Semen Diluents Research Articles

56 COMPARISON OF 4 DIFFERENT DILUENT AGENTS ON CRYOPRESERVATION OF SEMEN FROM UNIMPROVED INDIGENOUS SOUTH AFRICAN GOATS

The cryopreservation process is one of the most crucial factors that affect the post-thaw quality of frozen goat semen. The aim of this study was to compare 4 different semen diluents (Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®). A total of 5 indigenous bucks were used as semen donors. Semen was evaluated for macroscopic and microscopic characteristics. Semen was pooled and diluted randomly in each of the 4 different extenders. Semen samples were equilibrated for 3 h at 5°C. After equilibration, samples were loaded into 0.25-mL straws and placed into the controlled-rate programmable freezer using a customized freezing curve (from 5 to –5°C at 4°C min–1, –5 to –110°C at 25°C min–1, and then from –110 to –140°C at 35°C min–1). Data was analysed with the aid of the statistical program GenStat®, using a complete randomised design. Following fresh semen dilution with Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, the sperm progressive motility percentage was 41.5 ± 4, 43.5 ± 7, 48.6 ± 5, and 50.2 ± 7%, respectively (P > 0.05). In addition, the live normal sperm percentage following fresh semen dilution was 59.2 ± 5, 49.2 ± 6, 57.8 ± 5, and 54.4 ± 3% for Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, respectively (P > 0.05). After thawing semen samples frozen either with Tris-BSA, Tris-yolk, Bioxcell®, or Ovixcell, the sperm progressive motility percentage was 5.3 ± 2, 1.6 ± 1, 10.6 ± 2, and 13.3 ± 3%, respectively (P < 0.05). Furthermore, the live normal sperm percentage following thawing was 19.1 ± 3, 14.3 ± 3, 39.6 ± 6, and 37.1 ± 3% for Tris-BSA, Tris-yolk, Bioxcell®, and Ovixcell®, respectively. In conclusion, sperm progressive motility percentage was reduced following the freezing-thawing process, irrespective of the type of extender used. The Bioxcell® and Ovixcell® extenders showed to be better for South African indigenous goat semen cryopreservation purposes.

Semen collection and seminal characteristics of the Australian saltwater crocodile (Crocodylus porosus)

The development of a reliable non-invasive method of crocodile semen collection is fundamental for assessing male fertility and for implementing the benefits of artificial insemination for reproductive and genetic management of farmed populations and the captive breeding of endangered species. Following an initial description of male reproductive anatomy in the saltwater crocodile (Crocodylus porosus), this study developed a successful, reliable method of semen collection using cloacal digital manipulation of the terminal segment of the ductus deferens. Semen was recovered from 30 of 31 collection attempts from a total of 24 sedated males ranging from 197cm to 400cm in body length. Seminal characteristics (including abnormal sperm morphology) and bacteriology were documented for the first time. Mean (±SEM) seminal volume, pH, osmolality, sperm concentration, percentage of motile sperm and the percentage of sperm with an intact membrane measured 0.91±0.16mL, 7.3±0.1, 335.5±9.0mOsmkg-1, 2.29±0.26×109, 50.7±4.2%, and 79.9±3.6%, respectively. Sperm abnormalities included macro and microcephalic nuclei, teratoid spermatozoa, loose heads and a range of abnormal flagella. Most semen samples contained spermatozoa with what are presumed to be cytoplasmic droplets but the confirmation and then significance of this phenomenon as a sperm maturational pathology require further validation. In an attempt to develop targeted antibiotics for use in semen diluents, microflora of the penile shaft, sulcus and semen of a subset of crocodiles was analysed for culture and sensitivity. While a diverse range of bacteria were identified, the majority were sensitive to gentamicin. This study represents the first step towards assessment of breeding soundness examination and the use of artificial insemination technology in the saltwater crocodile industry.

