Effect of heterologous seminal plasma and semen extenders on motility of frozen-thawed ram spermatozoa

Ram seminal plasma increases the fertility of frozen-thawed ram spermatozoa deposited into the cervix. The aim of the current study was to compare the effect of ram seminal plasma to that of bull seminal plasma, dog prostatic fluid, protein-free TALP TrilEq (Triladyl with 0.5 mt of Equex STM paste added to each 100 mt) and heat-treated skim milk on longevity and percentages of progressively motile and aberrantly motile frozen-thawed ram spermatozoa. Three ejaculates from each of 6 rams were extended in TrilEq, pooled and frozen in straws as a single batch per ram. One hundred and eight straws (3 straws from each ram for each fluid) were thawed in random order. Once thawed, a straw was emptied into a tube with 0.85 ml of the appropriate fluid at 37 degrees C and kept at that temperature for 6 h. Motility was assessed at x200 magnification immediately (time zero) and 2, 4 and 6 h after thawing. Progressive motility decreased from each time to the next (P < 0.05) and was 39.0 % (0 h), 26.0 % (2 h), 19.6 % (4 h) and 12.6 % (6 h); SEM 1.24, n = 108 for each group. Ram seminal plasma resulted in higher progressive motility than bull seminal plasma, lower than milk, and similar to the other fluids. Ram seminal plasma resulted in lower aberrant motility than protein-free TALP and similar aberrant motility to other fluids. The effect of ram seminal plasma and dog prostatic fluid was very similar. The effect of ram seminal plasma on the fertility of frozen-thawed ram spermatozoa deposited into the cervix is not due an exceptionally beneficial effect on the motility of spermatozoa.     

Addition of Seminal Plasma to Post‐thawing Equine Semen: What is the Effect on Sperm Cell Viability?

Effect of seminal plasma addition after thawing on viability or cryocapacitation is not definitively established. This experiment was performed to verify the effect of adding seminal plasma, autologous or homologous (from an animal with good semen freezability). Five ejaculates from each of four stallions with proven fertility were collected and cryopreserved. The semen was subsequently thawed and divided into the following three treatment groups: no seminal plasma addition after semen thawing (NOSP); the addition of homologous seminal plasma after semen thawing (HSP) and the addition of autologous seminal plasma after semen thawing (ASP). The addition of 20% of seminal plasma led to an increase in the cell population that simultaneously show plasma and acrosomal membrane integrity (p < 0.05). The addition of seminal plasma did not alter the total motility, the amount of cells with mitochondrial membrane potential or the sperm velocities (average path velocity, straight-line velocity and curvilinear velocity). However, the beat/cross-frequency, straightness and linearity were reduced in ASP and HSP groups (p < 0.05). Unexpectedly, the addition of homologous seminal plasma reduced the proportion of cells with progressive motility (p < 0.05) and the addition of autologous seminal plasma reduced the amplitude of the lateral head displacement (p < 0.05). Based on the increase in the cell populations that had the plasma and acrosomal membrane integrity simultaneously identified in this study, we proposed that the addition of seminal plasma (autologous or homologous) into post-thawed semen before insemination could increase semen fertility.     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Effect of Post‐Thaw Addition of Seminal Plasma on Motility,Viability and Chromatin Integrity of Cryopreserved Donkey Jack (Equus asinus) Spermatozoa

Pregnancy rates in donkeys after artificial insemination with cryopreserved semen are still low, compared to the horse species. Addition of autologous seminal plasma to frozen‐thawed semen appeared to improve pregnancy rates. The aims of this study were to evaluate (1) sperm motility and plasma membrane integrity after thawing (T0) and after one and 2 h (T1 and T2) of post‐thaw incubation in either 0% (SP0) or 70% (SP70) autologous seminal plasma and (2) sperm motility, plasma membrane integrity and DNA quality (%COMP‐αt) after thawing (T0) and after 2 and 4 h (T2 and T4) of post‐thaw incubation in either 0% (SP0), 5% (SP5) or 20% (SP20) homologous seminal plasma. In experiment 1, seminal plasma decreased total and progressive sperm motility and plasma membrane intact spermatozoa immediately after dilution and at all following time points (p < 0.05). In experiment 2, total and progressive motility did not differ between treatments immediately after dilution and between SP0 and SP5 at T2, while they were lower in both SP5 and SP20 than in SP0 at T4. Plasma membrane intact sperm cells did not differ between SP0 and SP5 and were lower in SP20 at all time points. DNA quality was not affected by treatment immediately after dilution and was significantly worse for SP20 after 4 h of incubation (p < 0.05). The post‐thaw addition of seminal plasma at the tested concentrations did not improve donkey frozen semen characteristics in vitro over time.     

