Assessment of bull fertility using a mucus penetration test and a human chorionic gonadotrophin stimulation test

The relative predictive abilities of an in vitro mucus penetration test and a human chorionic gonadotrophin (hCG) stimulation test were compared for assessing bull fertility. Cervical mucus was collected from 22 Holstein cows and then stored in liquid nitrogen. Semen was collected from 13 Holstein bulls and evaluated for standard semen parameters. The mucus penetration test was performed with fresh ejaculates, and the results were expressed as the distance traveled by the vanguard spermatozoa during incubation at 38 degrees C for 10 min. Increases in plasma testosterone levels were determined by a ratio of testosterone concentration before and after hCG injection. Sperm motility and mucus penetration were significantly correlated with the conception rate. However, no significant correlation was confirmed between the increased plasma testosterone levels and conception rate. The results indicate that the mucus penetration test is an effective method for predicting the fertility of bulls.     

Estimation of the potential fertility based upon non-return rates of bulls: using polyacrylamide gel instead of cervical mucus in the sperm penetration test

In the present study, we aimed to develop a polyacrylamide gel that could be used instead of bovine cervical mucus in the cervical mucus penetration test (CMPT) to obtain coherent and replicable results in bulls. The frozen semen samples of six Holstein bulls, which were divided into two fertility groups as low and high according to their non-return rate (NRR), were used. In this study, the modified CMPT (mCMPT) was carried out within 0.25 mL transparent plastic straws with an inner diameter 1.7 mm. The penetration ability of spermatozoa to bovine cervical mucus and to polyacrylamide gels swollen with two different solutions [NaCl (G1) and PBS (G2)] was compared. For the penetration test, the straws filled with cervical mucus and both gels were dipped into thawed semen samples and incubated at 37 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, the frozen straws were cut at 1.5-1.75 cm (penetration distance range=PDR1), 3.25-3.5 cm (PDR2) and 5.0-5.25 cm (PDR3), beginning from open-end of the straws. The separated frozen parts were then immediately transferred onto special counting slides by pushing with a mandrel and left to thaw. Thawed samples were covered with cover glass and penetrated spermatozoa in these parts were counted. The relation between the results and fertility of bulls was determined. In the tests performed using mucus, the number of spermatozoa determined in the high fertility group was found to be higher at PDR3 (p<0.0001) compared to the low fertility group, while in G1 spermatozoa number was significantly higher at PDR1 and PDR3 (p<0.0001). However, in G2 medium, no significant difference was observed between either of the fertility groups with respect to spermatozoa number determined at all distance ranges. In the study, we have determined that the gel swollen with NaCl produces better results and this gel can be used instead of bovine cervical mucus for the CMPT. Therefore, we have concluded that the penetration test performed by polyacrylamide gel swollen with NaCl can be a suitable technique for estimation of the potential fertility of bull spermatozoa.     

Relationship between bovine fertility and the number of spermatozoa penetrating the cervical mucus within straws

In this study, by using a recently developed test technique, the relationship between the total spermatozoa number penetrating determined sites of bovine cervical mucus in straws and potential fertility of bulls, and other spermatological characteristics were investigated. Furthermore, we aimed to determine the effect on the test results, of two different incubation temperatures (37 and 41 degrees C) and two sperm penetration distance ranges (PDRs). Frozen semen samples of six Holstein bulls were used in the study. The bulls were divided into two fertility groups (high and low fertility) according to the "non-return rates" (NRR). For the penetration test, cervical mucus was drawn into transparent plastic straws and incubated with semen at 37 and 41 degrees C for 15 min. After the incubation, straws were frozen in liquid nitrogen vapour and stored at -20 degrees C. On the evaluation day, concentrations of spermatozoa penetrated to the PDRs, each of which was 2.5 mm, between 32.5 and 35 mm (first penetration distance range, PDR1), and 50 and 52.5 mm (second penetration distance range, PDR2) distance in the straws from the open end, were measured. When compared with the low fertility group, bulls from the high fertility group showed a higher number of spermatozoa at the determined PDRs, and a significant positive correlation was found between the total number of spermatozoa at the penetration distances and the NRR scores of the bulls.     

