Successful fertilization of Varicorhinus macrolepis eggs with sperm subjected to two freeze-thaw cycles

Although sperm from several fish species have been successfully cryopreserved, few studies have been done in small and/or endangered species. The aim of the present work was to develop a method of freezing and refreezing Varicorhinus macrolepis semen in 1.8 mL cryovials. The effect of extenders and cryoprotectants on the motility of post-thaw sperm was examined. The motility of frozen-thawed sperm in extender D-15 was higher than that in MPRS and fish Ringer solution (P<0.05). Dimethyl sulfoxide (DMSO) and glycerol provided greater protection to sperm than methanol during freezing and thawing; the most effective concentration of DMSO and glycerol was 10%. The fertilization rate of frozen-thawed sperm was not significantly different from that of fresh sperm. Furthermore, mean (+/-S.D.) hatching rate did not differ significantly between frozen-thawed (82.7+/-12.4%) and fresh sperm (90.7+/-4.5%). Although frozen-thawed sperm that was immediately refrozen had 0% post-thaw motility, frozen semen that was refrozen after dilution with D-15 (containing DMSO at a ratio of 1:2) had post-thaw motility of 38.3+/-2.9%. Motility was lower for refrozen than for frozen sperm (P<0.05). Furthermore, fertilization and hatching rates of refrozen sperm were 42.9+/-6.7 and 34.1+/-10.5%, respectively, which were lower than that of fresh sperm (P<0.05).     

Quality control of refrigerated and cryopreserved semen using computer-assisted sperm analysis (CASA), viable staining and standardized fertilization in African catfish (Clarias gariepinus)

A new integrated approach including computer-assisted sperm analysis (CASA), viability staining and fertilization was used to study the quality of cryodiluents used in fish sperm cryopreservation. As an example the sperm quality of an African catfish, Clarias gariepinus (Burchell, 1822), was assessed by its fertilizing ability, motility and viability at day 0 (fresh), after 2 days' storage at 4 degreesC and after 2 days, 5 months and 10 months frozen at -196 degreesC using solutions containing dimethyl sulphoxide (DMSO) or glycerol as permeating cryoprotectants. Four of the best freezing solutions were used, namely, Steyn's extender (S1, S4) and Mounib's extender (M3, M4) associating 10% hen's egg yolk. Progressive sperm movement measured by CASA and expressed by the straight line velocity (VSL), the average path velocity (VAP) and the curvilinear velocity (VCL) was highly correlated with hatching rates obtained from fertilization using minimal sperm:egg ratios. After 2 days, the motility of spermatozoa frozen with DMSO and 10% egg yolk had deteriorated less than that of spermatozoa stored at 4 degreesC. Post-thaw hatching rates reflected the post-thaw sperm viability, which was cryodiluent dependent: 14.9+/-2.0% (S4), 17.0+/-4.2% (S1), 25.9+/-3.7% (M4) and 52.1+/-3.4% (M3) after 5 months of cryopreservation. The percent motility of 10-months-frozen spermatozoa was high in M3 (70.7+/-11.4%) and M4 (64.0+/-2.0%) cryoprotected sperm when measured between 5 and 20 sec after activation, but decreased rapidly to 24.3+/-8.3% (M3) and 23.0+/-9.0% (M4) between 21 and 35 sec after activation. Mounib's extender (M3, M4) provided the best cryoprotection to the spermatozoa for all post-thaw sperm quality measurements and at all freezing durations. Sperm motility was positively related to fertility. Our method will make it possible to develop even better extenders and cryoprotectants.     

Comparisons between three different extenders for canine intrauterine insemination with frozen-thawed spermatozoa