Ram seminal plasma and fertility: results from an ongoing field study

The effects of partial replacement of ram semen diluent with ram seminal plasma on the fertility of ewes were studied. Crossbred Chios ewes (n = 152) were assigned to six groups. The oestrous cycles of the ewes were synchronised at the peak (Groups A, B, C and D) and at the end (Groups E and F) of the breeding season by means of intravaginal sponges impregnated with fluorogestone acetate (FGA) for 14 days. Four hundred IU of PMSG were injected intramuscularly at the time of sponge removal. Ewes of Groups A, C and E were artificially inseminated with ram semen diluted with skim milk extender, while those of Groups B, D and F with ram semen diluted with 50% skim milk and 50% ram seminal plasma. The addition of ram seminal plasma induced a significant increase (P < 0.05) in litter size in Groups B and D when compared with that of Groups A and C (1.85 and 1.88 vs. 1.39 and 1.52, respectively). This increase was not significant when insemination was performed at the end of the breeding season (2.0 vs. 1.4). These results indicate that the addition of seminal plasma can influence the fertility of ewes or the fertilising capacity of extended ram semen to some extent.     

Successful Low Dose Insemination of Flow Cytometrically Sorted Ram Spermatozoa in Sheep

The fertility of ram spermatozoa that had undergone flow cytometric sorting (MoFlo SX) and cryopreservation was assessed after low-dose insemination of synchronized Merino ewes. Oestrus was synchronized with progestagen-impregnated pessaries, PMSG and GnRH treatment. Ewes (n = 360) were inseminated with 1 x 10(6), 5 x 10(6) or 15 x 10(6) motile sorted frozen-thawed (S(1), S(5), or S(15) respectively) or non-sorted frozen-thawed (C(1), C(5) or C(15) respectively) spermatozoa from three rams. An additional group of ewes were inseminated with 50 x 10(6) motile non-sorted frozen-thawed spermatozoa (C(50)) to provide a commercial dose control. The percentage of ewes lambing after insemination was similar for C(50) (24/38, 63.2%), C(15) (37/54, 68.5%), S(15) (38/57, 66.7%), S(5) (37/56, 66.1%) and S(1) (32/52, 61.5%) groups (p > 0.05), but lower for C(5) (19/48, 39.6%) and C(1) (19/55, 34.5%) treatments (p < 0.05). This study demonstrates sorted ram spermatozoa are equally fertile to non-sorted spermatozoa even when inseminated at 2% of the dose. Furthermore, at very low artificial insemination doses (1 or 5 million motile) the fertility of sorted ram spermatozoa is superior to non-sorted spermatozoa inseminated in equal numbers. These results have significance for the future commercialization of sex-preselection technology in sheep as a reduction in the minimum effective sperm number will allow a corresponding decrease in the associated cost per dose.     

Characterization and Localization of Membrane Vesicles in Ejaculate Fractions from the Ram, Boar and Stallion

Membrane vesicles, separated by differential centrifugation from the seminal plasma, were detected in the sperm-rich ejaculate fractions of four boars and three stallions, and in the whole ejaculates of seven rams. The volume and percentage of vesicles, determined by a stereological technique, were higher in the sperm-rich than in the post-sperm-rich fractions of the boar and stallion ejaculates, and no vesicles were detected in the pre sperm-rich fractions. Vesicles were examined by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). For boar, stallion and ram semen, the mean (+/- s.e.m.) vesicle diameters were 130.9 +/- 3.22 (range 18-577), 164.1 +/- 4.42 (range 15-671) and 159.7 +/- 2.92 nm (range 22-986), respectively, although they were not significantly different (p = 0.709). The vesicles had approximately round (TEM) or spherical shape (SEM), were surrounded by single, double or multi-laminar membranes, and were trapped within ample amorphous material, sometimes containing short, flattened membranous elements. The majority of the vesicles had a clear interior but some contained granule-dense material. Ram membrane vesicles, purified from ultracentrifuged plasma by size exclusion chromatography, kept their round shape and the amorphous material was less evident compared with the sections taken before purification. This is the first report to identify seminal plasma membrane vesicles in the different fractions of ejaculated semen in the boar and stallion, and confirms their presence in ram seminal plasma. The origin and function of these vesicles are yet to be elucidated.     