Assessment of in vitro sperm characteristics after flow cytometric sorting of frozen-thawed bull spermatozoa

The effect of processing prior to sex-sorting, re-freezing and thawing of frozen-thawed bull spermatozoa on in vitro sperm characteristics was investigated. Frozen-thawed bull spermatozoa (three bulls; three ejaculates per bull) were prepared for sorting by washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility and forward progressive motility (FPM) after processing, staining, sorting and incubation (3 h; 37 degrees C). After frozen-thawed samples were processed and analyzed using a high-speed cell sorter, aliquots were removed and re-frozen and thawed (FTF-WASH; FTF-GRADIENT). Non-sorted frozen-thawed spermatozoa (FT-CONTROL) were also re-frozen and thawed (FTF-CONTROL). Spermatozoa from all treatments were assessed for penetration of an artificial cervical mucus at 0 h after sorting or thawing, and for motility, FPM and acrosomal status after 3-h incubation (37 degrees C). Frozen-thawed spermatozoa prepared by gradient centrifugation before sorting were sorted more efficiently than washed samples (P < 0.05). However, after sorting (FT) or thawing (FTF) and incubation, the percentage of motile spermatozoa and FPM rating was lower for GRADIENT than WASH (21.5 +/- 3.39%; 1.4 +/- 0.16 FPM versus 48.6 +/- 4.02%, 2.6 +/- 0.16 FPM; P < 0.01). Frozen-thawed sorted spermatozoa (FT) penetrated in greater numbers (151.0 +/- 19.50 spermatozoa) and distance (56.3 +/- 5.11 mm) in the artificial cervical mucus and had a higher proportion of motile spermatozoa (65.5 +/- 2.77%) and FPM rating (2.8 +/- 0.12) after incubation than spermatozoa that had been re-frozen and thawed after sorting (FTF: 14.0 +/- 3.67 spermatozoa, 21.6 +/- 3.05 mm, 12.2 +/- 1.31% and 1.2 +/- 0.10 FPM, respectively; P < 0.001). Regardless of processing prior to sorting, frozen-thawed sorted and non-sorted spermatozoa migrated similar distances in the artificial cervical mucus (FT-WASH: 60.0 +/- 1.2 mm; FT-GRADIENT: 57.2 +/- 0.76 mm; FT-CONTROL: 51.7 +/- 0.69 mm). The results of this preliminary study suggested that frozen-thawed bull spermatozoa can be efficiently sorted into high purity X- and Y-chromosome enriched samples with retained functional capacity.     

Influence of caffeine on movement characteristics, fertilizing capacity and ability to penetrate cervical mucus of human spermatozoa

When added to frozen-thawed human semen, the 3 doses of caffeine tested (2, 5 and 10 mM) induced a significant increase in the percentage of motile spermatozoa but did not influence the quality of movement. Considerable variability was noted between samples in their responsiveness to caffeine which, at the 5 and 10 mM doses, was significantly correlated with the degree of motility lost during cryostorage. Caffeine treatment of frozen-thawed human spermatozoa also increased the number of spermatozoa penetrating cervical mucus in unit time, by increasing the frequency rather than the success of collisions between spermatozoa and the cervical mucus interface. When caffeine-stimulated spermatozoa were washed free of seminal plasma containing this compound they were no longer at an advantage with respect to their motility or fertilizing ability. When 2 mM-caffeine was added to washed suspensions of capacitated spermatozoa it failed to stimulate motility but did significantly enhance the fertilizing ability of the spermatozoa, indicating a possible clinical role for this compound in in-vitro fertilization therapy.     