Effect of dialysis on the proacrosin/acrosin system and motility of turkey (Meleagris gallopavo) spermatozoa during liquid storage

1. The effect of dialysis on the proacrosin/acrosin system and motility of turkey spermatozoa were examined after 24 and 48 h of liquid storage at 4°C.2. Fifteen pools of semen diluted in extender were dialysed against Clemson Turkey Semen Diluent (dialysed semen) or stored in aerobic conditions (undialysed semen). Semen quality was assessed by measuring spermatozoa motility, amidase activity of spermatozoa suspension, spermatozoa extract and seminal plasma and anti-trypsin activity of seminal plasma.3. Extracted amidase activity of dialysed semen was lower than undialysed by 28%. Higher values for speed parameters of spermatozoa were found in dialysed semen in comparison to undialysed, for example, 81.6 µm/s versus 75.0 µm/s for straight-line velocity (VSL), 114.7 µm/s versus 110.3 µm/s for curvilinear velocity (VCL) and 86.6 µm/s versus 79.8 µm/s for average path velocity (VAP).4. It was concluded that dialysis caused lower amidase activity of spermatozoa and increased speed parameters of progressively motile turkey spermatozoa during storage. Lower extracted amidase activity of dialysed semen reflected better membrane integrity of dialysed semen and suggests that the proacrosin/acrosin system of dialysed spermatozoa is less susceptible to activation compared to undialysed semen.

Replacing chicken yolk with yolks from other sources in ram semen diluents and their effects on fertility in vitro

Over the past 70 years, egg yolk (EY) has been continuously used as a cryoprotectant in semen diluents. Earlier studies have shown that the composition of different EYs showed that the basic components were present in similar levels. However, further breakdown and analysis revealed significant differences between EY sources. The objective of this study was to evaluate and compare the effect of substitution chicken EY in ram semen diluents with yolks from other species on in vitro fertilization, subsequent embryonic development and total blastocysts formation. Through the breeding season, ejaculates were collected weekly from five Sarda breed rams of proven fertility, divided equally and diluted in Tris–citrate–fructose–glycerol based diluents containing 20% (v/v) of either partridge, ostrich, turkey, duck, tortoise, or chicken EYs (as a control) at 37°C. Extended semen was cooled to 4°C and preserved as a 0.25mL pellet in LN2. Adults Sarda ewe ovaries were collected from a local slaughterhouse and a total of 913 collected oocytes were divided randomly and matured oocytes were fertilized in vitro with frozen–thawed spermatozoa prepared as described above, and cultured until Day 8. The proportions of cleaved and expanded blastocysts/groups were evaluating after 46, and 144–192h post-insemination (hpi), respectively.Among all sources of EYs, semen cryopreserved in presence of partridge EY significantly increased the proportion of fertilized matured oocytes compared with semen preserved in presence of tortoise or chicken EYs (105/131, 80.1%), (74/123, 60.2%), and (189/270, 70.0%), respectively. Interestingly, using partridge, ostrich, turkey, and duck EYs in ram semen diluents increased significantly (P<0.001) the number of blastocysts formation on Day 6 after IVF versus tortoise or chicken yolks. However, there was no significant difference in total blastocysts yield between all sources of EY. In conclusion, the different compositions of yolks did offer different levels of in vitro production embryos. Specifically, semen diluents in presence of partridge yolks improved cleavage rates, and the numbers of early embryonic development. Whereas using ostrich, turkey, and duck EYs offered comparable fertilization and embryonic development versus conventional semen diluents with 20% chicken EY.