Ejaculates from 7 dogs were obtained on the same day and were pooled. This pooled semen was separated into 3 equal fractions and processed simultaneously, the only difference being in the extender used for freezing. The extenders were laiciphos (containing laiciphos, egg yolk, distilled water and glycerol- Group 1); Tes/Tris (containing Tes/Tris, egg yolk, distilled water and glycerol- Group 2); and biociphos (containing biociphos with glycerol in it, egg yolk and distilled water- Group 3). Spermatozoa were conditioned in 0.5ml French straws and presented normal characteristics before freezing and after thawing. The sperm concentration of the pooled was 683 x 10(6) sperm/ml; sperm motility was above 95%, the percentage of live spermatozoa was above 95% and was of good quality and mobility. Characteristics of the spermatozoa after thawing were the same for spermatozoa frozen with laiciphos and Tes/Tris. Mean sperm concentration was 201.5 +/- 4.95 x 10(6) sperm/ml, sperm motility was 65%, the percentage of live spermatozoa was 80% and the quality of motility.was good. Spermtozoa frozen with biociphos had the following post-thaw characteristics: sperm concentration was 201 x 10(6) sperm/ml, sperm motility was 50%, the percentage of live spermatozoa was 78% and the quality of mobility was medium. Abnormalities were less than 15% for all spermatozoa after thawing. Intrauterine artificial inseminations were performed by laparoscopic intrauterine insemination twice at Days 3 and 5 after the estimated LH peak in 15 normally cyclic Beagle bitches (5 per group) presenting normal hormonal profiles. There were no differences between groups. The females were inseminated with 1.0 ml of spermoatozoa (concentration of 200 x 10(6) sperm/ml) diluted with 1.0 ml of extender. A 60% pregnancy rate was obtained in bitches inseminated with frozen-thawed spermatozoa extended with laiciphos or Tes/Tris and 100% in bitches inseminated with spermatozoa extended with biociphos. Females inseminated with laiciphos, Tes/Tris and biociphos had a mean litter size of 5 +/- 2.6, 3 +/- 1 and 3.4 +/- 1.3 pups, respectively. This study demonstrated that post-thaw assessment of sperm characteristics is not the best technique for evaluating sperm fertility after freezing or for assessing different semen extenders.     

Motility and fertility of bull sperm in whole milk extender containing antioxidants

Bull sperm are exposed to aerobic conditions during processing before freezing, and they have little endogenous antioxidant to protect them against reactive oxygen species that may be present. Seventeen laboratory studies and two field trials were conducted with 174 semen collections from bulls in an artificial breeding cooperative. More than 250 combinations and concentrations of reduced glutathione (GSH), superoxide dismutase (SOD), ascorbic acid, hypotaurine (HPT), 2,2,6,6-tetramethylpeperidine-1-oxyl (Tempo) and 4-hydroxy-2, 2, 6, 6-tetramethylpeperidine (Tempol) were tested by adding these compounds to fresh semen, and to a whole milk (WM) glycerol extender. Semen packaged in straws in the WM extender was frozen with liquid nitrogen. The motility of frozen-thawed sperm during storage at 25 or 5 degrees C after freezing was compared with semen stored without freezing. Antioxidants generally were not beneficial, except the percentage of motile sperm was improved by 6-11% units (P<0.05) when sperm were stored unfrozen or after freezing when 0.5mM of GSH with or without SOD was added. In two field trials, non-return rates were 71.9, 69.5 and 70.9% (P>0.05) with WM containing 0.0, 0.5 and 1.0mM of GSH, respectively, and 74.0 and 73.9% with WM and WM plus 0.5mM of GSH and 100U/ml of SOD (P>0.05). WM contains an abundant supply of casein which is an antioxidant, and additional antioxidants were ineffective in improving motility of sperm immediately after freezing and thawing or in affecting fertility. However, sperm responses were different in egg yolk-Tris extender. Sperm in this egg yolk extender tolerated substantial concentrations of Tempo and Tempol compared with toxic effects in WM (P<0.05). Therefore, optimal combinations of antioxidants tested here may have more useful applications in organizations using an egg yolk-based semen extender.     

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.     

Influence of extender,temperature, and addition of glycerol on post-thaw sperm parameters in ram semen

