猪精液液态保存的研究进展

猪的人工授精主要使用液态保存精液 ,此法保存精子的存活率高、功能受损较小、授精繁殖率较高且操作简便。体外液态保存猪精液时 ,不仅要注意猪精子对温度变化敏感的特性 ,还要考虑酸碱度、离子的种类和强度、渗透压、过氧化损伤和微生物污染等对精子的影响 ,要向稀释液中添加各种保护成分 ,维持精子的存活和功能。目前主要是比较和改进各种稀释液 ,建立准确的精子质量评价方法 ,提高受精率     

Boar Semen Studies: II. Laboratory and Fertility Results of a Method for Deep Freezing

A successful method for low temperature preservation of bull semen was modified for use with boar semen and resulted in recovery of twenty to fifty per cent motile cells immediately after thawing. Recovered cells did not survive five hours incubation at 37° C. and no pregnancies resulted following insemination of twenty-four sows and gilts with frozen semen.     

稀释液中添加N-乙酰半胱氨酸对猪精液冷冻保存效果的影响

本文以手握法采集的猪精液为试验材料,以解冻后精子的活率、顶体完整率、低渗膨胀率、运动学参数(VAP、LIN、ALH、BCF)和膜脂质过氧化反应为评定标准,旨在探讨N-乙酰半胱氨酸对猪精液冷冻保存效果的影响。结果表明,在冷冻稀释液中添加1mmol/L的N-乙酰半胱氨酸可以显著地提高解冻后精子的活率和顶体完整率,降低精子的低渗膨胀百分率。在运动学参数方面,只有VAP显著升高,而对其它运动学参数没有影响。在冷冻稀释液中添加N-乙酰半胱氨酸对膜脂质过氧化反应没有影响。     

Cryo-scanning Electron Microscopy Discloses Differences in Dehydration of Frozen Boar Semen Stored in Large Containers

In general, freezing in flat plastic polyethylene terephthalate (PET) bags (FlatPacks) at 50°C/min gives better post-thaw viability, in terms of sperm motility and membrane integrity, than does freezing in plastic maxi-straws, probably owing to differences in cryobiology. To test the hypothesis that this better survival post-thaw relates to the degree of sperm dehydration during freezing, the present study investigated the structure of boar semen in a frozen state using cryo-scanning electron microscopy (cryo-SEM) to compare two different packages (FlatPacks and maxi-straws) for single artificial insemination (AI) doses, and three different freezing rates. The semen was split-sample frozen in maxi-straws or FlatPacks (both holding 5 ml) using 3% glycerol as cryoprotectant. Three freezing rates were applied from −5°C to −100°C, namely 2°C/min, 50°C/min and 1200°C/min, the lattermost by plunging the samples into liquid nitrogen (LN₂). The samples were thereafter fractured into LN₂ and larger areas of extra-cellular, unbound frozen water ('ice lakes') were measured to determine the degree of dehydration of the spermatozoa. These areas decreased in size with an increase in cooling rate, the differences in size being more dramatic for maxi-straws than for FlatPacks. Size of ice lakes was also influenced by location within package in relation to cooling rate, the central values being always smaller in maxi-straws than in Flatpacks (p < 0.05 at 2°C/min and 50°C/min) but not at 1200°C/min, which suggested the FlatPack allows for more homogenous freezing of boar semen.     

Artificial Insemination of Swine: Fecundity of Boar Semen Stored in Beltsville TS (BTS), Modified Modena (MM), or MR-A and Inseminated on One,Three and Four Days After Collection12