Effect of insemination with doses of 2 or 15 million frozen-thawed spermatozoa and semen deposition site on pregnancy rate in dairy cows

The effects of low-dose artificial insemination (AI) on pregnancy rates have seldom been studied in lactating dairy cows. We evaluated the pregnancy results after AI with doses of 2 and 15 million frozen-thawed spermatozoa and the effect of semen deposition in lactating dairy cows. A total of 284 first inseminations with 2 million spermatozoa and 312 first inseminations with 15 million spermatozoa were performed on 480 dairy farms. Low-dose inseminations (2 million spermatozoa) under field conditions in commercial dairy herds, without estrus synchronization, generally resulted in significantly reduced pregnancy rates compared with normal doses (15 million spermatozoa). The bull x technician effect on fertility was statistically significant. This finding indicates that there is a high variability in fertility among bulls using 2 million spermatozoa per dose. The semen deposition site did not influence pregnancy rates. It is concluded that a dose of 2 million frozen-thawed spermatozoa is probably too low for most bulls to achieve acceptable pregnancy rates in dairy cows.     

Cryopreservation of turbot (Scophthalmus maximus) spermatozoa

The aim of this study was to develop a method for cryopreserving turbot semen and to compare sperm motility characteristics, metabolic status and fertilization capacity of frozenthawed and fresh semen. The best results were obtained when spermatozoa were diluted at a 1:2 ratio with a modified Mounib extender, supplemented with 10% BSA and 10% DMSO. For freezing sperm samples, straws were placed at 6.5 cm above the surface of liquid nitrogen (LN) and plunged in LN. The straws were thawed in water bath at 30 degrees C for 5 sec. Use of this simple method resulted in a 60 to 80% reactivation rate of the thawed spermatozoa. Although the percentage of motile spermatozoa in the frozen-thawed semen samples was significantly lower than in fresh semen, spermatozoa velocity and respiratory rate remained unchanged. The process of cryopreservation significantly decreased intracellular ATP content. The fertilization rate of frozen-thawed spermatozoa was significantly lower than that of fresh spermatozoa, but it increased with sperm concentration.     

Correlation between the spermatozoal characteristics and sperm penetration distance in polyacrylamide gel and bovine cervical mucus

Correlation between the spermatozoal characteristics and the sperm penetration distance in polyacrylamide gel was assessed, utilizing frozen thawed semen samples obtained from 6 bulls, and it was compared with the correlation between sperm penetration in bovine cervical mucus and spermatozoal characteristics. In vitro sperm penetration tests were performed with mucus and gel. The sperm penetration in gel and mucus was significantly and positively correlated with post-thaw motility (r=0.81; r=0.89:P<0.01) and acrosome integrity (r=0.88; r=0.94:P<0.01). A significant negative correlation with abnormal spermatozoa (r=-0.84;r=0.83:P<0.01) was observed. Both sperm concentration and post-thaw live spermatozoa were not significantly correlated. A significant multiple regression between sperm penetration and the spermatozoal characteristics both in gel (R2=0.87; F=40.27; P<0.01) and mucus (R2=0.91; F=60.48; P<0.01) was observed. The major spermatozoal characteristics determining the capacity of spermatozoa to penetrate gel were post-thaw motility, percentage of abnormal spermatozoa and acrosome integrity. The acrosome integrity has a more significant contribution. The correlation established with sperm penetration in gel was very similar to that of sperm penetration in mucus. The utility of gel as a mucus substitute in in vitro sperm penetration tests was discussed.     

