Advances in cooled semen technology.

In the horse industry, milk or milk-based extenders are used routinely for dilution and storage of semen cooled to 4-8 degrees C. Although artificial insemination (AI) with chilled and transported semen has been in use for several years, pregnancy rates are still low and variable related to variable semen quality of stallions. Over the years, a variety of extenders have been proposed for cooling, storage and transport of stallion semen. Fractionation of milk by microfiltration, ultrafiltration, diafiltration and freeze-drying techniques has allowed preparation of purified milk fractions in order to test them on stallion sperm survival. Finally, a high protective fraction, native phosphocaseinate (NPPC), was identified. A new extender, INRA96, based on modified Hanks' salts, supplemented with NPPC was then developed for use with cooled/stored semen.Four experiments were conducted to compare INRA96 and milk-based extenders under various conditions of storage. The diluted semen was maintained under aerobic conditions when stored at 15 degrees C, and anaerobic conditions when stored at 4 degrees C. In experiment 1, split ejaculates from 13 stallions were diluted either in INRA96 extender then stored at 15 degrees C or diluted in Kenney or INRA82 extenders and then stored at 4 degrees C for 24h, until insemination. In experiment 2, semen from two stallions was extended in INRA96 then inseminated immediately or stored at 15 degrees C for 3 days until insemination. In experiment 3, semen from three stallions was diluted in INRA96 then stored at 15 or 4 degrees C for 24h until insemination, finally, in experiment 4, split ejaculates from four stallions were diluted in INRA96 or E-Z Mixin extenders then stored at 4 degrees C for 24h until insemination. Experiment 1 demonstrated that at 15 degrees C, INRA96 extender significantly improved pregnancy rate per cycle compared to Kenney or INRA82 extenders at 4 degrees C after 24h of storage (57%, n=178 versus 40%, n=171, respectively; P<0.01). Experiment 2 showed that semen stored at 15 degrees C for 3 days can achieve pregnancy at a fertility rate per cycle of 48% (n=52) compared to 68% (n=50, immediate insemination, P=0.06). Experiment 3 demonstrated that INRA96 extender can be as efficient at 15 degrees C (54%, n=37) as at 4 degrees C (54%, n=35) after 24h of storage. Finally, experiment 4 showed that INRA96 extender used at 4 degrees C (59%, n=39) seems to improve fertility per cycle compared to E-Z Mixin at 4 degrees C (49%, n=39, P=0.25), but this result has to be confirmed.These results demonstrate that semen diluted in INRA96 extender and stored at 15 degrees C can be an alternative to semen diluted in milk-based extenders and stored at 4 degrees C for "poor cooler" stallions. Furthermore, INRA96 extender can be as efficient at 15 degrees C as at 4 degrees C, for preserving sperm motility and fertility.     

Effect of different diluents on goat semen fertility

Skim milk (SM) is considered to be the most widely employed extender for goat sperm used for artificial insemination (AI). However, the fertilizing life span of sperm stored in milk or milk-based extenders does not exceed 12h. Besides some seminal plasma components, such as a protein fraction from the goat bulbourethral gland secretion (SBUIII), interacts with some milk fractions and inhibits the spermatozoa motility. The aim of this study was to prolong the survival of buck semen and its fertility. Buck ejaculates were diluted to a final concentration of 100x10(6)spermatozoa/ml with three different diluents: SM, TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl) and TEMPOL+hyaluronic acid (TEMPOL+HA). At 7h from dilution 42 goats were inseminated with semen diluted with SM (short-term semen) while after storage for 24h, 44 and 45 goats were inseminated with semen diluted with TEMPOL and TEMPOL+HA (long-term storage), respectively. At day 50 from AI the percentages of pregnant goats were 71.4% (30/42) with SM, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, with significant differences between SM and TEMPOL+HA. The kidding rate was 66.7% (28/42) with SM diluent, 61.4% (27/44) with TEMPOL and 48.8% (22/45) with TEMPOL+HA, without significant differences among treatment groups. In conclusion, it is possible to maintain good fertility in goats after AI with semen stored for 24h in TEMPOL.     

