluated in vitro, for motility, acrosomal integrity (NAR), plasma membrane integrity (PMI), and for sperm penetration (SPA) of zona-free hamster eggs. To evaluate in vivo fertilizing capacity, 52 sows were inseminated once, 30–34 hours after onset of estrus. Ova were recovered from the reproductive tract 2–4 days following insemination and evaluated for cleavage and sperm binding. The two boars differed significantly for all in vivo and in vitro parameters. Motility and PMI were higher (P < 0.05) for sperm frozen with 4% than 2% glycerol whereas NAR was higher with 2% glycerol. Higher numbers of sperm were bound to eggs in vivo for sperm frozen with 4% than with 2% glycerol (P < 0.05). The fertilization rate for sperm frozen with 4% glycerol was not significantly higher than the fertilization rate for sperm frozen with 2% glycerol (P = 0.12). On the basis of these data, 4% rather than 2% glycerol may be close to optimum when freezing boar semen in straws. However, more extensive in vivo fertility testing is required to confirm this trend. Low correlations were observed between in vitro (and in vivo) evaluation of sperm quality and fertility when the effect of boar and glycerol concentrations was kept constant. Inhalt: Die Brauchbarkeit von in vitro-Methoden für die Voraussage der in vivo-Befruch-tungskapazität von Eberspermatozoen nach Tiefgefrierung mit 2% oder 4% Glycerin Von 2 Ebern, die sich in der Qualität ihres Auftauspermas deutlich unterschieden, wur-den je 13 Ejakulate mit 2% oder 4% Glycerinanted (Endkonzentration — split sample) tiefgefroren. Die aufgetauten Samenproben wurden in vitro nach Motilität, akrosoma-ler Integrität (NAR), Plasmamembran-Integrität (PMI) und im Spermapenetrationstest (SPA) am zonafreien Hamstereiüberprüft. Zur Beurteilung der Befruchtungskapazität in vivo wurden 52 Sauen 30–34 h nach Brunstbeginn einmalig besamt. 2–4 Tage nach der Besamung wurden die Eier aus dem Genitaltrakt gewonnen und auf Teilung und auf Anheftung von Samenzellen an der Zona pellucida beurteilt. Die beiden Eber wa-ren in allen In vitro- und In vivo-Parametern signifikant verschieden. Motilitat und PMI waren besser (p <0,05) für Spermien, die in 4% Glycerin konserviert waren, NAR bes-ser in 2%igem Glycerin. Nach Konservierung in 4% Glycerin waren mehr Spermien an der Eihülle angeheftet als nach 2% Glycerin (p < 0,05). Aber die Befruchtungsraten waren nach 4% und 2% Glycerinkonservierung praktisch nicht verschieden (p = 0,12). Auf der Grundlage dieser Ergebnisse ist zu folgern, daβ die Tiefgefrierkonservierung von Pelletsperma mit 4% Glycerin günstiger ist als mit 2% Glycerin; dieser Trend muβ jedoch in umfangreicheren In vivo-Fertilitätstests noch bestätigt werden. Wenn die Ef-fekte Eber und Glycerinkonzentration konstant gehalten wurden, dann wurden nur niedrige Korrelationen zwischen In vitro- und In vivo-Beurteilung von Spermaqualität und Fruchtbarkeit erhalten.     

Relationship Between Motility and Membrane Integrity of Boar Spermatozoa in Media Varying in Osmolality

A study was conducted to evaluate the relationship between boar sperm motility and membrane integrity following exposure to media with 150–1120 mOsm. Total sperm motility was defined as the percentage of spermatozoa that had any form of motility was subjectively assessed under a light microscope. Sperm cell damage was expressed as a loss of membrane integrity as measured by a combination of fluorescent stains, carboxyfluorescein diacetate (CFDA) and propidium iodide (PI), and Hoechst 33258 (H33258). There were no significant differences between sperm motility and membrane-intact spermatozoa, as measured by CFDA-PI and H33258, in media with 250 and 300 mOsm. In anisosmotic conditions, a higher amount of membrane-intact spermatozoa than motile spermatozoa was observed. In hypo-osmotic conditions (150 mOsm), a high proportion of spermatozoa had curled or coiled tails and most of them retained their entire membrane integrity, as detected by CFDA-PI. In media with 350–1120 mOsm, some spermatozoa accumulated PI in the head region and CFDA in the mid-piece. These spermatozoa fluoresced blue at the lower region of the head, as detected by H33258. The ATP content in spermatozoa exposed to hypo- and hyperosmotic conditions was markedly reduced. There was no recovery of sperm motility on returning the spermatozoa to isosmotic conditions after 10 min incubation in anisosmotic conditions, indicating that the spermatozoa suffered an almost complete and irreversible loss of motility. This irreversible loss of motility may be a consequence of reduced ATP production in spermatozoa subjected to anisosmotic conditions. The results of this study demonstrate that plasma membrane integrity assessment in combination with sperm motility, using a range of media varying in osmolality, can give valuable information about the status and function of different sperm membranes, which might be relevant for semen preservation.     