Frozen bovine semen quality and bovine cervical mucus penetration test

Research has been carried out to test bovine cervical mucus penetration (penetration) as a means for evaluating frozen-thawed bovine semen. A commercially available cervical mucus penetration test kit (the kit) was used. A total of 158 previously frozen semen samples collected from 61 bulls were thawed in a 37 C water-bath for 2 minutes. Four ways to estimate penetration were compared using the distance traveled during 90 minutes 1) at 21 C, or 2) at 37 C, by 3) the first solitary mobile spermatozoon, or by 4) the front of the mass of the mobile spermatozoa. Penetration was measured using phase contrast microscopy and a millimeter grid. Spermatozoal quality parameters (concentration, total motility, progressive motility, acrosome integrity, total sperm integrity and cytoplasmic droplets) were measured and the correlation to penetration was calculated. The best way to assay penetration with the kit was by measuring the penetration of the first solitary mobile spermatozoon at 37 C. Semen quality variability was significant (P < 0.05) relative to penetration. Linear correlations between penetration and acrosome integrity r=0.42 as well as between penetration and total sperm integrity r=0.53 were highly significant (P < 0.001). There was significant linear multiple regression between penetration and acrosome integrity (expressed as percentage and number) and total sperm integrity (expressed as percentage and number) (r=0.62; F=23.5147; P<0.0001). There was a significant difference between the average progressive motility of samples with penetration > 20 mm and samples with penetration 20%), but it is not useful to define the fertility level of semen samples.     

On-Chip Cryopreservation: A Novel Method for Ultra-Rapid Cryoprotectant-Free Cryopreservation of Small Amounts of Human Spermatozoa

Cryopreservation of human spermatozoa free from cryoprotectant can avoid toxicity caused by highly concentrated cryoprotectant and a series of specific carriers have been previously explored, except for PDMS chip. Our study is aimed at exploring a novel device for ultra-rapid cryopreservation of small numbers of spermatozoa without cryoprotectant based on polydimethylsiloxane (PDMS) chips. Spermatozoa from 25 healthy men were involved in this study, comparing on-chip cryopreservation with different micro-channel height (group A: 10 µm height, group B: 50 µm height, group C: 100 µm height) and conventional freezing (group D) in liquid nitrogen for 72 h. The viability, motility, DNA integrity by comet assay and acrosome integrity by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA) staining of frozen-thawed spermatozoa of each group were compared. The motility and viability of post-thawed spermatozoa was significantly decreased than that of pre-freezing spermatozoa. There was no difference of viability and motility of frozen-thawed spermatozoa between group A and D, while viability and motility of group B and C decreased compared to group A. Comet assay showed that no matter for group A or D, there was no difference of CR, TL, TD and OTM between pre-frozen and post-thawed spermatozoa. There was no difference of CR, TL, TD and OTM of post-thawed spermatozoa between group A and group D neither, while spermatozoa DNA damage was more serious in group B and group C with increasing height of micro-channel compared with group A. The proportion of intact acrosome of post-thawed spermatozoa in group A was the highest when compared with group B and group C, though similar to that of group D. In conclusion, PDMS chip with 10 µm height micro-channel is ideal for ultra-rapid cryopreservation of small quantity of spermatozoa without cryoprotectant.     

Improvement in post-thaw viability of in vitro-produced bovine blastocysts vitrified by glass micropipette (GMP)

The present work was designed to study the in vitro and in vivo viability, as assessed by blastocyst formation, pregnancy rate and term delivery of bovine embryos produced under completely defined conditions with or without insulin-like growth factor I (IGF-I) following direct transfer after cryopreservation. Slaughterhouse-derived bovine oocytes were matured for 24h, fertilized with frozen-thawed spermatozoa and cultured in vitro under completely defined conditions with or without exposure to IGF-I (5 ng/ml). Only those embryos classified as excellent or good quality blastocysts were frozen. Each blastocyst was individually loaded into a straw, seeded and pre-cooled to -7 degrees C. After 10 min at -7 degrees C straws were frozen further to -30 degrees C at a rate of 0.3 degrees C/min and then plunged into liquid nitrogen. Synchronized recipient cows received one embryo in the horn ipsilateral to the corpus luteum (CL). Pregnancies were diagnosed by ultrasonography 35-45 days after embryo transfer (ET). IGF-I failed to improve cleavage rate, as well as blastocyst production, when added during in vitro culture (IVC). Pregnancy outcome was not significantly improved in cows that received an IGF-I-treated embryo compared with controls (4/10 versus 3/10, respectively). Five out of six calves delivered to date were born alive and healthy. We have shown that it is possible to obtain healthy live offspring from frozen-thawed embryos produced under chemically defined conditions after direct transfer.     