Contents: A field trial was conducted on 1,463 farms in The Netherlands to compare the fertilizing capacity of boar spermatozoa extended in Beltsville TS (BTS), Modified (MM), or MR-A, and inseminated in 2896 sows and gilts on the first, third or fourth day following collection. Semen was collected, extended, and stored at 18°C at six different AI centers and inseminated by inseminators on their regular routes. Sows inseminated with BTS and MR-A extended semen had higher farrowing rates than MM (79.3, 77.6 us 50.4, P <.0001), higher total pigs per litter (11.4, 11.1 vs 10.0, P <.0001) and higher total pigs born alive (10.7, 10.5 vs 9.4, P <. 0001). The farrowing rate of gilts inseminated with BTS extended semen was superior to MM (73.5 vs. 50.2%, P <.004), while MR-A gave farrowing rates greater than MM (64.1 vs 50.2%, P =.06). There was no difference in litter data for gilts. Farrowing rates for 1 and 4 day semen were superior to 3 day semen (73.2, 73.8 vs 60.3, P <.0001). The semen inseminated on day 4 contained 6 billion sperm per dose rather than the 3 billion per dose for 1 and 3 day semen. Based on the results of this study, BTS and MR-A are effective diluents for extension and storage of boar semen for use within the same week of collection. In addition, semen extended in either BTS of MR-A and stored and inseminated on the fourth day after collection can give fertility equal to first day insemination if the sperm per insemination dose is doubled. Inhalt: Künstliche Besamung beim Schwein: Fruchtbarkeitsergebnisse von Ebersamen nach Flussigkonservierung mit BTS, MR-A oder modifiziertem Modena-Verdünner (MM) und Besamung am ersten, dritten und vierten Tag nach Samengewinnung In einem in den Niederlanden durchgeführten Feldversuch wurden 3 verschiedene Verdünner-Medien für Ebersamen in vivo verglichen. Die Medien waren Beltsville TS (BTS), modifizierter Modena-Verdünner (MM) und das spanische MR-A-Medium. Insgesamt wurden 2896 Jung- und Altsauen in 1463 Herden mit 1, 3 oder 4 Tage altem Sperma besamt. Der Versuch wurde als “split-sample”-Versuch an 6 Eberstationen durchgeführt, der verdunnte Samen bei + 18°Cgelagert und von den Besamungsbeauftragten im Rahmen ihrer regulären Fahrten eingesetzt. BTS- und MR-A-konserviertes Sperma gab bei Altsauen gegenüber MM höhere Abferkelraten (79,3, 77,6 vs 50,4%, P < 0.0001), eine höhere totale Wurfzahl (11,4, 11,1 vs 10,0, P < 0.0001) und eine höhere Anzahl lebendgeborener Ferkel)10,7, 10,5 vs 9,4, P < 0.0001). Die mit BTS-uerdünntem Samen inseminierten Jungsauen wiesen gegenüber MM höhere Abferkelraten auf(73.5 vs 50,2%, P < 0.004). MR-A-Sperma lag ebenfalls hüher als MM-Samen (641 vs 50,2%, P=0.06). Die Wurfgröβen der Jungsauen reigten keine Unterschiede. Die Abferkelergebnisse nach Besamung mit 1 und 4 Tage altem Samen lagen höher als die des 3 rage alten Spermas (73,2, 73,8 vs 60,3%; P < 0.0001). Die am Tag 4 verwendeten Samenportionen enthielten 6 Milliarden Spermien gegenüber 3 Milliarden der nach 1 bzw. 3 Tagen inseminierten Portionen. Die Ergebnkse dieses Versuches zeigen, daβ BTS und MR-A effektive Verdünnermedien sind und die Lagerung von Eberfrischsperma zum Einsatz innerhalb der Entnahmewoche ermoglichen. Darüber hinaus können mit BTS oder MR-A verdünntes und am Tag 4 nach Gewinnung eingesetrtes Sperma Fruchtbarkeitsergebnisse erzielt werden, die ebenso gut sind wie nach Verwendung des 1-Tage-Spermas, wenn die Spermienzahl pro Besamungsdosis verdoppelt wird.     

Effects of Straw Volume and Equex-STM® on Boar Sperm Quality after Cryopreservation

The present experiments were designed to study the effect of adding the detergent Equex-STM^® to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM^®. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN₂) vapour approximately 3 cm above the level of LN₂ for 20 min and then were plunged into LN₂. Thawing was achieved in warm water at 50°C for 12 s and then was incubated in a 38°C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM^® was added to the freezing extender. There was no difference (p   =   0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM^® was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p   =   0.02, p   =   0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM^® (p   >   0.05). The results of these investigations suggested that Equex-STM^® exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.     