Using a two-step extension methodology, two experiments were conducted using a split-sample design to compare the effect on post-thaw ram sperm parameters of a milk-based extender (Experiment 1) containing four different egg yolk concentrations (5% [M5], 10% [M10], 15% [M15], and 20% [M20]), and a commercially available extender (Bioexcell); IMV, L'Aigle, France) free from additives of animal origin, containing two different final glycerol concentrations (3.2% [B] and 6.4% [BB]) (Experiment 2). In both experiments, glycerol was added either at 5 degrees C or at 15 degrees C together with the second fraction of each extender. The sperm characteristics assessed were motility (measured subjectively [SM] and by means of cell motion analysis (CASA), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline (CTC)/EthD-1). Results of Experiment 1 showed no significant positive effect of increasing the concentration of egg yolk above 10% on post-thaw motility, membrane integrity, or induction of sperm capacitation-like changes. In Experiment 2, Bioexcell (BB) yielded similar post-thaw results as did the milk extender (control). In both experiments, post-thaw sperm parameters were better preserved when glycerol was added at 5 degrees C, although the results were not always statistically significant for all variables studied. In conclusion, when using milk-based extenders for freezing ram semen, low (5-10%) concentrations of egg yolk and the addition of glycerol at 5 degrees C are recommended. Furthermore, the results indicate that when freezing ram semen, Bioexcell containing 6.4% glycerol may be used as an alternative extender to the conventional milk extender containing 5% egg yolk.     

Liposomes as an alternative to egg yolk in stallion freezing extender

Egg yolk is normally used as a protective agent to freeze semen of equine and other species. However, addition of egg yolk in extenders is not without disadvantages and the demand to find cryoprotective alternatives is strong. The objective of this study was to test the cryoprotective capacities of liposomes composed of egg yolk phospholipids. Two experiments were conducted: 1) the first to determine the optimal composition and concentration of liposomes to preserve post-thaw motility and membrane integrity of spermatozoa; 2) the second to assess in vivo the cryoprotective capacities of these liposomes. In Experiment 2, post-thaw motility and membrane integrity of spermatozoa were also analyzed. Experiment 1 demonstrated that liposomes composed of phospholipids E80 (commercial lecithins from egg yolk composed mainly of phosphatidylcholine and phosphatidylethanolamine) and of Hank's salts-glucose-lactose solution (E80-liposomes) were the most efficient in preserving post-thaw motility. The optimal concentration was 4 % (v/v). In Experiment 2, fertility rate after artificial insemination of semen frozen with E80-liposomes was 55 % (22/40) compared with 68 % (27/40) with the control extender containing egg yolk (EY) (p = 0.23). Post-thaw motility parameters were higher with EY than with E80-liposomes (p < 0.0001). For post-thaw membrane integrity no difference was observed between the two extenders (p = 0.08). Liposomes composed of egg yolk phospholipids appeared to be a promising alternative to replace egg yolk in semen freezing extenders in equine species.     

Evaluation of duck egg yolk for the cryopreservation of Nili-Ravi buffalo bull semen

This study was carried out to investigate if the substitution of chicken egg yolk (CEY) with duck egg yolk (DEY) in extenders can improve the quality of frozen-thawed semen of Nili-Ravi buffalo bulls and to study if reducing DEY level in extender affects the freezability results. Thirty semen samples collected from three buffalo bulls were diluted in extenders A, B, C, D and E containing tris, citric acid, fructose, egg yolk, glycerol and antibiotics. Extender A contained 20% CEY (control), while extenders B, C, D and E contained 5, 10, 15 and 20% DEY, respectively. After freezing and storage for 24h in liquid nitrogen, samples were evaluated for post-thaw quality. The post extension sperm motility did not differ between extenders A (control) and E (20% DEY). The same was true for post-thaw percentage of sperm with functional plasma membrane and percentage of sperm with abnormal heads or mid pieces. However, extender E showed higher (P<0.05) values for post-thaw sperm motility, livability and absolute index of livability of spermatozoa at 37 °C compared to extender A. Spermatozoa with abnormal tail were lower (P<0.05) in extender E compared to extender A. Values of these parameters of post-thaw semen quality were highest for extender E containing 20% DEY and decreased significantly with decrease in the concentration of DEY, except sperm abnormalities (head, mid-piece and tail) which increased with decrease in DEY level. These results showed that replacement of 20% CEY with 20% DEY in extenders significantly improved post-thaw sperm motility, livability and absolute index of livability of spermatozoa and reduced tail abnormalities. Reduction in the level of DEY in extenders from 20% adversely affected post-thaw semen quality of Nili-Ravi buffalo bulls.     