EFFECT OF GENETIC GROUP, SEMEN DILUENTS AND FREEZING REGIMENS ON SPERM FREEZABILITY AND GOATS REPRODUCTIVITY

Two experiments were designed to study the effect of bucks breeds, semen extenders and freezing regimens on post-thaw semen motility and viability index (experiment 1). In experiment 2, semen diluents effects on reproductive performance was conducted. Semen was collected from Aradi (A), Damascuss (D) and cross (½A½D) bucks breeds. Good quality semen was divided into 4 portions, each diluted with one diluent (Milk, Na. Citrate, Tris and Na. Bicarbonate). The diluted semen was packaged into 0.5 ml straws then cooled to 5°C. After equilibration, half of the packaged straws were suspended 15 cm above liquid nitrogen (LN) for 15 min. (Freezing regimen1; slow). Other half of straws was suspended at height 15 and 5 cm of LN for 10 and 5 min, respectively (Freezing regimen2; rapid) before plunged into LN. Frozen semen was thawed for post-thaw motility and viability. In the second experiment, semen with good quality was extended with three types of extenders (Milk, Na. Citrate and Tris). Diluted semen were cooled to 5°C and used for AI. Results revealed that, pre-freeze semen motility was significant higher in Tris, Na. Citrate and Na. Bicarbonate than milk diluent. Post-thaw semen motility and viability were highly significant for milk and Na. Citrate than Tris and Na. Bicarbonate diluents. Post-thaw semen motility was significantly higher in Aradi and Damascus than crossbred. Post-thaw semen motility and viability revealed, significant higher means for Freezing regimen 2 than regimen 1. Milk and Tris diluents were significantly higher than Na. Citrate for fertility, fecundity and prolificacy. Aradi does was higher than Damascus does in fertility, fecundity and prolificacy. Also, Aradi does was significantly higher than ½A½D does in fertility, while the reverse trend was observed in fecundity and prolificacy. It was concluded that, regarding to post-thaw semen motility, semen viability, fertility%, fecundity% and prolificacy%, milk diluent is preferable than Tris and Na. citrate. Fertility rate of Aradi goat breed is higher than Damascus and crossbred (½A½D).

The effect of temperature and pH on the motility and viability of ostrich sperm

As the chemical environment of semen diluents can have a profound effect on sperm quality, we examined the effect of temperature and pH on the motility and viability of sperm in the ostrich. Semen was collected from four males, each male being replicated three times. Ejaculates were diluted and incubated for 10min at 20°C and 40°C in four different buffers, temperature adjusted at pH 6, 7, 8 and 9 respectively. Average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), beat cross frequency (BCF) and amplitude of lateral displacement (ALH) were then recorded for each sample using CASA. The viability of sperm was assessed using nigrosin–eosin staining. Sperm incubated at 40°C had higher motility parameters, except for ALH. At 40°C, VAP, VSL and LIN increased with pH while VCL, BCF and ALH were higher for lower pHs. The viability of sperm was not affected by temperature but decreased at pH values>7. A pH in the neutral range appeared to yield higher quality sperm after in vitro storage at 20°C. However, the effect of different pH levels and temperatures on sperm longevity needs to be investigated further to develop viable ostrich specific diluents.

EFFECT OF GOAT BREEDS, SEMEN DILUENTS AND FREEZING METHODS ON SPERM FREEZABILITY AND REPRODUCTIVE PERFORMANCE

Two experiments were designed to study the effect of semen extenders and freezing regimens on post-thaw semen motility and viability index, effect of bucks breed was also included (experiment 1). In experiment 2, semen diluents effects on reproductive performance was conducted. Semen was collected from bucks breeds (Aradi (A), Damascuss (D) and cross (½A½D). Good quality semen was divided into 4 portions, each diluted with one diluent (Milk, Na.Citrate, Tris and Na.Bicarbonate). The diluted semen was packaged into 0.5ml straws then cooled to 5°C. After equilibration, half of the packaged straws were suspended 15cm above liquid nitrogen (LN) for 15min (Freezing regimen1; slow). Other half of straws was suspended at height 15 and 5cm of LN for 10 and 5min, respectively (Freezing regimen2; rapid) before plunged into LN. Frozen semen was thawed for post-thaw motility and viability. In the second experiment, semen with good quality was extended with three types of extenders (Milk, Na.Citrate and Tris). Diluted semen were cooled to 5°C and used for AI. Results revealed that, pre-freeze semen motility was significant higher in Tris, Na.Citrate and Na.Bicarbonate than milk diluent. Post-thaw semen motility and viability were highly significant for milk and Na.Citrate than Tris and Na.Bicarbonate diluents. Post-thaw semen motility was significantly higher in Aradi and Damascus than cross breed. Post-thaw semen motility and viability revealed, significant higher means for Freezing regimen 2 than regimen 1. Milk was significantly higher than Tris and Na.Citrate diluents for fertility and fecundity. It was concluded that, regarding to post-thaw semen viability, fertility%, kidding%, fecundity% and prolificacy%, milk is preferable than Tris and Na.citrate diluents.