Protocol Optimization for Long‐Term Liquid Storage of Goat Semen in a Chemically Defined Extender

A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of spermatozoa in extenders containing egg yolk or milk. The use of chemically defined extenders will have obvious advantages in liquid storage of buck semen. Our previous study showed that the self‐made mZAP extender performed better than commercial extenders, and maintained a sperm motility of 34% for 9 days and a fertilizing potential for successful pregnancies for 7 days. The aim of this study was to extend the viability and fertilizing potential of liquid‐stored goat spermatozoa by optimizing procedures for semen processing and storage in the mZAP extender. Semen samples collected from five goat bucks of the Lubei White and Boer breeds were diluted with the extender, cooled and stored at 5°C. Stored semen was evaluated for sperm viability parameters, every 48 h of storage. Data from three ejaculates of different bucks were analysed for each treatment. The percentage data were arcsine‐transformed before being analysed with anova and Duncan’s multiple comparison test. While cooling at the rate of 0.1–0.25°C/min did not affect sperm viability parameters, doing so at the rate of 0.6°C/min from 30 to 15°C reduced goat sperm motility and membrane integrity. Sperm motility and membrane integrity were significantly higher in semen coated with the extender containing 20% egg yolk than in non‐coated semen. Sperm motility, membrane integrity and acrosomal intactness were significantly higher when coated semen was 21‐fold diluted than when it was 11‐ or 51‐fold diluted and when extender was renewed at 48‐h intervals than when it was not renewed during storage. When goat semen coated with the egg yolk‐containing extender was 21‐fold diluted, cooled at the rate of 0.07–0.25°C/min, stored at 5°C and the extender renewed every 48 h, a sperm motility of 48% was maintained for 13 days, and an in vitro‐fertilizing potential similar to that of fresh semen was maintained for 11 days.     

Effect of Artificial Insemination Protocol and Dose of Frozen/Thawed Stallion Semen on Pregnancy Results in Mares

Deep intra‐uterine insemination is commonly accepted as a routine procedure for artificial insemination in horses. The motives and principles of deep insemination are well described, but the equipment used may differ. In this trial, the efficiency of two different insemination pipettes for deep intra‐uterine insemination in the mare was compared with insemination into the uterine body using commercially available frozen–thawed semen of two stallions of proven fertility. These inseminations were performed using two different doses. The semi‐flexible Minitube pipette was compared with a newly designed insemination device with a more flexible telescopic insemination catheter (Ghent device). The semi‐flexible Minitube pipette performed better than the newly designed insemination device with respect to pregnancy outcome (p = 0.008). The superiority of deep horn insemination over uterine body insemination was reflected by the better pregnancy rates obtained after deep insemination using the same low doses (30.6% better pregnancy rates) (p = 0.0123).     

Successful Timing of Ovulation Using Deslorelin (Ovuplant®) is Labour-saving in Mares Aimed for Single AI with Frozen Semen

To minimize the number of matings/inseminations, controlled ovulation has been practised since a long time ago. A potent short-term implant, releasing the GnRH analogue deslorelin (Ovuplant((R))) has been used in Australia and North America for several years for hastening the ovulation time in mares, but the product is not registered on the European market. This study was aimed to investigate: (1) ovulation time in mares implanted with Ovuplant when the largest follicle was 42 mm or more in size, (2) repeatability of ovulation time in successive oestruses when treated with Ovuplant, (3) pregnancy rate after single insemination with frozen-thawed semen near ovulation. This study included 11 mares, and altogether 17 timed ovulations. Follicular growth and ovulation were determined by palpation per rectum and by ultrasonography in the morning (at 7:00 hours) every second day until observation of a follicle of at least 42 mm in diameter. Then the mares were re-examined in the afternoon (at 19:00 hours), and an Ovuplant was inserted in the mucosa of the vulva. For detection of ovulation, the mares were palpated and ultrasounded repeatedly from 36-42 h after the insert. The mares were inseminated with frozen-thawed semen once at ovulation. All mares ovulated at 36-48 h after treatment and 94% at 38-42 h after treatment. The six mares that were treated at two oestruses ovulated at 39.9 and 39.7 h, respectively. Five of 11 mares (45.4%), inseminated with frozen-thawed semen at the first oestrous cycle were pregnant day 14-16 after ovulation. Using this protocol, there is no need of palpation/ultrasonography during night hours, and examination at 36 and 41 h after implantation might be enough for estimation of ovulation time.     