Insemination time and dilution rate of cooled and chilled ram semen affects fertility

Adult Merino ewes (n=448) were apportioned into two groups and inseminated with: extended at 30 degrees C with skim milk and stored for 6h at 15 degrees C (cooled semen) or extended with skim milk-citrate trisodium with egg yolk and stored for 24h at 5 degrees C (chilled semen). Each group was further subdivided according to the time of cervical insemination at 42, 46 and 50h after pessary (MAP-60 mg) removal and according to the dilution of the semen (120 x 10(6) spermatozoa in 0.05, 0.1 and 0.2 ml). The pregnancy rate after insemination with cooled semen was 50% better than that after chilled semen (56.7 vs. 37.5%; P<0.001). Pregnancy rate was not affected by the volume of insemination; however, there was a tendency of increased lambing rate with an insemination dose of 0.1 cc (1:2, dilution), especially when the ewes were inseminated with cooled semen. The effect of time on insemination was significant only in ewes inseminated with chilled semen at 5 degrees C (P<0.01). Insemination carried out 46 h after pessary removal resulted in higher pregnancy and lambing rate (36.5, 31.1; 52.0, 45.3; and 24.0, 20.0 at 42, 46 and 50h, respectively). Pregnancy of ewes inseminated with chilled semen at 46 h after pessary removal was similar to that obtained using cooled semen (52.0 vs. 56.7%). From this study, it is concluded that advancing the time of insemination with chilled semen at 5 degrees C improves pregnancy and that the lambing obtained under these conditions is similar to the one obtained with cooled semen.     

Effect of storage temperature, cooling rates and two different semen extenders on canine spermatozoal motility

Ejaculates were collected form three mixed-breed male dogs daily for 3 d. The semen was diluted in either a nonfat dried milk solid-glucose (NFDMS-G) or egg yolk citrate (EYC) extender at a concentration of 25 x 10(6) sperm/ml. The diluted samples were exposed to three different storage temperatures (35, 22 and 4 degrees C). Three cooling rates (-1.0, -0.3 and -0.1 degrees C/min) were also investigated at the lowest storage temperature (4 degrees C). The semen was evaluated for total motility, progressive motility and velocity at 0, 6, 12, 24, 48, 72, 96 and 120 h after collection by two independent observers. Interactions between extenders, temperatures and time after collection were found for each of the variables. Nonfat dried milk solid-glucose diluent was superior to EYC (P<0.05) in preservating sperm motility parameters that were evaluated for most of the observations. The evaluated sperm motility parameters were also significantly superior (P<0.05) in semen stored at 4 degrees C than at 35 or 22 degrees C for most of the observations. The progressive motility and velocity of sperm in semen cooled at 4 degrees C in NFDMS-G were higher (P<0.05) at the fast and medium cooling rates (-1.0 and -0.3 degrees C) than at the slow cooling rate (-0.1 degrees C/min) at 24 and 72 h, and at 48 h, respectively. In conclusion, the present study suggests that canine spermatozoal motility is well preserved when a NFDMS-glucose extender is added to the semen and the semen is cooled at a medium or fast rate to a storage temperature of 4 degrees C. Additional studies are needed to evaluate the fertility of semen stored in this manner.     

