Fertility of Deep Frozen Boar Spermatozoa

In the present investigation the results of two insemination trials with deep frozen boar spermatozoa are presented. The aim of the trials was to study the effect of different thawing diluents and to compare the fertility of deep frozen spermatozoa from four boars. The trials utilized a total of 139 gilts. The thawing diluents used were boar seminal plasma, protein free seminal plasma, the thawing diluent OLEP and isotonic glucose solution. The composition of OLEP was based on physical and biochemical analyses of boar seminal plasma. The electrolyte levels, pH and osmotic pressure of OLEP are similar to those of boar seminal plasma. From the results it is evident that thawing in boar seminal plasma, protein free seminal plasma and OLEP yielded equal results. Thawing in isotonic glucose solution yielded significantly poorer results concerning percentage of fertilized ova 24–48 hrs. after insemination and almost significantly poorer fertility results four weeks after insemination. The possible effects of the thawing diluents are discussed. With the freezing procedure applied, electrolyte levels, pH and osmotic pressure seem to be factors of importance for the survival of the frozen and thawed spermatozoa and for the maintenance of their fertilizing capacity. Almost significant differences were found in fertility of spermatozoa from different boars. These differences were reflected in pregnancy rates as well as ratio of foetuses to c. 1. in pregnant gilts. The differences were found to be independent of thawing diluent. The variation seems to be caused by differences in resistance of the spermatozoa to the freezing and thawing procedure. The need for laboratory methods for selection of boars with spermatozoa of good freezability is stressed.     

On the Fertility and Survival of Deep Frozen Boar Spermatozoa Thawed in Skim Milk

Satisfactory conception rates of deep frozen boar spermatozoa were obtained, with insemination by way of the cervix, after thawing the deep frozen spermatozoa in boar seminal plasma, both in preliminary trials (Crabo & Einarsson 1971, Crabo et al. 1972 b) and in a large field trial (Einarsson et al. 1972). Fertility with pellet frozen boar spermatozoa, thawed without dilution, was reported by Graham et al. (1971 a, b) and Pursel & Johnson (1971).     

Influence of Boars on the Relationship between Fertility and Post Thawing Sperm Quality of Deep Frozen Boar Spermatozoa*

The aim of this investigation was to evaluate the possibility of selecting boars for deep freezing by means of laboratory tests on frozen-thawed spermatozoa. Thirty-one randomly selected frozen ejaculates from four boars were investigated by a thermoresistance test after thawing in boar seminal plasma and in OLEP. Extracellular ASAT activity was measured in samples from 30 of the ejaculates after thawing in OLEP and in isotonic glucose solution. Twenty of the ejaculates were utilized for fertility tests by artificial insemination of 37 gilts preceding the laboratory investigation. Three of the boars proved fertile with frozen semen. One of these boars seemed to yield superior fertility to the other two boars. No fertility was obtained with frozen spermatozoa from the fourth boar. Prior to the freezing trial this boar had been used for fresh semen inseminations giving higher pregnancy rates than the average of Swedish A.I.-boars. This boar was therefore considered a case of “low freezability”. In the laboratory tests the samples from this boar showed the lowest motility after 3 hrs.’ storage at 37°C, the highest relative decrease of motility during the thermoresistance test, the highest release of ASAT after thawing in OLEP and the highest relative release of ASAT. Analyses of variance indicated significant and almost significant variation among boars in relative decrease of motility during the thermoresistance test and in relative release of ASAT. The results indicate that the boars were the main cause of variation in fertility as well as in outcome of the laboratory tests. These results do not permit a complete evaluation of the relationship between fertility and outcome of the applied laboratory tests. However, the results indicate a possibility of detecting boars producing spermatozoa with low freezability by means of laboratory tests.     

III: Ultrastructure of Boar Spermatozoa Frozen Ultra-Rapidly at Various Stages of Conventional Freezing and Thawing

Ejaculated boar spermatozoa subjected to a conventional freezing and thawing process, were ultra-rapidly fixed, freeze-substituted and examined by electron microscopy to monitor the presence of real or potential intracellular ice and the degree of cell protection attained with the different extenders used during the process. Numerous ice crystal marks representing the degree of hydration of the cells were located in the perinuclear space of those spermatozoa not in proper contact with the extender containing glycerol (i.e. prior to freezing). The spermatozoa which were in proper contact with the extenders presented a high degree of preservation of the acrosomes, plasma membranes as well as the nuclear envelopes. No ice marks were detected in acrosomes before thawing, indicating that the conventional assayed cryopreservation method provided a good protection against cryoinjury. The presence of acrosomal changes (internal vesiculization, hydration and swelling) in thawed samples however, raises serious questions about the thawing procedure employed.     