Effects of egg yolk during the freezing step of cryopreservation on the viability of goat spermatozoa

Four experiments were conducted to investigate the effects of egg yolk during the freezing step of cryopreservation (namely, the process except for the cooling step), on the viability of goat spermatozoa. The effects of egg yolk on sperm motility and acrosome integrity during the freezing step were investigated in Experiment 1. Spermatozoa diluted with Tris-citric acid-glucose (TCG) solution containing 20% (v/v) egg yolk were cooled to 5 degrees C, washed, and then frozen in TCG with egg yolk (TCG-Y), TCG without egg yolk (TGG-NY), 0.370 M trehalose with egg yolk (TH-Y), or trehalose without egg yolk (TH-NY). All extenders contained glycerol. In frozen-thawed spermatozoa, the inclusion of egg yolk in the freezing extenders increased (P<0.05) percentages of motile sperm, progressively motile sperm, and the recovery rate (ratio of post-thaw to pre-freeze values), but decreased (P<0.05) acrosomal integrity. Moreover, extenders with trehalose had better (P<0.05) post-thaw sperm viability. In Experiment 2, the effects of egg yolk on acrosome status before and after freezing were studied. Egg yolk significantly decreased the proportion of intact acrosomes before freezing, leading to fewer (P<0.05) intact acrosomes post-thaw and lower (P<0.05) recovery rates for intact acrosomes. In Experiment 3, including sodium dodecyl sulfate (SDS) in a diluent containing egg yolk tended to preserve the acrosome compared with the egg yolk containing diluent free of SDS, however, spermatozoa had a lower (P<0.05) proportion of intact acrosomes than those in a yolk-free diluent. However, after cooling, spermatozoa were diluted with a glycerolated extender containing egg yolk. Therefore, the objective of Experiment 4 was to explore whether the egg yolk or glycerol was responsible for the reduced intact acrosome percentage. In this experiment, after cooling and washing the spermatozoa were diluted in TCG with glycerol and/or egg yolk. The combination of glycerol and egg yolk in the extender reduced (P<0.05) the proportion of intact acrosomes compared with egg yolk or glycerol alone. In conclusion, the inclusion of egg yolk significantly improved sperm motility, indicating its beneficial effects during the freezing step of cryopreservation; trehalose appeared to synergistically increase its cryoprotective effects. Furthermore, although neither glycerol nor egg yolk per se affected the proportion of intact acrosomes, the combination of the two significantly reduced the proportion of acrosome-intact spermatozoa.     

Production and storage of goat semen for artificial insemination

Environmental influences on reproduction and semen production in the buck, the problem of interaction between seminal plasma and egg yolk or milk constituents in diluent, liquid storage and processing of semen for freezing are discussed. A review is given on the use of frozen-thawed semen for artificial insemination (AI) in spontaneous and induced oestrus and factors influencing the fertility.     

Duck egg yolk in extender improves the freezability of buffalo bull spermatozoa

We investigated the use of duck egg yolk (DEY), Guinea fowl egg yolk (GFEY) and Indian indigenous hen (Desi) egg yolk (IDEY) in extender for improving the post-thaw quality of buffalo (Bubalus bubalis) bull spermatozoa, and compared it with commercial hen egg yolk (CHEY; control). For this purpose, two consecutive ejaculates of semen from each of two Nili-Ravi buffalo bulls were collected on 1 day each week for 5 weeks (replicates; n=5) with artificial vagina (42 degrees C). Split pooled ejaculates, were diluted in tris-citric acid glycerol extender containing either DEY or GFEY or IDEY or CHEY at 37 degrees C. Extended semen was cooled to 4 degrees C in 2 h and equilibrated for 4 h at 4 degrees C. Cooled semen was then filled in 0.5 ml straws at 4 degrees C and frozen in programmable cell freezer. Thawing of semen was performed at 37 degrees C for 30 s. Sperm motility, plasma membrane integrity and sperm morphology (acrosome integrity, head, mid-piece and tail abnormalities) of each semen sample were assessed at 0, 3 and 6 h after thawing and incubation at 37 degrees C. Visual motility (%) and percentage of intact plasma membranes assessed at 6h post-thaw of buffalo bull spermatozoa were highest (P<0.05) due to DEY as compared to GFEY, IDEY and control. The percentage of spermatozoa with normal acrosomes at 0, 3 and 6 h post-thaw was highest (P<0.05) in DEY extender than GFEY, IDEY and CHEY. Sperm tail abnormalities (%) observed at 0, 3 and 6 h post-thaw in samples cryopreserved with freezing extender having DEY were lower (P<0.05) as compared to extender containing GFEY, IDEY and CHEY. In conclusion, DEY compared to other avian yolks in extender improves the frozen-thawed quality of buffalo bull spermatozoa.     