Effect of storage duration, storage temperature, and diluent on the viability and fertility of fresh ram sperm

Cervical artificial insemination (AI) in sheep with fresh semen yields a much higher pregnancy rate than when frozen-thawed semen is used, and consequently frozen semen is only acceptable for laparoscopic insemination. The short life span of fresh semen is a major constraint on the use of AI in genetic improvement programs for sheep. The main objective of this study was to examine the effects of storage conditions on viability and fertilization ability of fresh ram (Ovis aries) semen up to 72 h postcollection. Experiment 1 was designed to evaluate the effect of diluent type (standard skim milk, AndroMed, OviPro, and INRA 96) and storage temperature (5 °C and 15 °C) on the motility and viability of fresh ram semen. Storage temperature, irrespective of diluent, had a significant effect on both motility and viability. Storage at 5 °C maintained acceptable motility and viability up to 72 h compared with that of storage at 15 °C. In Experiment 2, the penetrating ability of fresh ram semen, diluted in either skim milk, AndroMed, or INRA 96, was assessed using artificial mucus. Flat capillary tubes containing artificial mucus were suspended in 250 μL semen at a sperm concentration of 20 × 10⁶/mL. Semen was stored at 5 °C and tested after 6, 24, 48, and 72 h. There was a significant diluent by time interaction. In Experiment 3, the fertilizing ability of fresh ram semen stored at 5 °C was evaluated in vitro. Fresh semen (diluted in either skim milk, AndroMed, or INRA 96) was added to matured ewe oocytes at 6, 24, or 72 h after semen collection. Cleavage rate was recorded at 48 h postinsemination, and blastocyst development was recorded on Days 6 to 9. There was a significant treatment effect on cleavage and blastocyst rates; insemination of semen stored for 24 h resulted in higher rates than those for storage at 72 h. In Experiment 4, the fertilizing ability of fresh ram semen was evaluated in vivo. Semen was diluted in INRA 96, stored at 5 °C, and used to inseminate ewes on the day of collection or at 24, 48, and 72 h postcollection. Multiparous ewes were cervically inseminated at a synchronized estrus. Fertility rate decreased linearly (P < 0.001) up to 72 h after semen collection.     

Effects of solid storage of sheep spermatozoa at 15 degrees C on their survival and penetrating capacity

This study was designed to evaluate the possible benefits of adding gelatin to a standard milk extender, for solid storage of sheep semen at 15 degrees C. Solid storage was assessed in terms of effects on sperm motility and membrane integrity up to 2 days (Study 1), and on in vitro penetration capacity after storage for 24h (Study 2). In both studies, semen was diluted in CONTROL (standard milk extender) and GEL (1.5 g gelatin/100ml extender) diluents to a final concentration of 400 x 10(6)sperm/ml. In Study 1, semen samples were stored at 15 degrees C, and sperm quality variables analyzed after 2, 24 and 48 h of storage. Motility and viability values were significantly lowered using the liquid compared to the gel extender for all storage periods, except for motility after 2h of storage, whose values were similar. After 2h of incubation at 37 degrees C, motile cell percentages and membrane integrity were significantly lower in the CONTROL group than in the GEL group for all storage periods. In Study 2, in vitro matured lamb oocytes were randomly divided into three groups and fertilized with CONTROL diluted semen stored for 2h or 24h, or with GEL diluted semen stored for 24h. After co-incubation, oocytes were evaluated for signs of penetration. Storage of semen in the GEL diluent for 24h gave rise to increased in vitro fertilization rates in comparison with the CONTROL diluent. Our findings indicate that the solid storage at 15 degrees C of ram spermatozoa by adding gelatin to the extender leads to improved survival and in vitro penetrating ability over the use of the normal liquid extender. A solid diluent could thus be a useful option for the preservation of fresh ovine semen for extended periods.     