Enhanced freezability of goat spermatozoa collected into tubes containing extender supplemented with bovine serum albumin (BSA)

The purpose of this study was to investigate the effects of semen collection into tubes containing extender supplemented with BSA on the cryosurvival of goat spermatozoa. Semen was collected from two goats into empty tubes or tubes containing 10 ml extender supplemented with 0, 0.1, 1, or 5% BSA, and the washed spermatozoa were frozen as pellets in egg yolk-trehalose extender with the addition of 0.04% SDS and 4% glycerol. Sperm motion parameters were evaluated after post-thawing and during a thermal resistance test. The acrosome status of frozen-thawed spermatozoa was also observed using FITC-PNA staining. In frozen semen that was collected into tubes containing extender supplemented with 5% BSA, the post-thawed spermatozoa exhibited a significant improvement in motion parameters and maintained high motility throughout incubation and acrosome integrity, as compared with semen collected into tubes containing extender supplemented with lower concentrations of BSA. In conclusion, semen collection into tubes with a large volume of extender containing high concentrations of BSA dramatically improves the motility and acrosome integrity of frozen-thawed spermatozoa. This suggests that the in vitro functional freezability of spermatozoa is abruptly modified by reducing contact with seminal plasma and by flash contact with BSA at ejaculation.     

稀释液中添加维生素E对绵羊冷冻精液品质的影响

采用两步稀释法 ,在绵羊冷冻精液中添加维生素E ,精液稀释平衡后精子活率 ,试验组 (0 .6 35 )高于对照组(0 .5 75 ) ,但无显著性差异 (P >0 .0 5 ) ;而解冻后 ,试验组活率 (0 .4 35 )极显著高于对照组 (0 .36 5 ) (P <0 .0 1) ;精子顶体总异常率 ,试验组 (33.72 % )极显著低于对照组 (4 4 .35 % ) (P <0 .0 1) ,其中试验组顶体膨胀率和脱落率与对照组分别为 1.2 8% ,2 3.0 8%与 2 .6 8% ,2 8.96 %。添加维生素E可以降低冷冻对精子顶体的损害程度 ,提高精液品质。试验也证明在解冻后保存 1h内 ,精子活率呈现缓慢下降的趋势 ,但 1h后活率急剧下降 ,因而 ,绵羊冻精颗粒解冻后应在 1h内输精完毕     

Effect of green buffer storage on the fertility of fresh camel semen after artificial insemination

Artificial insemination (AI) is one of the most widely used reproductive technologies, and there is considerably interest in commercializing this technology in camels. Storage of semen extender frozen (at -20 °C) is of considerable interest to scientists working with camels, as transportation of diluents at refrigeration temperature is not always possible given the hot, arid and remote conditions that dromedary camels exist in. Therefore, this study was conducted to compare the fertility of fresh camel semen, after dilution in fresh or frozen-thawed green buffer (GB), after AI into single and multiple ovulating female camels. No differences were observed in any sperm characteristics (motility, membrane integrity, acrosome integrity or morphology) when semen was diluted in fresh or frozen-thawed GB (p>0.05). Sperm motility was increased by dilution (fresh: 70.7 ± 4.9% and frozen: 68.8 ± 3.1%) compared with the motility of sperm in neat semen (35 ± 2.85%; p<0.05), and sperm motility changed from oscillatory to forward progressive after dilution. Pregnancy rates were higher (p<0.05) for single ovulating camels inseminated with semen diluted in fresh (72.7%) compared with frozen-thawed GB (27.3%), and fertilization rates were also higher (p<0.05) for multiple ovulating camels inseminated with semen diluted in fresh (83.3%) compared with frozen-thawed GB (11.1%). These results clearly demonstrate the detrimental effect of freezing and thawing semen diluent on the fertility of fresh camel semen. However, further studies are required to elucidate the mechanism responsible for this reduction in fertility. Moreover, these results demonstrate that the fertility of fresh camel semen diluted in fresh GB is high enough to be considered commercially viable.     