Effects of Dilution Rate on the Motility and Acrosome Morphology of Boar Spermatozoa Stored at 15 °C

The objective of this investigation was to establish the optimal extent of dilution for storage of boar spermatozoa at 15°C in Kiev diluent. The dilution titers used for the sperm-rich fraction of ejaculates from 8 boars ranged from 1:2 to 1:50. Seminal doses were stored for 10 days. Motility and acrosome morphology were evaluated after 1, 3, 5, 7, and 10 days of storage. The percentages of motile spermatozoa after 24 and 72 hours of storage were significantly higher for dilution rates between 1:7 and 1:11 than for dilution rates lower than 1:7 or higher than 1:11 (p < 0.05). The percentages of spermatozoa with normal acrosomes after 24, 72, and 120 hours of storage were significantly higher for dilution rates between 1:8 and 1:11 than for dilution rates lower than 1:8 or higher than 1:11 (p < 0.01 ).     

In vitro Fertilizing Capacity of Deep Frozen Boar Semen Packaged in 0.5 and 5 ml Straws

The aim of this study was to evaluate the straw size effect used for freezing on the in vitro fertilizing capacity. Twenty-one ejaculates from seven fertile boars were frozen under controlled conditions in 0.5 and 5 ml straws. Thawed semen was compared to fresh semen. For fresh and thawed semen in 0.5 and 5 ml straws, the results were: 92.18, 77.38 and 79.04% sperm penetration; 80.68, 66.89 and 69.33% monospermy; 11.51, 10.49 and 9.74% polyspermy; 86.19, 47.14 and 47.02% motility and 75.52, 48.19 and 46.81% normal apical ridge (NAR), respectively. Analysis of variance and test of multiple comparisons showed that under the conditions employed, penetration, monospermy, motility and NAR were significantly reduced by freezing–thawing, but polyspermy was much less affected. The results obtained suggest that frozen boar semen is adequate for in vitro fertilization. In addition freezing in 5 ml straws did not have any detrimental effect on either penetration, monospermy, polyspermy, motility and NAR, in comparison with freezing in 0.5 ml straws.     

Assessing in vivo Fertilizing Capacity of Liquid-Preserved Boar Semen According to the 'Hanover Gilt Model'

The goal of this study was to determine the ability of the Hanover gilt model to assess in vivo fertilizing capacity of preserved sperm and to consider whether any modifications to this model were needed. This model evaluates the fertilizing capacity of semen based on the fertilization rate, the rate of normal embryos and the accessory sperm count of 3–5‐day embryos. Its distinguishing characteristics are the use of one‐time insemination of sperm in reduced numbers, of spontaneously ovulating gilts and of ovulation detection through ultrasound examination of ovaries. Reduced sperm numbers allow for an accurate evaluation of the fertilizing potential of different semen treatments, thereby avoiding the compensatory effect of doses calibrated to maximize fertility. The model's usefulness was assessed in a trial run designed to compare the fertilizing capacity of liquid boar semen diluted into two different extenders. The diluent, the boar and the backflow, had no significant effect on any of the parameters studied. Gilts inseminated less than 24 h before ovulation had a significantly higher (p < 0.01) fertilization rate and accessory sperm cell count (p < 0.05) than those inseminated more than 24 h before ovulation. Very good/good embryos from homogeneous litters (only very good/good embryos were present) had a significantly higher (p < 0.01) accessory sperm count than those from heterogeneous litters (at least one embryo was of a different quality and/or oocytes were present). Both very good/good and degenerated/retarded embryos from heterogeneous litters had low accessory sperm numbers. This suggests that accessory sperm count is significantly related to the quality of the litter, but not to the quality of the embryo within gilts. It can be concluded that the Hanover gilt model is sensitive enough to show fertility differences (in this study, those associated with in vivo ageing of semen), while using relatively few gilts and little time.     