Fertilizing capacity of equine spermatozoa stored for 24 hours at 5 or 20 degrees C

A breeding trial was conducted to evaluate the effect of in vitro storage time and temperature on fertilizing capacity of equine spermatozoa. Semen obtained from one stallion and diluted with skim milk-glucose extender was used to artificially inseminate 45 estrussynchronized mares. The mares were assigned to one of three treatment groups (15 mares per group): 1) insemination with fresh semen (collected within 0.5 h of use), 2) insemination with semen stored for 24 h at 20 degrees C or 3) insemination with semen stored for 24 h at 5 degrees C. The mares were inseminated daily during estrus, from the detection of a 35-mm follicle until ovulation, with 250 x 10(6) progressively motile spermatozoa (based on initial sperm motility of fresh semen). Semen samples (n = 35) were evaluated prior to insemination for percentages of total sperm motility (TSM), progressive sperm motility (PSM) and sperm velocity (SV). Single-cycle 15-d pregnancy rates. resulting from insemination with fresh semen, from fresh semen stored for 24 h at 20 degrees C or from semen stored for 24 h at 5 degrees C were the same (11 15 ; 73%). Mean diameters (mm) of 15-d embryonic vesicles were not different (P>0.05) among these three treatment groups (21.5 +/- 2.9, 19.6 +/- 2.6 and 20.5 +/- 3.6, respectively). Ten pregnant mares were aborted on Day 15 of gestation for use in another project. The pregnancy status of the 23 remaining pregnant mares was again determined at 35 to 40 d and 55 to 60 d of gestation. No pregnancy losses occurred during this time period. Mean TSM percentages were different (P<0.05) among the three groups: the fresh semen percentage was 89 +/- 2, semen stored for 24 h at 20 degrees C was 57 +/- 11 and semen stored for 24 h at 5 degrees C was 80 +/- 6. Similar differences were found for mean PSM and SV. Semen storage at either 20 or 5 degrees C for 24 h had no apparent effect on the fertilizing capacity of the extended semen samples; however, the reduction in all motility parameters tested was more dramatic in semen stored at 20 degrees C than that stored at 5 degrees C.     

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.     

Delayed insemination is successful with a new extender for storing fresh equine semen at 15 degrees C under aerobic conditions

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage before artificial insemination. Milk is a biological fluid with a complex composition and contains components which are beneficial or harmful to spermatozoa. The aim of this study was to test the fertility of stallion semen after long-term storage using different milk diluents (INRA 82 or Kenney's diluent) vs one diluent chemically defined (INRA 96), which is composed of efficient milk components and optimized for sperm survival and storage temperature. The milk fraction used was that which best maintained spermatozoal survival based on motility measured in previous studies. Four breeding trials were conducted to determine the influence of combination of new diluent and storage conditions on fertility of the stallion. We compared the standard protocol of storing semen in a skim milk diluent (INRA 82 or Kenney's diluent) at 4 degrees C under anaerobic conditions with the experimental protocol which consisted of storing in a chemically defined, milk-free diluent (INRA 96), at 15 degrees C, under aerobic conditions. After 4 breeding trials, in which the semen was stored for 24 h under the 2 protocols, we obtained 57% (n = 178) and 40% (n = 173) of fertility per cycle using the experimental and the standard protocol respectively (p < 0.001). Another breeding trial was conducted to determine the influence of storage time on the fertility of spermatozoa. We have compared the fertility of semen inseminated immediately (68% of fertility per cycle, n = 50) vs the fertility of semen stored under the experimental protocol for 72 h before insemination (48% of fertility per cycle, n = 52). The experimental protocol improved sperm fertility compared to the standard protocol and seems to be a particular alternative for stallions with cold shock sensitive spermatozoa. Storing semen for 72 h under the experimental protocol seems to be useful in the field.     

Effects of cooling rate and storage temperature on equine spermatozoal motility parameters