Evaluation of the protective effect of duck egg yolk during cooling of buck semen

The present study aimed to evaluate if substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in diluents could improve and/or affects buck semen quality preserved at 5°C for 120 hr. and to study if changing level of DEY either by increasing or decreasing could affects buck semen preserved at 5°C for 120 hr. Semen samples collected twice weekly from 9 healthy mature bucks were diluted with 6 different diluents containing tris, citric acid, glucose, egg yolk and antibiotic. Diluent 1 contained 2.5% CEY (control), while diluents 2, 3, 4, 5 and 6 contained 1, 2.5, 5, 7.5 and 10% DEY, respectively. After cooling to 5-6°C, semen samples from each diluent were examined and evaluated each 12 hr. for motility, viability, total abnormalities, acrosomal damage and plasma membrane integrity. The results showed that at 1st 24 hr. diluents 1 (CEY 2.5%), 3 (DEY 2.5%) and 4 (DEY 5%) were significantly higher (P < 0.05) than other diluents in terms of motility, viability and plasma membrane integrity, and significantly lower (P < 0.05) than other diluents in terms of total abnormalities and acrosomal damage. However, after 36 hr. and till 120 hr. diluent 3 (DEY 2.5%) was better than other diluents in the aforementioned parameters. Results also showed that decreasing DEY level in semen diluents as in diluent 2 (DEY 1%) was not satisfactory. Similarly, increasing DEY level in semen diluents as in diluent 4 (DEY 5%), 5 (DEY 7.5%) and 6 (DEY 10%) was not satisfactory and adversely affect spermatozoa which dead after 60 hr. and 84 hr. in diluents 5 (DEY 7.5%) and 6 (DEY 10%) respectively. The present study showed that inclusion of DEY at level of 2.5% rather than CEY 2.5% in diluents could improve bucks semen quality preserved at 5°C.

Influence of addition of different antibiotics in semen diluent on viable bacterial count and spermatozoal viability of Awassi ram semen

The objectives of the present study were to determine the effects of six different antibiotics in controlling the growth of semen contaminating bacteria and if these antibiotics have any adverse effect on Awassi ram spermatozoa. Semen samples from six mature Awassi rams were used in this study. A total number of 120 ejaculates were collected from the rams using an artificial vagina once a week. Semen ejaculates were evaluated for volume, sperm concentration, mass motility, individual motility, percentage live sperm, sperm abnormalities, and viable bacterial count. Semen samples were diluted by sodium citrate-fructose-egg yolk. The diluted semen sample was divided into 7 parts. Six types of antibiotics were added to the semen diluent parts including; penicillin G 1000 IU ml-1 with streptomycin 1 mg ml-1, gentamicin sulphate 250 mg ml-1, tetracycline 0.5 mg ml-1, lincomycin 1 mg ml-1, cefoperazone sodium 1mg ml-1, cefdinir 1 mg ml-1 and the seventh part considered as a control group without antibiotic addition. The diluted semen samples were cooled and preserved at 5 Co for 5 days. Cooled diluted semen samples were examined for individual motility, percent of live sperm, sperm abnormalities, acrosomal defects and bacterial count every 24 h until 5 days. Comparing with the control, all the antibiotics examined were effective in controlling bacterial growth (P