Effects of cooling and warming conditions on post-thawed motility and fertility of cryopreserved buffalo spermatozoa.

The principal objective of this study was to derive an improved procedure for cryopreservation of swamp buffalo (Bubalus bubalis) spermatozoa. Experiments were conducted to determine effects of cooling rate, intermediate plunge temperature and warming rate on motility and acrosome integrity of spermatozoa. Spermatozoa were obtained from three bulls (three ejaculates/bull) and were subjected to nine cooling conditions before being frozen in liquid nitrogen: cooling at 10, 20, or 30 degrees C/min each to -40, -80, or -120 degrees C before being plunged into liquid nitrogen. The spermatozoa frozen under a given condition were then thawed either at 1000 or 200 degrees C/min. Cooling rate, intermediate temperature and warming rate significantly affected survival of spermatozoa obtained from the three bulls. Cooling spermatozoa from 4 to -120 degrees C either at 20 or 30 degrees C/min yielded better progressive motility compared to other cooling conditions (50 versus 30%). Rapid warming was superior to slow warming. In an additional study, motility and fertility of spermatozoa frozen after being cooled to -120 degrees C at 20 degrees C and 30 degrees C/min and those frozen by a standard protocol used routinely for semen processing were assessed. Progressive motility of cryopreserved spermatozoa cooled at 20 degrees C and 30 degrees C/min was 40%, while that of spermatozoa cryopreserved using a standard protocol was 25%. A total of 178 buffalo cows were inseminated with cryopreserved spermatozoa obtained from one bull, and their pregnancy status was assessed 60 days later by rectal palpation. Out of the 60, 26 (43%) and 23 of 58 (40%) cows inseminated with sperm cooled at 20 and 30 degrees C/min, respectively, became pregnant, whereas 17 of 60 (28%) cows inseminated with sperm frozen by a standard protocol became pregnant. This study demonstrates that an effective cryopreservation procedure for buffalo spermatozoa can be derived by systematic examination of various cryobiological factors.     

Intra- and interspecies comparison of sperm migration through polyacrylamide gel as an index of spermatozoal viability

Sperm migration distance through a polyacrylamide gel mimicking cervical mucus was compared among seven species: dog, cat, goat, ram, clouded leopard, dorcas gazelle, and Eld's deer. Freshly collected spermatozoa were evaluated microscopically and then assayed for migration distance in gel-filled capillary tubes in raw or diluted concentrations or after longevity declines in sperm motility and progressive status. Sperm count per milliliter of ejaculate and sperm percent motility ratings varied (P < 0.05) among selected species. Undiluted ejaculated spermatozoa from all species, except the clouded leopard, penetrated the polyacrylamide gel. Migration distance (mm) was different (P < 0.05) among species and not clearly correlated to concentration or motility factors. Although sperm concentrations in the dog and Eld's deer were similar, average migration distance of deer sperm was more than fourfold greater (P < 0.01) than that of dog sperm. The sperm motility rating for the gazelle was greater (P < 0.05) than that for the cat; however, penetration distance in the cat was more than twice as great (P < 0.05) as that in the gazelle. Species also varied in migration response after altering sperm concentration: Halving the sperm count of the dog, gazelle, and Eld's deer had no effect, but the same procedure decreased (P < 0.05) penetration distance in the goat and ram. No migration was observed in any species at a sperm concentration of 25 × 10⁶ cells or less. Within species a decline in sperm percent motility-progressive status ratings was correlated (P < 0.05) to a subsequent decrease in penetration distance. These results provide a comparative assessment of sperm cell migration through a synthetic cervical mucus and suggest that this test may be a useful adjunct in evaluating reproductive potential. However, the assay is specific in interpretive merit, as sperm penetration distance within a single batch of gel is markedly variant among species and in some species dependent on sperm concentration. This interspecies specificity in sperm migration through a homogeneous gel suggests intrinsic species variance in sperm cell-cervical mucus interaction, suggesting that this assay could be valuable in future studies of the mechanism of sperm transport.     