Two experiments were conducted to examine the effects of cooling rate and storage temperature on motility parameters of stallion spermatozoa. In Experiment 1, specific cooling rates to be used in Experiment 2 were established. In Experiment 2, three ejaculates from each of two stallions were diluted to 25 x 10(6) sperm/ml with 37 degrees C nonfat dry skim milk-glucose-penicillin-streptomycin seminal extender, then assigned to one of five treatments: 1) storage at 37 degrees C, 2) storage at 25 degrees C, 3) slow cooling rate to and storage at 4 degrees C, 4) moderate cooling rate to and storage at 4 degrees C, and 5) fast cooling rate to and storage at 4 degrees C. Total spermatozoal motility (TSM), progressive spermatozoal motility (PSM), and spermatozoal velocity (SV) were estimated at 6, 12, 24, 48, 72, 96 and 120 h postejaculation. The longevity of spermatozoal motility was greatly reduced when spermatozoa were stored at 37 degrees C as compared to lower spermatozoal storage temperatures. At 6 h postejaculation, TSM values (mean % +/- SEM) of semen stored at 37 degrees C, slowly cooled to and stored at 25 degrees C or slowly cooled to and stored at 4 degrees C were 5.4 +/- 1.1, 79.8 +/- 1.6, and 82.1 +/- 1.6, respectively. Mean TSM for semen that was cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a moderate rate for four of seven time periods (6, 24, 72 and 120 h), and it was greater (P<0.05) than mean TSM of semen cooled to 4 degrees C at a fast rate for five of seven time periods (6, 12, 24, 72 and 120 h). Mean TSM of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean TSM of semen cooled to 25 degrees C for five of seven time periods (24 to 120 h). A similar pattern was found for PSM. Mean SV of semen cooled to 4 degrees C at a slow rate was greater (P<0.05) than mean SV of semen cooled to 25 degrees C for all time periods. A slow cooling rate (initial cooling rate of -0.3 degrees /min) and a storage temperature of 4 degrees C appear to optimize liquid preservation of equine spermatozoal motility in vitro.     

Milk caseins decrease the binding of the major bovine seminal plasma proteins to sperm and prevent lipid loss from the sperm membrane during sperm storage

Milk is used as a medium for sperm preservation. Caseins, the major proteins of milk, appear to be responsible for the protective effect of milk on sperm. Recently, we have shown that egg yolk, which is also widely used to preserve semen, protects sperm functions by preventing the binding to sperm of the major proteins of bull seminal plasma (BSP proteins), thereby preventing BSP protein-mediated stimulation of lipid loss from the sperm membrane. In the present study, we investigated whether milk caseins protect sperm in the same manner as egg yolk. Bovine ejaculates were diluted with skimmed milk permeate (skimmed milk devoid of caseins) or permeate that was supplemented with caseins and stored at 4 degrees C for 4 h. In the semen diluted with permeate, sperm viability and motility decreased in a time-dependent manner. However, in semen diluted with milk or permeate supplemented with caseins, sperm functions were maintained. In addition, lower amounts of the BSP proteins were associated with sperm in semen diluted with milk or permeate supplemented with caseins, as compared to semen diluted with permeate. No milk proteins were detected in the sperm protein extracts. Furthermore, sperm diluted with milk or permeate supplemented with caseins showed 3-fold lower losses of cholesterol and choline phospholipids than sperm diluted with permeate during storage. Thus, milk caseins decreased the binding of BSP proteins to sperm and reduced sperm lipid loss, while maintaining sperm motility and viability during storage. These results support our view that milk caseins prevent the detrimental effects of BSP proteins on the sperm membrane during sperm preservation.     

Improvement of rooster semen quality using coenzyme Q10 during cooling storage in the Lake extender