Influence of boars on the relationship between fertility and post thawing sperm quality of deep frozen boar spermatozoa.

The possibility of selecting boars for deep freezing of spermatozoa was evaluated by tests of frozen-thawed spermatozoa. 20 of 31 ejaculates were used for artificial insemination of 37 gilts. Boar seminal plasma and OLEP were used as thawing diluents. The thermoresistance test and the extracellular concentration of aspartate aminotransferase (ASAT) were used as indicators of fertility. Spermatozoa from 4 boars were utilized. 1 of the boars appeared to be of superior fertility, while the spermatozoa from another was infertile. The latter animal had been used for fresh artificial insemination trials, and had shown higher pregnancy rates than the average. Samples from this boar exhibited the lowest degree of motility after 3 hours storage at 37 degrees C, the greatest relative decrease of motility during the thermoresistance test, the greatest release of ASAT after thawing by OLEP, and greatest relative release of ASAT. These variations were statistically significant (p less than .05). The results show that there is a possibility of detecting boars with spermatozoa with potential low freezability.     

A preliminary study on using autologous and heterologous boar sperm supernatant from freezing processes as post-thawing solution: its effect on sperm motility

The aim of this study was to evaluate the effect of post-thawing dilution with autologous and heterologous sperm supernatant on motility of frozen-thawed boar spermatozoa. During the cryopreservation, sperm supernatant (a combination of seminal plasma and semen extender, 50% v/v) or seminal plasma from nine boars (Duroc, Large White, and Landrace; three in each) was collected by centrifugation and stored frozen until use as post-thawing solution. Sperm pellets were further processed and cryopreserved using control-rate freezer and was thawed at 50°C for 12 s. After thawing, frozen thawed semen samples were diluted with seminal plasma (group A), supernatant from Landrace (group B), supernatant from Large White (group C), supernatant from Duroc (group D), and Modena™ semen extender (group E). Post-thawing motility was evaluated using a phase-contrast microscope after thawing at 1, 10, 20, and 30 min. The present results show that at 1 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (53.3%) and C (53.9%) than the other groups. At 10 min, the highest (P ≤ 0.001) progressive motility was found in groups B (65%) and C (61%). At 20 and 30 min, a significantly higher percentage (P ≤ 0.001) of progressive motility was found in groups B (58.9%), C (53.5%), and D (45.6%) than groups A (3.9%) and E (20.6%). It can be stated that supernatant from the freezing processes (consisting of seminal plasma and Modena™, 50% v/v) had a beneficial effect on post-thawing progressive motility of frozen boar semen.     

牛冷冻精液质量评估研究进展

人工授精(AI)是第一代动物繁殖生物技术,最近几十年以来,研究人员已经探索出了许多评估冷冻-解冻后精液品质的新技术。由于精子质膜在低渗条件下的功能状态、离子分布及浓度和各种细胞器的功能都与冷冻解冻后精子的存活能力相关,冻精质量的评定也从最初的形态和主观活率分析,发展到更精确的染色质、质膜完整性及分解代谢活性的分子变化分析。但是,在实际应用当中,许多方法花费很大,况且单个的评估参数都与动物精子实际的受精力相关性不强。因此,要提高牛冻精质量的预言强度,应该将这些独立的参数结合起来,利用多元回归分析,建立一套更完美更精确的评估体系。     

Fertility of Mares Inseminated With Frozen-Thawed Semen Processed by Single Layer Centrifugation Through a Colloid