Effect of a Gonadotropin-releasing Hormone Vaccine (ImprovacTM) on Steroid Hormones, Boar Taint Compounds and Performance in Entire Male Pigs

The objective of this study, comprising two trials, was to evaluate the effect of a gonadotropin‐releasing hormone (GnRH‐vaccine Improvac^TM; Pfizer Ltd) in a sample of the Swedish pig population. The pigs (n = 120) were assigned to three groups: control (entire male pigs), surgical castration and immunization against GnRH. Surgically castrated pigs did not express detectable levels of either testosterone or estrone sulphate (E1S) in plasma, or androstenone in fat and had lower skatole and indole levels in fat than entire male pigs. Immunization significantly reduced testes weight and bulbourethral gland length, plasma levels of the testicular hormones testosterone and E1S, and fat levels of androstenone, skatole and indole. Skatole levels in plasma were significantly lower than in entire male pigs in the second trial, but not in the first due to overall low skatole levels. All immunized pigs and surgically castrated pigs expressed skatole concentrations in fat below the level of 0.2 μg/g, above which meat is regarded as tainted. In contrast, eight entire male pigs exceeded this level. Indole levels in plasma from immunized pigs were lower than those from entire male pigs. Surgical castration caused lower daily weight gain in the suckling period compared with piglets raised intact, whereas in the post‐weaning period no difference was observed. Immunization resulted in higher feed intake and daily weight gain after the second injection. The estimated lean meat content was improved in comparison with the castrated pigs, but was lower than for entire male pigs. Dressing percentage was lower in immunized pigs than in surgically castrated and entire male pigs. The frequency of skin damage did not differ between immunized and entire male pigs or between immunized and surgically castrated pigs.     

Comparison of Cryoprotective Effects of Lycopene and Cysteamine in Different Cryoprotectants on Bull Semen and Fertility Results

The objectives of this study were to compare glycerol and ethylene glycol at different concentrations as cryoprotectants and lycopene or cysteamine (with/without) as antioxidants in Tris extender for bull semen. Twenty‐four ejaculates were obtained from three bulls. Each ejaculate was split into four equal aliquots and diluted using both of the Tris extenders with glycerol (5% or 7%) or ethylene glycol (3% or 5%). After that, each extenders were split into three equal aliquots and added using both of the cysteamine 5 mm or lycopene 500 μg/ml, and control (without additives). The addition of 7% glycerol with cysteamine, 5% ethylene glycol with cysteamine and 3% ethylene glycol with cysteamine groups gave the lowest CASA motility than the other groups. However, 7% glycerol and 7% glycerol with lycopene resulted in a better rate of CASA progressive motility compared with that of other groups. Generally, all the lycopene groups signed better protective effects on acrosome and total morphology than the other groups. Glycerol 7% and 3% ethylene glycol with lycopene groups yielded to slight higher percentages of membrane integrity assessed by HOST than that of the other groups, but 7% glycerol with cysteamine and 3% ethylene glycol with cysteamine showed the worst percentages of membrane integrity. Glycerol 7% and 5% glycerol with lycopene gave rise to a higher value of VAP, VSL and VCL compared with that of the other groups. On the contrary, adding to 5% glycerol with cysteamine showed negative effect for VAP, VSL, VCL and ALH values. All cryoprotectant groups with lycopene decreased chromatin damage than the other groups. Ethylene glycol 3% led to lower non‐return rates of inseminated cows. However, this result was not considered to be statistically important.     

Use of Cholesterol‐Loaded Cyclodextrin in Donkey Semen Cryopreservation Improves Sperm Viability but Results in Low Fertility in Mares

The use of cholesterol‐loaded cyclodextrin (CLC) on semen cryopreservation has been related with better sperm viability in several species; however, the effect on fertility is not known in donkey semen. Ejaculates (n = 25) from five donkeys were diluted in S‐MEDIUM with 0, 1, 2 or 3 mg of CLC/120 × 10⁶ spermatozoa. Semen was frozen, and thawed samples were evaluated by computer‐assisted sperm analyser system (CASA), supravital test, hyposmotic swelling test and fluorescent dyes to assess the integrity of sperm membranes. Mares (n = 60) were inseminated with frozen‐thawed semen treated with the doses of 0 or 1 mg CLC. Percentages of sperm with progressive motility and with functional plasma membrane were greater (p < 0.05) in the CLC‐treated groups than in the control. Percentages of intact plasma membrane and intact plasma membrane and acrosome detected by fluorescent dyes were also greater (p < 0.05) in CLC‐treated groups. Although no difference (p > 0.05) in conception rates was detected between groups (control, 3/30, 10%; CLC‐treated, 1/30, 3.3%), fertility was low for artificial insemination programs in mares. Therefore, we firstly demonstrated that frozen semen treated with CLC in S‐MEDIA extender before freezing improves the in vitro sperm viability, but semen treated or not with CLC in S‐MEDIUM extender results in a very low conception rate in mares inseminated with thawed donkey semen.     