Sensitivity of rooster semen to stressful condition of cooling restricts the semen storage in commercial flocks for artificial insemination. This study was accomplished to investigate the effect of coenzyme Q10 (CoQ10) addition to the Lake extender during chilled-storage on the parameters of sperm quality and fertility performance. Roosters’ pooled semen samples were assigned into equal parts and diluted with Lake extender supplemented with different concentrations of CoQ10 (0, 1, 2, 5 and 10 μM CoQ10). Then, semen samples were cooled to 5 °C and stored over 48 h. Total and progressive motilities, abnormal morphology, viability, membrane functionality, lipid peroxidation (LPO) and mitochondria active potential of diluted sperm were evaluated at 0, 24 and 48 h of cooling storage. Fertility performance of cooled stored semen was examined at 24 h of cooling storage. Although CoQ10 did not affect sperm quality at the starting time of cooling storage (0 h), extender supplementation with 5 μM of CoQ10 showed higher (P ≤ 0.05) sperm total and progressive motilities, membrane functionality, viability and mitochondria active potential at 24 h as well as total motility, viability and membrane functionality at 48 h in contrast with other groups. Moreover, lipid peroxidation was lower (P ≤ 0.05) in semen samples diluted with 5 μM CoQ10 at 24 and 48 h compared to others. After artificial insemination with 24 h chilled-stored sperm, fertility efficiency was higher (P ≤ 0.05) in treatments contained 5 μM CoQ10 compared to the control group. According to the results, using optimum dose of CoQ10 could be helpful to save rooster semen against chilled storage structural and functional damages.     

Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15 degrees C

Artificial insemination (AI) in rabbits is not extensive in the breeding programs of the rabbit meat industry. A limiting factor is related to the semen preservation. In order to improve the use of AI, two experiments have been conducted to evaluate sperm viability and fertility of rabbit semen chilled and stored at 15 degrees C after dilution in Tris-based extenders. In Experiment 1, pooled semen samples were diluted 1:10 (semen/extender) in four different Tris-based extenders (Tris-citric-glucose (TCG), TES-Tris-glucose (TTG), Tris-citric-fructose (TCF) and TES-Tris-fructose (TTF)) and stored at 15 degrees C. Sperm viability was evaluated at 0, 24, 48, 72 and 96 h following dilution for total sperm motility (TSM), forward progressive motility (FPM), plasma membrane integrity (PMI) and acrosome integrity (NAR). Viability of spermatozoa declined with time of storage (P<0.05), irrespective of the extender used. There were interactions between extender and time of storage (P<0.05) in all viability parameters evaluated. After 96 h of storage, TCG provided the highest sperm viability (P<0.05) and TTG the lowest (P>0.05). In Experiment 2, a field trial was conducted at a commercial farm to evaluate the conception and farrowing rates of rabbit spermatozoa extended in TCG. After synchronization of oestrous and induction of ovulation, 3713 does with different physiological conditions (nulliparous, primiparous, lactating and re-breeding) were inseminated one time (15x10(6) sperm per doses) with semen stored at 0 (n: 1275), 24 (n: 1503) and 48 h (n: 935) at 15 degrees C. Overall conception and farrowing rates were 77.1+/-0.7 and 70.4+/-0.7, respectively, and the mean litter size was 7.6+/-0.1. Fertility results were unaffected by the time of semen storage (P>0.05). Regardless of time of semen storage, fertility results were affected by the physiological conditions of does (P<0.05). Nulliparous and lactating does showed the highest fertility and primiparous the lowest. In summary, these results indicate that Tris-buffer extenders are effective for preserving viability and fertilizing capability of rabbit spermatozoa stored at 15 degrees C.     

Motility and fertility of alginate encapsulated boar spermatozoa

Ejaculated boar spermatozoa are vulnerable to cold shock. Prolonged storage of boar spermatozoa at low temperatures reduces survival rate, resulting in a bottleneck for the extension of artificial insemination in pig husbandry. This study evaluated whether alginate microencapsulization processing can improve the longevity of boar spermatozoa stored at 5 degrees C and the fertility of microencapsulated spermatozoa in vivo. Sperm-rich fraction semen from three purebred boars were concentrated and microencapsulated using alginate at 16-18 degrees C, and then were stored at 5 degrees C. Following storage for 1, 3 and 7 days, the microcapsule was taken out to assess sperm release under 37 degrees C incubation with or without 110 rpm stirring. The percentage of sperm released from microcapsules with 110 rpm stirring was higher than without stirring (81 versus 60%) after 24h of incubation. In another experiment, semen was also microencapsulated to evaluate the sperm motility. The motility of spermatozoa was assessed at 10 min, 8, 24, 32, 48, 56 and 72 h following incubation at 37 degrees C for nine consecutive days. The fertility of the free and microencapsulated semen was assessed by inseminating sows, and the reproductive traits (conception rate, farrowing rate, and litter size) were recorded. The motility of encapsulated spermatozoa was significantly higher than that of free semen after 8h incubation at 37 degrees C after storing for over three days (P<0.05). No significant difference existed in conception rate, farrowing rate, and litter size between the microencapsulated and non-encapsulated semen after four days of storage. In conclusion, microencapsulation can increase the longevity of boar spermatozoa and may sustain in vivo ova fertilization ability.     