The aim of this study was to determine whether there was an increase in pregnancy rates when frozen-thawed stallion semen was processed by single layer centrifugation (SLC) through a colloid before insemination. In addition, changes in semen parameters, including motility, were determined before and after SLC. Twenty light-horse mares (aged 3-16 years) and one Thoroughbred stallion (aged 16 years) having average fertility with fresh and cooled semen (>50% per cycle) and displaying a postthaw motility of >35% were used. Control mares were inseminated using 4- × 0.5-mL straws (200 × 10⁶/mL) of frozen-thawed semen. Treatment mares were inseminated with 4 × 0.5 mL of frozen-thawed semen after processing by SLC. Pregnancy rates were compared using Fisher exact test, and continuous parameters were evaluated by a Student t test. The pregnancy rates at day 14 were not different for the mares inseminated with control versus SLC-processed semen, despite the difference in sperm number (171 × 10⁶ ± 21, 59 × 10⁶ ± 25 progressively motile sperm). After frozen-thawed semen was processed by SLC, the percentage progressively motile sperm improved (P < .05), and SLC processing resulted in a 21.8% recovery of spermatozoa. In summary, centrifugation of frozen-thawed semen through a single layer of colloid increased the percentage of motile spermatozoa, but did not improve pregnancy rates after deep horn insemination.     

Gefrierkonservierung von Schafbockspermien in Gegenwart oberflächenaktiver Substanzen

Inhalt Ein Zusatz von Alkylsulfaten bzw. von TRITON X 45 in sehr geringer Konzentration zum Medium für die Gefrierkonservierung von Schafbocksperma führt zu verbesserten Auftauergebnissen. In einem Medium ohne Kryoprotektiva wird die Empfindlichkeit gegenüber Kälteschock bzw. osmotischen Schock verringert. Der vermutlich membranstabilisierende Effekt von Natriumhexadecylsulfat hatte keinen Einfluβ auf das Befruchtungsergebnis nach Insemination gefrierkonservierten Schafbockspermas.
Ein Zusatz von N-Acetyl-cystein bei der Gefrierkonservierung von Schafbocksperma verbessert Motilität und Resistenz nach dem Auftauen signifikant. Andere Vitalitätsparameter werden nicht beeinfluβt. Im Besamungsversuch konnte der positive Effekt dieses Zusatzes bestätigt werden, ohne daβ damit praxisrelevante Ergebnisse erreicht wurden.
Contents: Cryopreservation of ram spermatozoa in the presence of surface active substances
The addition of alkylsulphates or TRITON X 45 in very low concentrations to diluents for the cryopreservation of ram semen leads to an improvement of after-thawing results. In a diluent without cryoprotectives the sensitivity against cold shock as well as osmotic shock is diminished. The membrane stabilising effect of sodium-hexadecylsulphate has no influence on the fertility rate after insemination of cryopreserved ram semen.
The addition of N-acetyl-cysteine in cry opreservation of ram spermatozoa increases the motility and lifetime in a resistance test after thawing significantly. Other parameters of viability are not influenced. In insemination experiments the positive effect of this addition could be verified, but, on the whole, the effect is too small to be relevant in practice.     

CASA Assessment of Kinematic Parameters of Ram Spermatozoa and their Relationship to Migration Efficiency in Ruminant Cervical Mucus

Sperm motility is an indicator of male fertility because of its importance for sperm migration through the female genital tract and for gamete interaction at fertilization. This study analyses the relationship between computer assisted semen analysis (CASA) motility patterns and sperm migration of rams in ruminant cervical mucus. In experiment 1, spermatozoa extended with sperm analysis medium (SAM) and seminal plasma were compared in terms of motility. In experiment 2, 56 semen samples were collected either with artificial vagina (AV) or electroejaculator to be compared in terms of motility performance. In experiment 3, 104 ejaculates collected by AV from 26 males were analysed via the CASA system to characterize their motility patterns. In experiment 4, ejaculates from pairs of rams (20 rams in total) were simultaneously assessed for mucus migration (ovine, caprine, bovine) and motility patterns to evaluate the correlations between both parameters. Semen collected by AV and extended in SAM allows the most reliable assessment for sperm motility. Ram spermatozoa move fast and follow a linear trajectory compared with other ruminants. Continuous line velocity (VCL) and average path velocity (VAP) are the only sperm kinematic parameters that presented significant positive correlations with the ability to migrate in sheep cervical mucus (p < 0.05). Continuous line velocity, VAP, straight line velocity and linearity are highly significantly related with migration efficiency in goat cervical mucus (p < 0.01) and only lateral head displacement is negatively related to sperm migration in bovine cervical mucus (p < 0.05). These results suggest that specific kinematic parameters confer the ability of spermatozoa to colonize and migrate through epithelial mucus with different rheological properties.     