Comment identifier le spermatozoïde vivant?

Testicular sperm extraction (TESE) has been used to retrieve spermatozoa in patients with secretory azoospermia for intracytoplasmic sperm injection (ICSI). However, testicular spermatozoa have poor motility that significantly decreases after cryopreservation and thawing. The major difficulty with testicular spermatozoa is to distinguish between living and dead spermatozoa, as most spermatozoa are immotile. The aim of this study was firstly to report the various methods used to explore spermatozoa vitality. Most tests assess the functional and structural integrity of the sperm membrane, such as staining methods and hypo-osmotic swelling test (HOS-test). We then evaluates the potential of pentoxifylline (PTX), a phosphodiesterase inhibitor of the methylxanthine group, to improve the distinction between living and dead immotile testicular spermatozoa by increasing the number of post-thawed motile spermatozoa. We also analysed the results of 100 ICSI cycles performed with frozen-thawed testicular (n=72) and epididymal (n=28) spermatozoa treated with 3.5 mM PTX. To test the effect of PTX on motility, 14 samples of frozen-thawed testicular spermatozoa from eight patients with secretory azoospermia and six patients with excretory azoospermia were divided into three equal samples: one sample treated with 3.5 mM PTX, one sample initially migrated on two-layer Percoll gradient and then divided into two aliquots (one treated with 3.5 mM PTX, one without treatment), and the last sample without migration and without PTX treatment. The number of motile spermatozoa was evaluated in 10 μL of each sample with an inverted microscope at 15, 30, 60, 120 minutes and 24 hours. We also compared the outcome of ICSI in 100 cycles using frozen-thawed epididymal or testicular spermatozoa between secretory and excretory patients. PTX significantly increased the number of motile frozen-thawed testicular spermatozoa in secretory and excretory azoospermia. In excretory azoospermia, the number of motile spermatozoa was further increased when PTX was associated with migration on Percoll gradient, while PTX alone gave the best results in secretory azoospermia. Fertilization and pregnancy rates as well as embryo quality and division stages were comparable in the two groups. By increasing the number of motile frozen-thawed testicular spermatozoa, PTX improves the selection of living spermatozoa.     

Chemotactic response of frozen-thawed bovine spermatozoa towards follicular fluid

The aim of this study was to verify whether cattle spermatozoa respond by chemotaxis to follicular fluid (FF). The experimental conditions were defined to maintain a frozen-thawed sperm population with great motility and capacitation, and lesser sperm agglutination. Several sperm preparation conditions were studied: sperm separation from the seminal plasma by Sephadex column or migration-sedimentation, incubation under capacitating conditions in the presence or absence of a superficial layer of mineral oil, and different pH of the culture medium. The percentage of motile and agglutinated spermatozoa was determined in plate dishes under inverted phase contrast microscope. The percentage of capacitated spermatozoa was calculated as the difference between the percentages of acrosome reacted spermatozoa with and without lysophosphatidylcholine stimulation. The most ideal experimental conditions to evaluate chemotaxis in frozen-thawed cattle spermatozoa were: to separate the cells from the seminal plasma by migration-sedimentation and to incubate them under oil, in culture medium at pH 7.2, for less than 2h. The chemotaxis assays were conducted with spermatozoa treated as mentioned above which were confronted to several dilutions of FF (1:10(3), 1:10(4), 1:10(5), 1:10(6)) in a chemotaxis chamber by videomicroscopy and computer image analysis. A subpopulation of capacitated spermatozoa ( approximately 10%) that responded chemotactically to a concentration gradient generated by FF (1:10(4) to 1:10(5)) was observed. Since cryopreserved spermatozoa are regularly used to artificially inseminate the cows, the sperm chemotactic response towards FF would be potentially used to diagnose the bull sperm sample or to select the spermatozoa in the most functional state.     