Effect of different extenders and storage temperatures on sperm viability of liquid ram semen

Semen was collected with an artificial vagina from four adult rams. The ejaculates were pooled and diluted, using a split-sample technique, in four different extenders: one for milk (Mi), one for sodium citrate (Na), and two for Tris-based extenders (T1 and T2) including egg yolk. Thereafter, the diluted semen was stored at 5 and 20 degrees C, respectively. We evaluated sperm viability after 0, 6, 12, 24 and 30 h of storage. We assessed sperm motility subjectively, and we determined sperm membrane integrity using both the hypo-osmotic resistance test (ORT) and a fluorophore staining (SYBR-14 and propidium iodide) technique. We evaluated acrosomal status with Spermac and capacitation status with Chlortetracycline (CTC assay). All sperm viability parameters were influenced by storage time and extender, while sperm motility was the only evaluated parameter that was influenced by the interaction between extender and temperature. Semen that was diluted and stored in the commercially available Tris-based extender (T2) maintained sperm motility for a longer period of time, and acrosome and membrane integrity was higher during storage for up to 30 h as compared to the other extenders independent of storage temperature. In general, however, storage of ram semen at 5 degrees C seemed to influence sperm viability parameters less than storage at 20 degrees C. In conclusion, the results of the present study indicate that Tris-based extenders, especially T2, preserved sperm viability better than both the sodium citrate- and the milk-based extender did when liquid ram semen was stored up to 30 h at 5 and 20 degrees C. Whether the differences found between the extenders will be reflected in the fertility results after AI is yet unknown and needs to be further studied.     

Bivalent response to long-term storage in liquid-preserved boar semen: a flow cytometric analysis

The fertility of liquid-preserved boar semen declines during storage at 17°C, insemination trials even indicating early losses in fertilizing ability within the first 24-48 h of storage. Standard semen parameters barely reflect these changes in semen quality, and new approaches for assessment of functional changes in stored spermatozoa are needed. Capacitation, the essential prefertilization step for spermatozoa in the female genital tract, is specifically induced in vitro by bicarbonate. Therefore, we have investigated changes in responsiveness of boar spermatozoa to bicarbonate during storage. Ejaculates of 14 boars were diluted in Beltsville thawing solution, cooled to 17°C and stored for 12, 24, 72, 120, and 168 h before investigation. At each time, basic semen quality was characterized by sperm motility and viability. Subsequently, washed subsamples were incubated in variants of an in vitro fertilization (IVF) medium and assessed for kinetic changes of viability (plasma membrane integrity) and intracellular calcium concentration using flow cytometry in combination with propidium iodide and Fluo-3. By this means, it was possible to determine specific effects of bicarbonate and calcium on sperm subpopulations over incubation time. During storage, standard semen parameters remained on a high level. However, flow cytometric analysis of sperm responses to capacitating and control media revealed two opposing effects of storage. There was a loss of response to bicarbonate in part of the live sperm population but an increasing degree of instability in the rest. Assessment of response to capacitating media by flow cytometry appears a markedly more sensitive way of monitoring sperm functionality during storage than the standard semen parameters of motility and viability.     