Evaluation of a new diluent and different processing procedures for cryopreservation of ram semen

Ram semen was processed for freezing after initial dilution with a modified Tris-fructose diluent. Two aliquots were processed by cooling gradually to 5 degrees C, further dilution, equilibration and freezing in 0.5 ml straws either in pressurized liquid nitrogen (LN(2)) vapor (Method A) or on a block of dry ice (Method B). A third aliquot was cooled rapidly to 16 degrees C and then slowly to 5 degrees C, diluted further, equilibrated and frozen in straws in pressurized LN(2) vapor (Method C). The second dilution was carried out using a new diluent based on dextran-lactose. The diluted semen was equilibrated for 2 h before freezing. Semen was evaluated by artificial insemination (AI). The fertility of ewes bred by a double insemination with frozen-thawed semen processed by Methods A, B and C was 73% (n = 33), 67% (n = 30) and 80% (n = 30), respectively. In comparison, the fertility of ewes inseminated with fresh semen was 93% (n = 31). These preliminary data indicate an acceptable fertility can be achieved by AI with frozen-thawed semen processed using improved procedures.     

Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males

This study aimed to establish artificial insemination (AI) protocols to predictably initiate pregnancy during the breeding season in the European brown hare (EBH) (Lepus europaeus PALLAS, 1778). Semen was collected from seven captive and eight free-ranging males by means of electroejaculation. Semen from the free-ranging males was cryopreserved using directional freezing. Total motility/integrity of fresh and frozen-thawed semen was 91.6%/87.7% and 46.9%/53.8%, respectively. Ovulation was induced in ultrasonographically preselected females using a gonadotropin-releasing hormone analogue. Each female was inseminated with 1 mL fresh (Group A, n = 16) or frozen-thawed semen (Group B, n = 9) at a concentration of 100 × 10⁶ spermatozoa/mL. The use of ultrasonography (10 to 22 MHz) confirmed the intracervical semen deposit, the success of artificial ovulation induction (formation of postovulatory corpus luteum), and permitted the monitoring of individual pregnancies. Although sperm motility/integrity was significantly different between groups, no significant difference was detected in conception rates (A, 87.50%; B, 77.78%). Because of embryonic resorption, there was a slight difference in fertility rate between groups (A, 62.5%; B, 77.78%). Overall, AI in captive EBH using fresh and frozen-thawed semen achieved successful fertility rates. Long-term cryopreserved semen was used to bring new genetic material from the wild into a genetically limited captive population without extensive animal transport. Therefore, AI has the potential to enhance breeding programs for EBH especially when cryopreserved semen from wild donors is used.     

Effect of Bradykinin on Murrah buffalo (Bubalus bubalis) semen cryopreservation

Keeping in view the poor freezability of bubaline semen in conventionally used extenders, this study was conducted on three Murrah bulls to improve semen cryopreservation with the incorporation of Bradykinin (0.5, 1.0 and 2.0 ng ml(-1)) in routinely used egg yolk tris-glycerol (EYTG) extender. Bradykinin (2.0 ng ml(-1)) had significant (P<0.05) beneficial effect on live sperm % (81.6+/-1.8) and hypo osmotic swelling (HOS) % (63.0+/-1.3) as compared to their respective control values of 73.4+/-2.1 and 56.3+/-2.0 at 0 h post freezing. The post-thaw progressive sperm motility in semen samples diluted with EYTG containing 2.0 ng ml(-1) Bradykinin (65.5+/-1.4) was also significantly (P<0.01) higher than control (60.3+/-1.9) at 0 h post freezing. Thus incorporation of 2 ng ml(-1) Bradykinin in buffalo semen diluted in EYTG extender may be useful in improving the quality of cryopreserved bubaline semen.     