Deep Uterine Insemination of Cattle: A Fruitful Way Forward with Smaller Numbers of Spermatozoa

After describing the site of fertilisation and that of the functional sperm reservoir in the female tract, proposals are made concerning a modified site of sperm deposition in cattle. By means of a deep pre-ovulatory insemination into the ipsilateral uterine horn, the chances should be raised of establishing viable spermatozoa in the isthmus where they would undergo a form of physiological encapsulation and storage. Release and activation of such spermatozoa would be prompted by imminent ovulation. Potential advantages of this approach include those of raising the overall fertility of genetically valuable bulls whose non-return rates are sub-optimal; reducing the number of spermatozoa in each insemination dose; using effectively the limited numbers of sex-selected sperm cells (X and Y chromosome bearing spermatozoa) currently available from flow cytometry. Putative disadvantages might include rectal palpation of the ovaries to locate the pre-ovulatory follicle; perforation of the uterine wall by the deep insemination catheter; risk of Polyspermie fertilisation; and the inappropriateness of the technique for non-clinically qualified inseminators. Each of these reservations is responded to in a rational manner. Given a change of attitude, a modified technique of insemination would be feasible under commercial conditions and might give a welcome boost to a sagging artificial insemination industry. kw|Keywords|k]uterus; k]cow; k]fertilisation; k]sperm reservoir; k]Fallopian tube; k]isthmus; k]polyspermy; k]glycoproteins     

Viability and Acrosome Staining of Bull, Boar and Rabbit Spermatozoa

A practical and reliable staining procedure was developed to distinguish the viability and acrosomal status of bull, boar and rabbit spermatozoa. The first stain with trypan blue or Congo red is rapid and avoids artifacts. This stain is precipitated by neutral red during the 2 min required for fixation. The precipitate gives a high contrast black color, resistant to the subsequent rinsings and persists during the time required for staining the acrosome with Giemsa. Ten classes of spermatozoa are distinguished (live or dead with intact acrosomes, loose acrosomes, damaged acrosomes, no acrosome, or with no acrosome and no postacrosomal ring). The intact acrosomes are purple, the loose acrosomes are dark lavender and the damaged acrosomes are pale lavender. The anterior part of the head of live spermatozoa with no acrosome is white or light pink and the same area of dead spermatozoa is white or pale gray. The postacrosomal ring is red. The postacrosomal area of the head of live spermatozoa is white or light pink and the same part of dead spermatozoa is black, dark violet or gray. The procedure did not give satisfactory results for stallion spermatozoa.     

Isolation and Characterization of Membrane Vesicles from Human and Boar Spermatozoa: Methods Using Nitrogen Cavitation and Ionophore Induced Vesiculation

Abstract

A method for isolation of plasma membrane vesicles from human and boar spermatozoa using nitrogen cavitation is described. The purity of the preparations were assessed by electron microscopy, marker enzyme assay and the sedimentation characteristics of fused plasma membrane-acrosomal membrane vesicles in sucrose gradients. PAGE-SDS profiles of plasma membrane polypeptides from boar spermatozoa were significantly different from those of human spermatozoa. Differences in electrophoretic profiles of polypeptides from different regions of the spermatozooon were also observed.     

Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season

In the spermatozoa of Asterias amurensis , patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate. The type II spermatozoa were also immotile and the respiratory rate remained quite low for about 5 min after the dilution. Thereafter, they spontaneously became motile and the respiratory rate increased. The type III spermatozoa became motile upon their dilution and exhibited high respiratory rate. Differences in the motility and respiratory rate of spermatozoa among batches probably result from degree of their maturation. In moving spermatozoa, the ADP and AMP levels increased at the expense of ATP. 2,4-Dinitrophenol elevated the respiratory rate only in immotile spermatozoa, which showed a high ATP level and quite low ADP level, but did not made them motile. Oligomycin inhibited the respiration of both motile and immotile spermatozoa. Probably, the respiratory rate is made low by a shortage of ADP in immotile spermatozoa and is enhanced by ADP production due to the initiation of their movement.     

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

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

Initiation of Sperm Motility Induced by Cyclic AMP in Hamster and Boar

When the plasma membrane of hamster and boar spermatozoa was extraced by treatment with Triton X-100 and the demembranated spermatozoa were transferred to a reactivating medium containing only ATP, axonemes were initially immotile, and then gradually became motile. Under these experimental conditions, the cAMP content in the reactivating medium increased soon. This suggests that cAMP is synthesized from ATP by adenylate cyclase involved in incompletely removed or solubilized residual sperm membrane and that the autosynthesized cAMP causes the delay in motility initiation. This delayed initiation of motility did not occur when phosphodiesterase was added to the reactivating medium and the phosphodiesterase-dependent quiescent sperm became motile instantaneously at any time when excess cAMP was supplemented. Furthermore, demembranated sperm which were diluted in the reactivating medium containing ATP and cAMP, immediately became motile. cAMP levels in the cell increased during the initiation of sperm motility in both species. These results suggest that cAMP is the real factor indispensable for the initiation of sperm motility at ejaculation in mammals.     