Role of Ca2+ in corticosterone-induced muscle growth retardation

The increased plasma glucocorticoid concentration in stressful conditions stimulates muscle protein degradation, and results in growth retardation in animals. However, the mechanism is still to be clarified. The present study was undertaken to examine the participation of Ca²⁺ in the glucocorticoid action, using nifedipine (NIF), a Ca²⁺ channel antagonist. The effects of NIF on growth, differentiation, and protein degradation were examined in glucocorticoid-treated primary cultured chick muscle cells. Muscle cell growth and cell differentiation were assessed by protein content and creatine kinase (CK) activity, respectively, and the rate of myofiblillar protein degradation was estimated by the release of N ^τ-methylhistidine (MeHis). Creatine kinase activity was increased by corticosterone (CTC) and this effect was minimized by NIF. Protein content was decreased by CTC and normalized by NIF. N ^τ-methylhistidine release was significantly increased by CTC and tended to be minimized by NIF. The present results indicate that CTC increases skeletal muscle proteolysis followed by muscle growth retardation partially because of enhanced Ca²⁺ influx through the NIF-sensitive Ca²⁺ channel. Enhanced muscle differentiation by CTC is mediated also by the NIF-sensitive Ca²⁺ channel.     

临沂市奶山羊鲜精人工授精技术应用

临沂市具有较好的农业资源,适合发展奶山羊养殖业。但是,目前奶山羊传统的自然交配繁育速率较慢,且受胎率低,不利于奶山羊养殖业的快速发展。近年来,由于人工授精技术具有增强繁殖速率、提升受胎率、提高种公羊利用率和降低成本等优点,在畜牧养殖业中的运用和推广十分广泛。但是人工授精的成功率受到母羊发情期、精液采集和保存、输精方式等多种因素的影响,因此增强对该技术的了解,对促进当地奶山羊的良种繁育具有重要意义。     

Artificial Insemination with Frozen Semen in Ewes at Different Times of the Breeding Season

Totally 13575 ewes of two different breeds, Dala and Spel, were inseminated with semen, frozen in straws and thawed at 70°C for 8 sec. An insemination dose of 0.2 ml containing approx. 150 × 10⁶ spermatozoa with at least 45 to 50% progressive motility was imerted 5 to 12 mm into the cervix. The insemination was performed once between 12 and 30 h after the onset of heat. The NR rate of the Dala ewes increased significantly during the season. The NR rate of the ewes inseminated before 15. November was 44.3%, from 15. to 20. November 52.2%. from 20. to 25. November 55.3% and from 25. November and later 61.4%. The corresponding values for the ewes of the Spel breed were 57.3, 58.7, 61.5 and 71.0% respectively, and only the difference between the two last values was statistically significant. The difference between the fertility of the two breeds was significant within each of the periods .     

Preservation of canine and feline corneoscleral tissue in Optisol® GS

Objective The objective of the research was to determine whether preservation of corneal tissue of dogs and cats in Optisol^® GS (OGS, Bausch & Lomb Surgical, Irvine, CA, USA) is feasible for subsequent use in penetrating keratoplasty. Animals The study subjects were 33 dogs and 31 cats with no gross corneal pathology, which had been euthanised by pentobarbital overdose for reasons unrelated to this project. Procedure One cornea of each pair was evaluated immediately and the other was evaluated after storage in Optisol^® GS for either 5, 10, 15 or 20 days. The most important criterion was the preservation of the endothelial cell layer. Results Corneoscleral tissue of cats survived longer, when preserved in Optisol^® GS at 4 °C, than that of dogs. Light and scanning electron microscopy revealed good preservation of the endothelial cell layer for up to 10 days in dogs and up to 15 days in cats.