Successful pregnancies with directional freezing of large volume buck semen

Artificial insemination with frozen-thawed buck semen shows variable results which depend on many factors related to semen quality and the cryopreservation processing. We conducted experiments based on a new freezing method, directional freezing, of large volumes (8 ml). In the first experiment semen from three Saanen bucks, ages 1-2-years-old and genetically selected for milk improvement, was frozen individually. Two to three-years-old Saanen females (n = 164) were synchronized with controlled internal drug release (CIDR), pregnant mare serum gonadotrophin (PMSG) and prostaglandin. Double cervical inseminations were performed with frozen-thawed semen and fresh semen as control. In the second experiment we used pooled, washed frozen semen to examine the effect of washed seminal plasma. The motility after washing was 80-90% and after thawing was 55-65% for all bucks. The sperm concentration increased with the collections and the advance into the breeding season from 1.9 x 10(9) to 4.4 x 10(9) cell/ml average. Two inseminations were carried out at 8h intervals. The first insemination was performed at 32 h after CIDR withdrawal with fresh and frozen-thawed semen. Pregnancy rates were assessed by ultrasonography conducted 40 and 90 days post-insemination (from three bucks). Results were 58, 67, 50% with fresh semen, and for frozen semen were 33, 37 and 53%; these results were significantly different in one of the three bucks (P < 0.005). In the second experiment with pooled, washed semen the pregnancy rate was 41.6%, which compared with the average results of the frozen semen in the first experiment 38.9% no significant difference was found. We conclude that freezing buck semen in large volumes (8 ml) is possible. Cryobanking of buck semen will facilitate a genetic breeding program in goats and preservation of biodiversity. Washed semen did not improve the fertility of the semen when Andromed bull extender is used.     

Rhesus monkey sperm cryopreservation with TEST-yolk extender in the absence of permeable cryoprotectant

Recently, there has been increased interest in ultra-rapid freezing with mammalian spermatozoa, especially for vitrification in the absence of cryoprotectants. Sperm cryopreservation in non-human primates has been successful, but the use of frozen-thawed sperm in standard artificial insemination (AI) remains difficult, and removal of permeable cryoprotectant may offer opportunities for increased AI success. The present study intended to explore the possibility of freezing rhesus monkey sperm in the absence of permeable cryoprotectants. Specifically, we evaluated various factors such as presence or absence of egg yolk, the percentage of egg yolk in the extenders, and the effect of cooling and thawing rate on the success of freezing without permeable cryoprotectants. Findings revealed that freezing with TEST in the absence of egg yolk offers little protection (<15% post-thaw motility). Egg yolk of 40% or more in TEST resulted in decreased motility, while egg yolk in the range of 20-30% yielded the most motile sperm. Cooling at a slow rate (29 °C/min) reduced post-thaw motility significantly for samples frozen with TEST-yolk alone, but had no effect for controls in the presence of glycerol. Similarly, slow thawing in room temperature air is detrimental for freezing without permeable cryoprotectant (<2% motility). In addition to motility, the ability of sperm to capacitate based on an increase in intracellular calcium levels upon activation with cAMP and caffeine suggested no difference between fresh and frozen-thawed motile sperm, regardless of treatment. In summary, the present study demonstrates that ejaculated and epididymal sperm from rhesus monkeys can be cryopreserved with TEST-yolk (20%) in the absence of permeable cryoprotectant when samples were loaded in a standard 0.25-mL straw, cooled rapidly in liquid nitrogen vapor at 220 °C/min, and thawed rapidly in a 37 °C water bath. This study also represents the first success of freezing without permeable cryoprotectant in non-human primates.     