Motility of Fresh and Frozen-Thawed Stallion Sperm from Three Segments of the Epididymal Cauda and the Effect of Post-Thaw Seminal Plasma Addition on Motility

Preservation of epididymal spermatozoa is an advantageous method to preserve genetic material of endangered species or valuable breeding animals after sudden death and injuries. Despite lower pregnancy rates, fertilization with epididymal sperm has been proven successful. Variable sperm quality after cryopreservation among individual stallions and the usually terminal chance to preserve epididymal sperm are opportunities for the development of a freezing procedure suitable for the majority of stallions. To evaluate the effect of the preservation procedure, we analyzed the sperm motion characteristics after every step of processing. In addition, we investigated the influence of seminal plasma on motility of frozen-thawed semen. We compared three segments of the cauda epididymidis and harvested spermatozoa by retrograde flushing (most caudal part) and mincing (more cranial segments) to augment the number of spermatozoa. During processing, there were differences in sperm motion characteristics depending on the segment of the cauda epididymidis. Distinct increases in motility due to different extenders and the effect of seminal plasma suggest that motion characteristics of raw and frozen-thawed spermatozoa are strongly influenced by microenvironment and must be interpreted with caution.     

Seminal plasma components in camelids and comparisons with other species

Camelid semen is characterized by a highly viscous, low-volume ejaculate with a low concentration of spermatozoa that exhibit low progressive motility. The viscous seminal plasma is currently the major impediment to the development of assisted reproductive technologies (ARTs) in camelids. To advance ARTs such as sperm cryopreservation and artificial insemination in camelids, it is necessary to identify the cause of the viscosity and gain an understanding of the role of seminal plasma components on sperm function and fertility. Numerous compounds and proteins have been identified as mediators of sperm function and predictors of fertility in other livestock species, and understanding the importance of specific proteins has progressed the success of ARTs in these species. Current knowledge on the components of camelid seminal plasma is outlined, together with the implications of these components for the development of ARTs in camelids. The cause of semen viscosity, as well as proteins that are present in camelid seminal plasma, is described for the first time. Seminal plasma components are compared with those of other species to hypothesize their role in sperm function and fertility.     

Birth of puppies after intrauterine and intratubal insemination with frozen-thawed canine semen

The present study was performed to assess the fertility of frozen-thawed dog semen prepared by freezing with 6% glycerol and thawing at 70℃ for 8 sec, and to evaluate the least number of post-thaw spermatozoa necessary to achieve pregnancy by intrauterine or intratubal artificial insemination. It was found that the pregnancy rate of intrauterine artificial insemination was 100% using 6% glycerol buffer and thawing at 70℃ for 8 sec with 5 × 10⁷ spermatozoa. Even though the pregnancy rate (80%) and the whelping rate (24.5%) in the 5 × 10⁶ spermatozoa inseminated group were lower than those of the 5 × 10⁷ spermatozoa group, conception was confirmed with 5 × 10⁶ spermatozoa. Although the pregnancy rate of intratubal insemination was low (20%) with 4 × 10⁶ spermatozoa, this study is the first report to show the pregnancy rate of intratubal insemination with frozen-thawed ejaculated canine semen. In order to improve the pregnancy rate with intratubal insemination of canine spermatozoa, it is necessary to investigate the optimal insemination site of the uterine tube, the appropriate number of sperm, and the direct effect of buffer on oocytes.     

The Origin of Membrane Vesicles in Ram Seminal Plasma

The hypothesis tested in this study was that the membrane vesicles present in ram seminal plasma are of testicular origin, rather than being secreted by the accessory sex glands as has been previously reported for a number of species. Membrane vesicles were present in cellular extracts from reproductive organs and accessory sex glands of six rams, and in the seminal plasma of a further eight rams. When four of the latter rams were subjected to vasectomy, to isolate ejaculate contents to only the secretions of the accessory sex glands, the vesicles were largely eliminated from their ejaculates, while vesicles were still present in the ejaculates of the four control rams. The constituents of the cytoplasmic droplets and membrane vesicles derived from the seminal plasma were compared by transmission electron microscopy (TEM). Vesicles present in the cytoplasmic droplets were similar in morphology but smaller on average than those in the seminal plasma. It was concluded that the membrane vesicles in ram seminal plasma originate from either the cytoplasmic droplets, or a combination of vesicles from the droplets and the epididymis.