Use of a defined diluent increases the sex-sorting efficiency of stallion sperm

The low efficiency of flow cytometric sex-sorting of stallion sperm has been attributed to the use of an opaque skim milk-based diluent during Hoechst 33342 ( H33342) staining. Three experiments were conducted to formulate an optically clear stallion semen diluent for use during H33342 staining, and to determine whether a clear diluent improved resolution during sorting. For Experiment 1, sperm were incubated at 34 °C in each of five diluents containing either no protein, skim milk, 0.25% Cohn's Fraction V BSA, 0.5% BSA, or 1% BSA, following an 18 h storage (15 °C) period, or shortly after collection. Sperm incubated in both skim milk and 1% BSA-supplemented diluents had equivalent total (47 and 49.5%, respectively) and progressive (4.73 and 5.67%, respectively) sperm motilities after 45 min, and comparable acrosome integrity (65.9 and 67.9%, respectively). For Experiment 2, the protein source was optimised by comparing the characteristics of sperm stored and incubated in five diluents supplemented with skim milk, BSA, fatty acid and endotoxin free BSA (I-BSA), KnockOut™ Serum Replacement, and β-lactoglobulin, respectively. The I-BSA diluent was superior to skim milk for motility maintenance during incubation (74.0 vs 63.7%). The effect of diluent on sorting was investigated in Experiment 3 using a range of H33342 concentrations and incubation durations. The clear (1% BSA) diluent improved the split ratio compared with the opaque (skim milk) diluent (0.17 vs 0.08), with an optimum staining time of 45 min using 0.09 mM H33342. In conclusion, a diluent containing 1% fatty acid free, low endotoxin BSA in lieu of skim milk improved the sorting efficiency and motility characteristics of stallion sperm after storage for 18 h.

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.

Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future

Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

A Method for Cryopreserving Semen from Yakido Roosters Using N-Methylacetamide as a Cryoprotective Agent

A method of freezing semen using N-methylacetamide (MA) as a cryoprotective agent was applied to Yakido, a chicken breed designated as a “Natural Monument” under the “Law for the Protection of Cultural Property” (Law No. 214, May 30, 1950) in Japan. Semen was collected from Yakido roosters, and pooled semen was diluted 1: 1 using semen diluent containing no cryoprotective agent. After equilibration at 5°C for 30min, diluted semen was further diluted 1: 1 using semen diluent containing MA at a final concentration of 9%. Diluted semen was packaged into 0.5-mL plastic straws and frozen by placing the straws in liquid nitrogen vapor followed by plunging into liquid nitrogen at -196°C. After storage in liquid nitrogen for about one month, frozen semen was thawed in ice water. Intravaginal artificial insemination (AI) was performed immediately after thawing semen without removing the cryoprotective agent. In Fertility Trial 1, AI was performed once a week for four consecutive weeks. Weekly fertility rates using frozen semen were 83.8±3.8%, 77.1±8.8%, 88.6±5.1%, and 77.3±5.7% for weeks 1, 2, 3, and 4, respectively. Hatchability was over 90% throughout the experimental period. In Fertility Trial 2, the duration of fertility was examined after intravaginal insemination for 2 consecutive days. Daily fertility rates using frozen-thawed semen were 90.0%, 90.0%, 91.7%, 88.9%, 100.0%, 100.0%, 100.0%, 55.6%, 60.0%, 25.0%, 30.0%, 30.0%, and 9.1% for days 1 through 13 after AI, respectively. Overall hatchability was 89.5%. These results indicated that freezing chicken spermatozoa using MA as a cryoprotective agent is a practical method of preserving chicken semen collected from genetically valuable chicken breeds.

Optimization of the cryopreservation of African clawed frog ( Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg 2SO 4, 1 mmol/L CaCl 2, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10 6 to 8 × 10 6/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.

59 THE INFLUENCE OF DILUENT EGG YOLK PARTICLES ON THE ACCURACY OF FLOW CYTOMETRIC ANALYSES OF SPERM FUNCTIONAL CHARACTERISTICS