Prognostic value of canine frozen-thawed semen parameters on in vitro sperm-oocyte interactions

Combining the data from conventional semen analysis with oocyte penetration assays should improve the assessment of the fertilizing ability of a semen sample. Thus, the objective of the present study was to evaluate the prognostic value of various semen parameters on the in vitro interactions between frozen-thawed canine sperm and homologous oocytes. Ten ejaculates from five stud dogs (two ejaculates/dog) were collected by digital manipulation. Semen samples were evaluated, extended in Tris-egg yolk-glycerol, frozen and stored in liquid nitrogen, and thawed several weeks later. Samples were evaluated for motility and sperm populations by computer-aided semen analysis (CASA), plasma membrane integrity (carboxy-fluorescein diacetate and propidium iodide), and sperm morphology (Bengal Rose). Thawed spermatozoa were also incubated with homologous oocytes for 18 h in an atmosphere of 5% CO(2) and 95% air at 38 degrees C and sperm-oocyte interactions were evaluated. Simple linear regression models were calculated, with sperm parameters as independent variables and sperm-oocyte interactions as the dependent variable. There were significant associations between: percentage of oocytes bound to spermatozoa and beat cross frequency (BCF; R(2)=63%); percentage of oocytes that interacted with spermatozoa and BCF (R(2)=73%); and number of penetrated spermatozoa and velocity average pathway (VAP; R(2)=64%) and velocity straight line (VSL; R(2)=64%). Although plasma membrane integrity and sperm morphology had little prognostic value for in vitro interactions between canine frozen-thawed sperm and homologous oocytes, some motility patterns (evaluated by CASA) were predictive of in vitro sperm-oocyte interactions.     

Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla)

We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.     

Attempts on freezing the Greylag (Anser anser L.) gander semen

Semen of Greylag (Anser anser L.) ganders was frozen according to a method previously elaborated by the authors for freezing the White Koluda gander semen. Semen was collected from five to eight Greylag ganders, twice a week during three succeeding reproductive cycles, by dorso-abdominal massage. Semen samples were diluted in the ratio of 1:1 or 2:1 (two parts semen: one part diluent) with EK diluent, supplemented by 6% DMF, equilibrated and pre-frozen to -140 degrees C at a rate 60 degrees C/min, before being transferred into liquid nitrogen container. Semen samples thawed in a water bath of 60 degrees C were used for twice a week insemination in a volume of 200 microl. Three Greylag and three White Koluda geese were involved in frozen-thawed semen fertilizing ability test. The reproductive cycle of wild geese lasts usually about 6-7 weeks. The ejaculate volume (30-140 microl) and sperm concentration (10x10(6) to 150x10(6) ml(-1)) are much lower than these of domestic ganders, but spermatozoa morphology is similar, particularly while compared to 1-year-old White Koluda ganders semen. There are about 90% of live spermatozoa and about 30% of live morphologically normal cells in Greylag gander fresh semen. The Greylag gander spermatozoa susceptibility to cryopreservation procedure is as high as in domestic ganders. Dilution ratio 2:1 resulted in higher number of live spermatozoa, which withstood cryoinjury stress. In relation to fresh semen about 60% of spermatozoa remained intact (on the basis of light microscope examination) in the frozen-thawed semen. Insemination of frozen-thawed semen resulted in 37.5% of fertile eggs in Greylag and 25.0% in White Koluda geese. Low fertility rate was caused by an insufficient number of live normal spermatozoa used for insemination (about three million in every dose).     