精液冷却过程中应激蛋白90对猪精子活动力的影响

公猪的精子对冷休克高度敏感,在迅速冷却时失去活力,称之为“冷休克”,而应激蛋白90(HSP90)外源的低表达阻碍细胞周期和减弱冷休克反应。对受精来说,精子运动性是一个重要的参数,HSP90和精子运动有很大的联系。因此,研究猪精子冷却过程中HSP90与精子运动力的关系是很必要的。Geldanamycin(GA)是一种特效的HSP90抑制剂(抗生素),能快速和选择性地抑制HSP90的活性。研究使用此种抗生素抑制HSP90功能,评价HSP90对猪精子活动力的影响。1材料与方法1.1供试精液试验用猪精液采自长白猪和约克夏猪,采取手握法采集精液。精液过滤、离心后,…     

In vitro Evaluation of the Quality and Fertilizing Capacity of Boar Semen Frozen in 0.25 ml straws

Twenty-two boar ejaculates were frozen in 0.25 ml straws using a controlled cooling rate, then evaluated in vitro in order to assess: (i) the extent to which a range of semen evaluation parameters accurately characterize sperm quality, (ii) the value of quality assessment in the characterization of long-term sperm survival and fertility and (iii) the suitability of the cryopreservation protocol used for yielding semen with good quality and fertilizing capacity. Motility with or without caffeine, plasma membrane integrity (PMI) evaluated with both propidium iodide (PI) and Hoechst 33258, and acrosome morphology were studied, the ejaculates being then classified into five quality groups. A thermoresistance test and a homologous in vitro fertilization test were applied to selected ejaculates of these groups. Caffeine-stimulated motility and PMI evaluated with PI provided better estimations of semen quality than the other tests of motility, PMI, or acrosome morphology, but this quality assessment could not reveal differences in fertilizing capacity or thermoresistance among ejaculates. Over 43% spermatozoa survived cryopreservation in 19 of the 22 ejaculates, with inter-boar and inter-ejaculate variability in the freezing success being observed. The fertilizing capacity, however, was seriously affected by the process regardless of the semen quality. It is concluded that caffeine-stimulated motility and PMI evaluated with PI give accurate information on sperm quality, but important aspects to the valuation of semen such as thermoresistance and fertilizing capacity are not revealed by this quality study. Moreover, the approach of selecting suitable protocols of cryopreservation does not appear to be sufficient for guaranteeing systematically good quality and fertilizing capacity in the frozen-thawed semen.     

In vitro Fertilizing Capacity of Frozen-thawed Bull Spermatozoa Selected by Single-layer (Glycidoxypropyltrimethoxysilane) Silane-coated Silica Colloidal Centrifugation

Barriers to the use of density gradient centrifugation for preparing animal spermatozoa for artificial insemination (AI) include the scarcity of animal-specific formulations and the daunting prospect of processing large volumes of ejaculate in small aliquots (1.5 ml extended semen). Recently, new colloid formulations have been tested in vitro in a modified procedure, centrifugation on a single layer of colloid. The present study investigated the fertilizing ability during in vitro fertilization (IVF) of frozen-thawed bovine spermatozoa following centrifugation through a single layer of glycerolpropylsilane (GS)-coated silica colloid with a species-specific formulation (patent applied for; treatment, T). Controls (C) included centrifugation through gradients of either the same colloid (C1) or Percoll™ (C2). Sperm recovery surpassed 50% for both C1–C2 and T (n.s.). Mean values of various parameters of computerized analysis of sperm motility did not differ between T and C1 (n.s.), and only the proportions of path straightness and linearity were lower in T vs C2 (p < 0.05). In T, the mean (±SD) percentages of fertilization rate, blastocyst development rate and the total number of blastomeres were 58.1 ± 23.3%, 24.5 ± 14.3% and 94.6 ± 23.4%, respectively. The proportions did not differ significantly from controls (C1/C2). Therefore, centrifugation through a single layer of colloid offers an alternative method to density gradient centrifugation for selection of viable, potentially fertile frozen-thawed bull spermatozoa. This single-layer technique is gentle, versatile and convenient because it facilitates scaling-up the process of sperm preparation to allow larger numbers of spermatozoa (for instance, whole ejaculates) to be processed for AI.     

Analysis of In vitro Fertilizing Capacity to Evaluate the Freezing Procedures of Boar Semen and to Predict the Subsequent Fertility

A porcine in vitro fertilization (IVF) system and seminal quality parameters of frozen–thawed boar semen were used to assess the effectiveness of two different thawing rates of frozen boar semen, and to address the question of whether differences between fertility of ejaculates could be predicted in a limited field trial. In the first experiment, two thawing procedures were analysed (37°C, 30 s; 50°C, 12 s) and no differences in sperm quality were found. However, when the procedure was 50°C, 12 s the IVF results showed a higher number of sperm per penetrated oocyte and a near 10 points higher rate of pronuclear formation. In the second experiment, the fertility results obtained in the limited field trial show to be efficient enough for application in a commercial use, especially for three of the employed boars (fertility ≥80%). In this limited study, the conventional seminal parameters are not accurate enough to discriminate good and bad boars in relation to fertility. On the contrary, parameters of in vitro penetrability are more precise to predict subsequent fertilities. As conclusion, the IVF fertilization system seems to be a good tool to evaluate the quality of frozen–thawed boar semen previous to its commercial way, to verify the bank semen storage quality and a good way to assay new sperm freezing procedures, as it is the more precise evaluating method in estimating the potential fertilizing ability.     