Flow cytometry is an important tool to assess semen quality and sperm function, especially after cryopreservation, but the presence of egg yolk particles in many semen diluents can distort the results. The aims of the present study were to investigate the effect of the presence of egg yolk in the semen diluent on the outcome of flow cytometric analysis of dog spermatozoa and to explore the use of Laser Dye Styryl-751 (LDS-751), a permeant DNA stain with an emission wavelength of 712 nm, to exclude egg yolk particles from the analysis of plasma membrane fluidity (Merocyanine 540 and Yo-Pro-1) and intracellular Ca2+ (Fluo-3 and PI). Flow cytometric analyses were carried out on a Coulter Epics XL flow cytometer (Beckman-Coulter Inc., Fullerton, CA). Data were analyzed using EXPO32TM ADC software (Beckman-Coulter). A paired t-test was used to test whether gating out egg yolk particles using LDS-751 affected the analysis. Method agreement analysis was undertaken to assess the agreement between the 2 staining protocols (i.e. with or without LDS-751). The proportion of spermatozoa with high plasma membrane fluidity was significantly (P &lt; 0.05) greater after gating out egg yolk particles; the measurements of high membrane fluidity spermatozoa were on average 7.7 percentage points greater than without gating out egg yolk as shown by the method agreement analysis (SD = 9.35). Similarly, the proportion of spermatozoa with high intercellular Ca2+ was significantly (P &lt; 0.01) greater after gating out egg yolk particles; the proportions of spermatozoa with high intracellular Ca2+ were 5.95 percentage points greater when egg yolk particles were gated out compared with measurements in the presence of egg yolk particles (SD = 5.43). The method agreement shows that both methods show close agreement with and without egg yolk particles (i.e. results mostly fall within the mean of differences between pairs of repeated measurements ±2 SD). Therefore, the 2 methods are measuring the same phenomena. However, the change in measurement values with exclusion of egg yolk particles is significant. Therefore, accuracy is increased; that is, closer to the true value. In conclusion, using MC540/Yo-Pro-1 and Fluo-3/PI staining alone in the presence of egg yolk particles resulted in underestimation of the proportions of high plasma membrane fluidity and high intracellular Ca2+ spermatozoa, respectively. LDS-751 could be used with other fluorescent stains that target organelles such as mitochondria and acrosomes with multicolor flow cytometric analysis to exclude egg yolk particles.

Testing Usability of Butylated Hydroxytoluene in Conservation of Goat Semen

The objective of this study was to investigate whether butylated hydroxytoluene (BHT) could be used as a suitable supporter or alternative of egg yolk during preservation of goat spermatozoa. Three in vitro experiments and a fertility test were conducted to evaluate the effect of BHT on viability of chilled-stored semen as well as motility and kidding rate of frozen-thawed spermatozoa. In the first two experiments, ejaculates (n = 30/experiment) were collected from 10 bucks, split, diluted with egg yolk-based and egg yolk-free extenders supplemented with or without 0.3, 0.6, 2, 5 and 8 mM BHT and stored at 5 degrees C for 168 h. In the third experiment, 30 ejaculates were collected from the above-mentioned bucks, split and diluted with egg yolk-free extenders supplemented with or without 0.3, 0.6 and 0.9 mM BHT and egg yolk-based extenders supplemented with or without 5 mM BHT. Diluted semen was cooled to 5 degrees C over a period of 4 h, frozen and thawed in the form of 0.3-ml pellets. In the fertility test, 75 ejaculates were collected from two proven fertile bucks, split, diluted with egg yolk-free extenders containing 0.6 mm BHT and egg yolk-based extenders supplemented with or without 5 mM BHT, frozen and thawed as described above. An insemination volume of 0.6 ml containing 120-140 x 10(6) progressively motile spermatozoa was used for a single cervical insemination of cloprostenol-synchronized does (n = 230). The results showed that addition of 5 mM BHT to egg yolk-deficient (2.5%) extenders significantly improved viability of chilled-stored semen together with motility (48.5%) and fertility (62.5%) of frozen-thawed spermatozoa. Replacement of egg yolk in semen extenders by 0.6 mm BHT could sustain not only viability of chilled-stored semen but also post-thaw motility (47.5%) and fertility (53.75%) of frozen-thawed spermatozoa. In conclusion, supplementation of semen diluents with BHT can ameliorate preservability of goat sperm.