外源激素诱导大熊猫发情排卵及受孕效果

圈养的适龄雌性大熊猫,通常每年会出现发情特征显著、一般、不显著等行为表现;但也有少数育龄雌性大熊猫有的年度根本就小发情。据本研究中心1991～1999年对16只育龄雌性大熊猫的59次发情行为统计,有19次发情行为不显著或一般;有3次属不正常发情,发情行为不显著或不正常所占的比例较大(约占35％)。     

Inactivation of Salmonellae on Chilled and Deep Frozen Broiler Carcasses by Irradiation

Chilled and deep-frozen broiler carcasses were examined for total counts of salmonellae using pooled samples taken from 76 batches each of five birds. By means of a most probable number technique (MPN) it was found that counts expressed/100 g of skin or /500 ml of thaw water varied between < 2 and 1400 with 90% being < 100. Irradiation of carcasses using a dose of 250 krad was found to be highly effective in destroying salmonellae whether the birds had been chilled or deep-frozen.     

度他雄胺对大鼠附睾精子成熟和生育的影响

目的通过度他雄胺对大鼠附睾精子和生育的影响,探索调节雄性生育的睾丸后作用靶点。方法使用度他雄胺20和40 mg/(kg.d)大鼠灌胃给药,连续2周。给药结束后雄雌鼠按1∶2合笼,计算生殖指数;采用计算机辅助精子分析系统分析精子活力和形态;采用SYBR-14和PI双重荧光染色计算精子存活率;采用Elisa法测定大鼠睾酮(T)和双氢睾酮(DHT)血清浓度;采用HE染色法对各组睾丸、附睾进行组织学分析。结果度他雄胺低、高剂量组双氢睾酮浓度均显著下降,分别为0.54和0.28 nmol/L(P<0.01),精子活力明显降低,分别为39.0%和28.7%(P<0.01),畸形率分别增加为10.3%和15.6%(P<0.05),最后受孕率分别降为62.5%和38.4%。而睾酮水平和交配指数均无明显变化(P>0.05),睾丸和附睾亦无明显病理学改变。结论度他雄胺通过抑制DHT生成,影响附睾精子成熟而导致大鼠不育,为今后男性避孕和不育药物研发提供了新思路。     

Interrelations between Time Intervals of a Kinetocardiogram and a Pulsogram

Kinetocardiography and pulsography (sphygmography) of the radial artery were used to determine the phase structure (time intervals) of the cardiac cycle, and the data obtained with the two methods were compared. This analysis showed that the time intervals of the cardiac cycle (such as protodiastole, isovolumetric relaxation, and atrial systole) can be determined more accurately from the first- and second-order derivative curves of the pulse wave than from the real-time kinetocardiogram. Hence, the pumping function of the heart can be assessed more correctly with radial artery pulsography.     

Equilibrium between Genetic Drift and Migration at Various Mutation Rates: Simulation Analysis

Rates of approach to equilibrium values of F _ST /R _ST at various mutation rates and using different mutation models (K-allele model KAM and stepwise model SMM) were analyzed numerically for the finite island model and the one-dimensional stepping stone models of migration, using simulation. In the island model of migration and the KAM mutation model, the rate of approach to the equilibrium F _ST value was appreciably higher and the equilibrium value was almost twofold lower at μ (mutation rate) = m (migration rate) than at μ ≪ m. In the one-dimensional stepping stone model of migration and the KAM model of mutation, the mutation rate significantly affected both the rate of approaching F _ST equilibrium and the equilibrium value. In both island and one-dimensional stepping stone models and SMM, R _ST was not influenced by various mutation rates. The rate of approach to the equilibrium values of both F _ST and R _ST was lower for the stepping stone model than to the island model. R _ST was rather resistant to deviations from the SMM mutation model. __________ Translated from Genetika, Vol. 41, No. 9, 2005, pp. 1283–1288. Original Russian Text Copyright ? 2005 by Efremov.     

II: Effect of Cooling Rate and Duration of Freezing Point Plateau on Boar Semen Frozen in Mini- and Maxi-Straws and Plastic Bags

The post-thaw motility and the acrosome integrity of semen from 4 boars frozen with a programmable freezing machine, in mini (0.25 ml) and maxi (5 ml) plastic straws and in 10 × 5 cm TeflonR FEP-plastic bags (0.12 mm thick, 5 ml), were compared. The freezing of the semen was monitored by way of thermocouples placed in the straws and the bags. Three freezing programmes were used, namely A: from + 5° C, at a rate of 3° C/min, to −6° C, held for 1 min at –6° C, and followed by a cooling rate of 20° C/min to −100° C; B: a similar curve except that there was no holding time at −6° C and that the cooling rate was 30° C/min, and C: from +5°C to −100° C, with a cooling rate of 35° C/min, followed by storage in liquid N2. Despite the treezing curve assayed, both the mini-straws and the bags depicted much shorter freezing point plateaus as compared to the maxi-straws. Post-thaw sperm motility as well as the amount of normal apical ridges were equally significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. Significant differences in these post-thawing parameters were obtained between the freezing curves used. The stepwise freezing procedure A appeared as the best alternative for boar semen, considering this in vitro evaluation.     