Protein tyrosine phosphorylation and zona binding ability of in vitro capacitated and cryopreserved buffalo spermatozoa

Many similarities between the changes associated with normal capacitation and cryocapacitation have been demonstrated. The present study was undertaken to determine whether similarities exist in the protein tyrosine phosphorylation pattern and zona binding ability between in vitro capacitated (heparin induced; 20 μg/ml) and frozen-thawed (cryocapacitated) buffalo spermatozoa. Semen from seven buffalo bulls (eight ejaculates each) was divided into two parts. Part I was used as fresh semen and part II was extended in Tris-egg yolk extender, equilibrated and frozen in liquid nitrogen. Localization of phosphotyrosine-containing protein was determined using an indirect immunoflourescence assay with anti-phosphotyrosine antibody. For zona binding assay, good quality oocytes collected by aspiration technique from fresh buffalo ovaries were used. The bound spermatozoa were stained with Hoechst 33342 dye and observed under fluorescent microscope. The results revealed sperm head associated protein tyrosine phosphorylation in both in vitro capacitated and frozen-thawed spermatozoa. In the zona binding assay, the mean number of bound spermatozoa was 90.6 ± 1.9 and 104.7 ± 2.2 in fresh semen after incubation in non capacitating media at 0 h and 3 h, respectively. But after incubation in capacitating media with heparin for 3 h, the mean number of spermatozoa attached to zona pellucida was 138.4 ± 2.6. The in vitro capacitated spermatozoa had significantly (P < 0.05) higher binding ability than that of fresh spermatozoa. After freezing and thawing, 2.5 fold reductions in the zona binding ability of cryopreserved spermatozoa was observed compared to in vitro capacitated spermatozoa. The binding ability of in vitro capacitated spermatozoa was significantly (P < 0.01) higher than that of frozen-thawed (cryocapacitated) spermatozoa. The study concluded that both in vitro capacitated and frozen-thawed (cryocapacitated) spermatozoa had similar immune-localization of tyrosine phosphorylated protein pattern, however, differed in the zona binding ability.     

Effect of preincubation of cryopreserved porcine epididymal sperm

Preincubation of spermatozoa is important for capacitation and successful fertilization in vitro. The effects of preincubation time on frozen-thawed boar epididymal spermatozoa as measured by sperm motility, acrosomal integrity and fertilization ability in vitro were examined. Epididymal spermatozoa were collected from three Large White boars and frozen. The thawed spermatozoa were preincubated for 0, 15, 30, 60 and 120 min. Their motility was evaluated by a sperm motility analyzer and then the sperm motility indexes (SMIs) were calculated. The status of their acrosomal integrity was evaluated by triple-staining. Then, their fertilization ability was examined by in vitro fertilization (IVF) using porcine oocytes matured in vitro. SMIs of spermatozoa and the incidences of acrosome-intact live spermatozoa from the three boars were high (21-39 for SMI and 50-61% for acrosome-intact live spermatozoa) just after thawing, but both decreased as the duration of preincubation was prolonged (2-10 and 23-40%, respectively). The incidences of sperm penetration were high (61-89% of inseminated oocytes) when the sperm were preincubated for 0-60 min. However, sperm penetration decreased as the preincubation period was prolonged to 120 min. The degree of this decrease differed depending upon the boar from which the spermatozoa were obtained (10-72%). When the two parameters, sperm motility and acrosomal integrity, were analyzed statistically, the latter parameter rather than the former one showed a significant effect on penetration ability in vitro after each duration of preincubation. These results suggest that preincubation of frozen-thawed boar epididymal spermatozoa is not required for IVF and also that the maintenance of acrosomal integrity in unreacted status, rather than the maintenance of sperm motility, is important for fertilization ability after thawing and during preincubation of boar epididymal spermatozoa.