Thermotemporal dynamics of contaminant bacteria and antimicrobials in extended porcine semen

Bacterial contamination of extended porcine semen has been associated with deleterious effects on both semen quality and sow fertility. Retrospective, prospective and in vitro studies were performed to delineate the prevalence and behavior of certain bacterial contaminants in extended semen, and antimicrobial pharmacodynamics in various semen diluents. Retrospective review of extended semen samples submitted from North American boar studs for microbiological screening at the University of Pennsylvania Reference Andrology Laboratory in 2005 and 2006 yielded bacteriospermia prevalence rates of 17% (144/832) and 26% (256/984), respectively. In a prospective study of regional boar studs, of 91 extended semen samples tested over 1-y, 29% were positive for bacteriospermia. Retrospective and prospective studies both showed that the preponderance of contaminant positive samples occurred during the fall months ( P < 0.05). To better understand behavior of select contaminant bacteria, generation intervals were determined for Serratia marcescens (SM) and Achromobacter xylosoxidans (AX) at 16, 22 and 37 °C. Generation times were temperature-dependent, with intervals decreasing two- to four-fold as incubation temperature increased. Growth patterns for SM, AX and Burkholderia cepacia were evaluated in various semen diluents. The different diluents exhibited constant or episodic patterns of growth within and among bacteria throughout the 5-d test period. Kill-time kinetics at 37 °C of several genera of bacteria in four semen diluents containing amoxicillin, gentamicin, tylosin, and lincomycin/spectinomycin (single drug or combination) ranged from 75 to over 360 min, and was highly dependent ( P < 0.05) upon both type of bacteria and semen diluent.

263. Substitution of skim milk with bovine serum albumin in a stallion semen diluent

Skim milk has long been utilised as a source of protective proteins in stallion semen diluents. However, skim milk is also thought to contain components that are toxic to sperm and reduces the clarity of sperm suspensions, which impedes sperm assessments. This may also reduce the effectiveness of staining procedures used to process sperm for flow cytometric sex-sorting. The aim of this study was to ascertain the optimal concentration of bovine serum albumin (BSA) to replace skim milk in a traditional stallion semen diluent, Kenney's Modified Tyrode's (KMT) Medium1, for handling and processing stallion sperm before flow cytometric sex-sorting. Two ejaculates were collected from each of three pony stallions. Each ejaculate was divided into five aliquots and diluted in either KMT with skim milk or KMT supplemented with 0, 0.25, 0.5 or 1% BSA. Diluted samples were further divided into two aliquots and either stored at 15°C for 18 h before incubation and assessment, or incubated and assessed immediately upon arrival. Samples were incubated at 34°C and evaluated at 0, 45 and 90 min for objective motility and acrosome integrity. No interactions were observed between any treatments over time. There was a lower percentage of intact and a higher percentage of detached acrosomes for sperm incubated in KMT containing 0% BSA than all other treatments. A greater proportion of sperm incubated in KMT with skim milk had partial acrosome damage compared with other treatments. There was no difference in % total motility for sperm incubated in KMT with skim milk, and KMT containing 0.5 and 1% BSA (Table 1). These results indicate that BSA may be suitable as an alternative protein source in stallion semen diluents. Further studies are required to compare sex-sorting rates and sperm quality after sex-sorting, incubation and staining in skim milk compared with BSA-based media. (1) Padilla, A W and Foote, R H 1991, ‘Extender and centrifugation effects on the motility patterns of slow-cooled stallion spermatozoa’, Journal of Animal Science, vol. 69, no. 8, pp. 3308–331

Risks of transmitting mycoplasmas by semen and embryo transfer techniques in cattle, sheep, goats and pigs.