An effective method for freezing White Italian gander semen

Efficiency of freezing method, worked out for the White Italian gander semen was evaluated by comparing motility, morphology and fertilizing ability of spermatozoa in fresh and frozen-thawed semen. A part of pooled semen, collected from 25 White Italian ganders by dorso-abdominal massage was used immediately for artificial insemination of 10 geese (the control group) with a dose of 80 microl. This insemination was performed six times at weekly intervals. The remainder of the semen was diluted 1:0.5 (v/v) with EK diluent, equilibrated for 15 min at +4 degrees C, mixed with 6% (v/v) of dimethylformamide (DMF) and frozen to -140 degrees C at a rate of 60 degrees C/min. Frozen semen was thawed in a 60 degrees C water-bath and inseminated twice weekly in a dose of 100 microl (10 females of the experimental group, 12 inseminations were made). The freezing process affected spermatozoa motility and morphology, but had no effect on their fertilizing ability. Positive movement was observed in 50-60% of the spermatozoa in fresh semen and about 40% of the frozen-thawed cells. The average percentage of total live and live normal spermatozoa decreased due to freezing from 92.2 to 68.4% and from 34.7 to 14.1%, respectively. After the fresh semen insemination with average 12 million of the live normal spermatozoa per week average fertility was 88.24%; hatchability of set eggs was 80.88% and hatchability of fertile eggs was 91.67%. For frozen-thawed semen inseminated with average 9.5 million of the undamaged spermatozoa per week, the average fertility and hatchability rate was 83.78, 73.87, and 88.17%, respectively. Fecundity rates obtained after insemination with the frozen-thawed gander semen allow for the application of the freezing technique into breeding practice, in place of natural mating or to assist natural mating in periods of lowered fertility level.     

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.     

In vitro and in vivo comparison of egg yolk-based and soybean lecithin-based extenders for cryopreservation of bovine semen

Semen diluents containing egg yolk as a cryoprotectant may pose hygienic risks and are difficult to standardize. Although a new generation of semen diluents free of animal ingredients is available, egg yolk-containing extenders are still widely used for cryopreserving semen. We compared the effects of using different extenders on bovine sperm function in vitro and on fertility in vivo. A soy lecithin extender (SL; AndroMed) and an egg yolk-containing (TRIS-EY) extender were tested. No differences (P>0.05) were detected between the two extenders for sperm-zona pellucida binding capacity (HZI=115+/-13). Assessment of the inducibility of the acrosome reaction with progesterone showed no differences (P>0.05) between extenders for live acrosome-reacted sperm (15+/-2.36 and 14.42+/-2.02%, respectively, for SL and TRIS-EY). However, post-thaw sperm motility was significantly lower (P<0.05) when semen was extended in the TRIS-EY diluent. Field trials revealed that nonreturn rates of SL-extended semen showed significantly higher insemination success (P<0.0001) compared with the nonreturn rates for the TRIS-EY extender (70.45 and 67.85%, respectively). We suggest that consistent with quality standards that should be required for cryoprotectant media and because of the superior quality of the egg yolk-free extender, a defined soybean lecithin-containing diluter might be the better choice as a semen extender in the future.     

Comparing sugar type supplementation for cryopreservation of boar semen in egg yolk based extender

Cryopreservation of boar semen is still considered suboptimal due to lower fertility when compared to fresh semen. The aim of this study was to evaluate the effects of the addition of different sugars (lactose, trehalose and glucose) on boar spermatozoa cryopreserved in an egg yolk based extender. Ejaculates were collected from a boar previously selected and semen samples were processed using the straw freezing procedure. In experiment 1, subsamples of semen were frozen in three different extenders: recommended lactose egg yolk extender (LEY); trehalose egg yolk extender (TEY) and glucose egg yolk extender (GEY). Sperm quality was assessed for motility, viability, acrosome integrity and hypoosmotic swelling test response upon collection, after freezing and thawing and then every hour for 3 h. Results showed that total motility at 1 and 3 h, progressive motility at 3 h, positive hypoosmotic response at 2 and 3 h and acrosome integrity at all times were significantly improved when trehalose was added to the extender. In experiment 2, sugar influence was also demonstrated in vitro fertilization. A total of 1691 oocytes were in vitro matured and inseminated with frozen-thawed sperm at 2000:1 sperm:oocyte ratio and coincubated for 6 h. Presumptive zygotes were cultured in NCSU-23 medium to assess fertilization parameters and embryo development. Both penetration and monospermy rates were significantly higher for trehalose frozen semen. A significant increase was observed in efficiency and blastocyst formation rates from TEY to the other groups. Our results demonstrated that trehalose extender enhances spermatozoa viability and its in vitro fertilization parameters in boar ejaculates with good sperm freezability. Further studies are necessary to assess the impact of sugars on the entire population.