Fertility comparison between breeding at 24 hours or at 24 and 48 hours after collection with cooled equine semen

It has become a common practice in the equine breeding industry to send 2 insemination doses for breeding with transported cooled semen, one to be used for the initial insemination upon arrival, and the other to be held a second insemination the next day. One fertile stallion and 36 fertile mares were used to determine if breeding once with 1 dose of semen cooled for 24 h would improve fertility compared with breeding twice, 1 d apart, with half the dose of semen cooled for 24 h on the first day of breeding and half cooled for 48 h on the second day of breeding. Mares were given two intramuscular injections of 10 mg PGF2 alpha 14 d apart. Following the second injection, mares were teased with a stallion and their ovaries were scanned by transrectal ultrasonography daily. When a dominant follicle (> 35 mm diameter) was detected, 1500 units hCG were injected intravenously, and the mares were inseminated. Semen was collected in advance of anticipated breeding, mixed in nonfat dry milk solids-glucose extender to a concentration of 25 million sperm/mL, and placed in 2 commercial cooling containers for 24 or 48 h of storage prior to breeding. Mares were randomly assigned to 1 of 2 insemination treatment groups: 1) Group T1 (n = 18), in which mares were inseminated on the day of hCG injection with 500 million spermatozoa cooled for 24 h, or 2) Group T2 (n = 18), in which mares were inseminated on the day of hCG injection with 250 million spermatozoa cooled for 24 h, and again on the following day with 250 million spermatozoa cooled for 48 h. Pregnancy status was confirmed by transrectal ultrasonographic examination at 14 and 16 d after ovulation. Pregnancy rates were the same for both insemination treatment groups (12/18; 67%). There was no advantage to holding half of the insemination dose for rebreeding on the following day.     

Circadian Rhythm Variations in the Adult Rat Induced By Low and High Protein Diets Administered at Various Stages of Development

Hypothalamic tryptophan (TP), serotonin (5HT) and 5-hydroxy-indoleacetic acid (5HIAA), serum tryptophan (free and total), and circulating corticosterone and aldosterone were measured over 24 hr in 60-day-old male rats fed high (60%) and low (6%) protein diets. The diets were administered during specific stages of rat growth: fetal phase and suckling (when the process of mitosis is most rapid), post-lactation, and from the fetal phase through to the adult stage. In all groups, there was a direct correlation between protein intake and hypothalamic and serum TP levels. Remarkable decreases in the amplitude and mesor of the aldosterone rhythm were observed when the hyperprotein diet was administered in the fetal and suckling phases, or from the fetal phase until the adult stages. The rhythm of the serotonin system was significantly altered by a low protein diet: decreased 5-HT amplitude (low protein diet in the fetal stage and suckling), decreased 5-HT amplitude and deranged S-HIAA rhythm (low protein in the post-lactation period), deranged 5-HIAA rhythm (low protein diet throughout the experiment). Lastly, there was a remarkable increase in the corticosterone mesor in rats administered a low protein diet after weaning and in rats constantly given this diet. Growth was greatly inhibited in these two groups of animals, therefore, it cannot be excluded that the high levels of circulating hormone resulted from their debilitated condition.     

Cell Adhesion and Fusion Induced by Insemination in Zona-Free Hamster Eggs: Electrophysiological Monitoring of the Fusion Process

Cell adhesion and fusion were found to occur in zona-free hamster eggs placed in contact and then inseminated. A cytoplasmic fusion occurred in 7% of paired eggs mainly between 10 and 50 min after insemination. All other eggs that failed to fuse adhered tightly to one another within about 1 hr. Electrophysiological monitoring of the fusion process revealed that an electrical coupling (EC) suddenly appears between apposed eggs and completed within 2–15 min as the first step of cell fusion. Periodic hyperpolarizing responses (HRs), which have been found previously in fertilized hamster eggs to reflect a periodic increase in cytoplasmic Ca²⁺ ions, become gradually synchronized and completely coincide in paired eggs 10–30 min after the establishment of EC. Thus the paired eggs become an electrically single cell as the second step. Then the boundary between the eggs breaks down, resulting in formation of a single, spherical egg in several minutes. On the other hand, most of adhered eggs showed neither EC nor synchronization of HRs. Some adhered eggs showed only a low efficiency EC.