Abstract This object of this review is to consider whether mycoplasmas, particularly those that are important pathogens in cattle, sheep, goats and pigs, are likely to be transmitted by semen and embryo transfer techniques, and if so, what can be done to prevent such transmission. The first part gives a brief overview of semen and embryo transfer techniques, focusing on general risks of disease transmission and on routine disease control strategies. Subsequent parts provide information about mycoplasmas, including their classification, pathogenesis, diagnosis and control. Details are then given of the specific mycoplasmas of cattle, sheep, goats and pigs, and the seriously pathogenic species are distinguished from the opportunist pathogens and harmless commensals. It is emphasized, however, that within species the virulence of different strains can vary widely. An important feature of the mycoplasmas is that they often reside in the reproductive tracts of their hosts and therefore tend to pose a higher risk of being transmitted by semen and embryos than do many other micro-organisms. They also tend to adhere to cells and tissues, including semen and embryos, and can be difficult to remove. Some mycoplasmas can also invade and reside within non-phagocytic cells. Their resistance to routine disease control procedures such as semen and embryo washing, and to the inclusion of antibiotics in semen diluents and in embryo media, adds further challenges. It remains true, nevertheless, that semen and embryo transfer techniques offer great advantages over the movement of live animals for transferring genetic material, and will minimize the risks of disease transmission between different farms or countries. The review concludes by recommending control strategies, uniquely rigorous ones in this case, to avoid the transfer of mycoplasmas via semen and embryos.

78 CRYOPRESERVATION OF FROG (RANA PIPIENS) SPERM CELLS COLLECTED BY NON-LETHAL METHODS

There are few reported studies of cryopreservation of frog spermatoazoa. None have used non-lethal methods for collection, which involves a simple IP injection, followed by collection of sperm from the urine. Our study compared post-thaw survival of spermatozoa frozen using three different diluents: Salamon's ram semen diluent (SSD) (Evans and Maxwell 1987 Salamon's Artificial Insemination of Sheep and Goats, Sydney, Australia: Butterworths, pp. 122–141); our modification of Salamon's diluent (MSSD), containing an additional 5.9 mL glycerol and 6.6 g of sucrose per 100 mL; and a diluent reported to be currently the most successful for freezing frog sperm: Browne's frog diluent (BFD) (Browne et al. 2002 Cryobiology 44, 251–257). The 1 � SSD was composed of 2.9 g Tris, 0.4 g glucose, 3.2 g citric acid monohydrate, 12 mL egg yolk, and 4.5 mL glycerol per 100 mL. The composition of 1 � BFD was 10 g sucrose and 15 mL DMSO per 100 mL. Ten male frogs of unknown age were injected with 200 IU hCG (Chorulon; Intravet, Millsboro, DE, USA) in 500 µL PBS. Urine specimens were collected by cloacal lavage 30, 60, and 90 min post-injection. Specimenswere stored in a cold room at 8�C until all collections were finished. Diluents requiring addition of egg yolk were completed with that from a freshly laid chicken egg. Sperm cells were pooled and concentrations were measured using microscopy. Sperm cells were divided and combined with diluents in 1.5-mL microcentrifuge tubes. Tubes were suspended in a 500-mL beaker, which was placed in an ice bath to allow slow cooling to 2�C over 45 min. Aliquots of 100 µL were pipetted into indentations on dry ice and left to freeze for 3 min. Frozen pellets were plunged into liquid N2, and while under LN2, pellets were placed in microcentrifuge tubes, which were then stuffed with cotton wool. Upon thawing, pellets were removed from LN2 and dropped into 200 µL of thawing solution (0.9085 g Tris, 0.25 g fructose, and 0.45 g citric acid per 15 mL final volume) at 21�C while gently vortexing. Spermatozoa were assessed using live/dead cell stain (Molecular Probes, Eugene, OR, USA) and fluorescent microscopy. Percentages of live spermatozoa from the three treatments were 58.5%, 70.5%, and 44.1% in SSD, MSSD, and BFD, respectively. When considering the number of sperm cells lysed along with live/dead cell stain data, 40%, 55.2%, and 34.4% in SSD, MSSD, and BFD, respectively, survived post-thaw. All values were significantly different (P &lt; 0.001). Superior results were observed with MSSD; use of SSD also resulted in better results than BFD. The most prevalent method for collection of frog sperm is to pith the frog and then macerate the testis. With frog populations dwindling worldwide, use of a technique that is virtually harmless such as used here in conjunction with cryopreservation of specimens could be useful for preserving genetic variation in endangered frog species.