鸵鸟卵黄对猪精子冷冻后质量的影响

为了提高猪冷冻精液品质和精子抵抗低温打击的能力,本研究以5%、10%、15%、20%和25%等不同浓度的鸵鸟卵黄作为冷冻保护剂,以20%的鸡蛋卵黄和20%的鸽蛋卵黄为对照,将冷冻-解冻后的精子活率、质膜完整率和顶体完整率作为评价指标,分析鸵鸟卵黄对猪精子的抗冷冻保护作用。结果表明：稀释液中添加20%鸽蛋卵黄时,精子活率、顶体完整率和质膜完整性分别为52.11%、55.62%和54.94%,显著高于其他组（P〈0.05）。虽然稀释液中添加15%鸵鸟卵黄时,冷冻-解冻后精子活率、顶体完整率和质膜完整率显著高于5%、10%、20%和25%鸵鸟卵黄组,但仍然显著低于稀释液中添加20%鸽蛋卵黄处理组。本研究表明,鸵鸟卵黄在冷冻过程中对猪精子具有一定的保护作用,但相对于鸽子蛋和鸡蛋卵黄效果并不理想。     

Relationship Between Motility and Mitochondrial Functional Status in Canine Spermatozoa

Inner mitochondrial membrane potential (IMM) is considered a sensitive indicator for the energetic status and motility of spermatozoa. The relationship between sperm motility parameters evaluated by Computer Assisted Sperm motility Analyzer and plasma membrane integrity and IMM assessed by triple staining (PI/SYBR-14 and JC-1) was evaluated in 10 dogs of unknown fertility. Sperm motility showed large variations ranging from 10% to 98%. Proportion of viable sperm cells and of spermatozoa with high IMM ranged from 74% to 99% and from 53% to 87%, respectively. The presence of a high IMM assessed by JC-1 was more strongly correlated to sperm viability ( r  = 1) than to sperm motility ( r  = 0.778). Our results indicate that JC-1 is suitable for detection of IMM changes in canine spermatozoa, but it should always be associated with an objective motility analysis to avoid incorrect evaluation of potential sperm fertility. Ejaculates with a low motility rate showed an unexpectedly high proportion of sperm with high IMM, suggesting that mitochondrial respiration could not be sufficient to support sperm motility, although it may be important for sperm survival in the female genital tract.     

Distinct Effects of Boar Seminal Plasma Fractions Exhibiting Different Protein Profiles on the Functionality of Highly Diluted Boar Spermatozoa

The aim of this study was to evaluate how different protein profiles of seminal plasma (SP) fractions affect sperm functionality in vitro. Ejaculates from three boars were separated into six fractions. The fractions differed from each other in their sperm content, in their total SP protein content, and their spermadhesin PSP-I/PSP-II and heparin-binding protein (HBP) concentrations. Spermatozoa were mainly recovered in fraction 2 (sperm-rich fraction, >1800 × 10⁶ spermatozoa/ml), whereas the pre-sperm fraction 1 and the post-sperm fractions 4–6 contained low numbers of spermatozoa (<500 × 10⁶/ml). Except in fraction 2, the total SP protein concentration and the concentration of both, spermadhesin PSP-I/PSP-II and the HBPs increased with fraction order. Distinct time-dependent effects were observed on motility characteristics and membrane integrity of highly diluted boar spermatozoa upon incubation with a 10% dilution of the SP from each fraction. The highest sperm viability was recorded after exposure for 5 h to fraction 2, followed by fractions 1 and 3. The percentages of motile spermatozoa also differed significantly among fractions after 5 h of incubation. Spermatozoa incubated with SP of fractions 1–3 showed the highest percentage motility. We conclude that different SP fractions exert distinct effects on the functionality of highly diluted boar spermatozoa. Fractions 1–3 appear to promote sperm survival, whereas fractions 4–6 seem to be harmful for preserving the physiological functions of highly diluted boar spermatozoa.     

鸵鸟卵黄低密度脂蛋白对猪精子冻后品质的影响

在家畜精液冷冻中,卵黄被广泛应用,且其中的低密度脂蛋白(LDL)对精子起主要保护作用。本研究利用含6%、7%、8%和9%鸵鸟卵黄LDL配制的稀释液制作猪细管冷冻精液,分析鸵鸟卵黄LDL对冷冻-解冻后猪精子质量参数的影响。结果表明:在含不同浓度鸵鸟卵黄LDL的稀释液中,8%LDL的稀释液冷冻效果最好,冻后精子活率平均可达52.13%,显著高于其他组(P<0.05);精子顶体完整率平均为58.33%,质膜完整率为72.38%,与其他处理组相比差异显著(P<0.05)。但与鸡蛋卵黄LDL和鸽子蛋卵黄LDL处理组相比,鸵鸟卵黄LDL处理组冷冻-解冻后猪精子质量参数相对较低。本研究表明,虽然鸵鸟卵黄LDL在冷冻过程中对猪精子具有一定的保护作用,但相对于鸽子蛋和鸡蛋卵黄LDL效果并不理想。     

Quality and Fertilizing Ability In Vivo of Sex‐Sorted Stallion Spermatozoa

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex‐sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX^® flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14‐propidium iodide), mitochondrial function (JC‐1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 × 10⁶ X‐bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non‐sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post‐thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.     

Exogenous DNA Uptake of Boar Spermatozoa by a Magnetic Nanoparticle Vector System

The sperm‐mediated gene transfer method is applicable to transgenesis in many species that use spermatozoa for reproduction recently, which has been shown various results. In the current study, we show that transgenic porcine embryos can be efficiently produced by employing a simple transfection method that uses magnetic nanoparticles (MNPs). The complexes formed between plasmid DNA and MNPs were bounded on ejaculated boar spermatozoa at a higher efficiency compared to methods using DNA alone or lipofection. Using confocal microscopy, rhodamine fluorophore‐labelled MNPs were detected on external surfaces of the spermatozoa membrane, which were bounded on zona pellucida of in vitro maturated oocyte during in vitro fertilization. Electron microscopy revealed that clusters of MNPs were detected in inside of plasma membrane and nucleus of the spermatozoa head. Additionally, we found that magnetofected boar spermatozoa could be fertilized with oocytes in vitro and that the resulting gene of green fluorescent protein was detected in fertilized eggs by genomic PCR analysis. Taken together, these results suggest that MNPs can be used to efficiently introduce a transgene into embryo via spermatozoa.     

Motility and Plasma Membrane Integrity of Spermatozoa in Fractionated Stallion Ejaculates after Storage

With the aim of investigating properties of stallion seminal plasma to eventually improve semen-handling techniques, sperm motility and plasma membrane integrity were analysed in different fractions of the ejaculates after storage. Semen was collected using a computer-controlled automated phantom that separates the ejaculates into five successive cups. Samples containing seminal plasma and skim milk extender were compared with samples stored in skim milk extender after the removal of seminal plasma by centrifugation. Fractionated ejaculates were stored cooled for 24 h after dilution with extender (Expt 1) or frozen in liquid nitrogen (Expt 2). In Expt 1, cup 1 was pre-sperm fluid, cups 2 and 3 sperm-rich fractions, and cup 4 sperm-poor fractions. In Expt 2, cups 1 and 2 were sperm-rich fractions, and cups 3 and 4 sperm-poor fractions. One sample (WE) represented the whole ejaculate in both experiments. Motility parameters were determined with a Hamilton-Thorn Motility Analyzer, and plasma membrane integrity was assessed using carboxyfluorescein diacetate and propidium iodide staining and fluorescence microscopy. The removal of seminal plasma lowered motility values, but not plasma membrane integrity, in both experiments. No significant differences between cups were observed after cooled storage. The cups differed significantly in most post-thaw motility parameters, and the sperm-rich fraction showed higher post-thaw motility than the whole ejaculate.     

不同添加剂及剂型对猪冷冻精液品质的影响

试验旨在研究咖啡因、维生素E、超氧化物歧化酶(SOD)及剂型对猪冷冻精液品质的影响。采用添加了不同浓度咖啡因、维生素E、超氧化物歧化酶(SOD)的冷冻稀释液和3种剂型的冻精管进行精液冷冻。3种添加剂对冻精解冻后的精液品质都有一定的促进作用,试验中其添加最适浓度分别为:咖啡因为0.2 mg/mL,维生素E为0.4 mg/mL,SOD为300 IU/mL;0.25mL剂型的冻精解冻后精子品质最好。咖啡因、抗氧化剂及冻精管剂型对冷冻精液的效果有一定的影响。