Effect of solid storage at 15 degrees C on the subsequent motility and fertility of rabbit semen

We conducted two studies to improve preservation of rabbit semen. The objective of the first study was determine whether a glucose- and fructose-based extender with two different amounts of gelatin would solidify at 15 degrees C, and to evaluate the influence of gelatin supplementation on sperm motility parameters after storing semen up to 10 days at 15 degrees C. The fertility of rabbit semen diluted in the best gelatin-supplemented extender established in Study 1 and stored for up to 5 days was evaluated in the second study. In Study 1, semen was collected with an artificial vagina from 40 bucks. Each ejaculate was diluted to (80-100) x 10(6) spermatozoa/mL (1:3, semen/extender) at 37 degrees C in one of the three following glucose- and fructose-based extenders: control (standard liquid extender), semi-gel or gel (0.7 or 1.4 g gelatin in 100 mL extender, respectively). Pools of semen were allocated among 0.6 mL plastic artificial insemination (AI) guns. Thirty (10 per extender group) AI doses were immediately analyzed (0 h) and the remainder stored in a refrigerator (15 degrees C) for 12, 24, 36, 48, 72, 96, or 240 h. All doses with gelatin extenders solidified at 15 degrees C. Semen samples, prewarmed to 37 degrees C, were evaluated with a computer-assisted sperm analysis (CASA) system. The percentage of motile cells was significantly lower using the liquid compared to the gel extenders during semen storage from 0 to 96 h. Although significance was lost, these differences persisted after 240 h of storage. Motility of spermatozoa in the semi-gel extender was intermediate between that of liquid and gel extender throughout the study. Study 2 was performed on 1250 multiparous lactating does. Five homogeneous groups of 250 does previously synchronized were inseminated using semen previously stored for 120, 96, 72, 48 or 24 h, respectively. Rabbit does receiving 24 h-stored semen (diluted with the control extender used in Study 1) served as controls. The remaining females received seminal doses supplemented with 1.4 g/100mL gelatin (gel extender used in Study 1). Kindling rates for rabbit does inseminated with gelatin-supplemented (solid) semen doses stored for 48 h (88%) or 72 h (83%) were similar to those recorded for liquid controls stored for 24 h (81%), whereas rates significantly decreased when the semen was solid and stored for 96 h (64%) or 120 h (60%) before AI. In conclusion, rabbit spermatozoa were effectively stored in the solid state at 15 degrees C, with fertility preserved for up to 5 days. Solid storage of rabbit semen would facilitate commercial distribution.     

Capacitation and acrosomal exocytosis are enhanced by incubation of stallion spermatozoa in a commercial semen extender

Preserved stallion semen often has decreased spermatozoal motility and fertility that can vary significantly between individual stallions. It is not known whether the medium used for extending equine sperm contributes to these decreases by inducing premature capacitation during storage. If spermatozoa undergo capacitation or acrosome reaction prior to insemination, this could result in a diminished capacity to penetrate the cumulus mass and fertilize the egg. We hypothesized that skim milk-based semen extenders, similar to those used in cooled storage, stabilize sperm membranes and prolong sperm motility and longevity. However, this could decrease the efficiency of sperm to undergo subsequent capacitation in vivo. This study was designed to evaluate the effects from two media on sperm function. Spermatozoal motility was analyzed, intracellular calcium was measured, and the ability of sperm to undergo acrosome reaction was compared after incubation in a skim milk extender (SME) and Tyrode's medium containing albumin, lactate, and pyruvate (TALP) at 37 degrees C. Results suggest that the SME facilitated capacitation as detected by an increase in both intracellular calcium and acrosome reactions, and a decrease in motility, as compared to TALP. Our data support a shortened functional lifespan for equine sperm in skim milk extender, which indicates that further refinements in cooled semen preservation are required to improve fertility of transported equine semen.     

Motility and fertility of equine spermatozoa in a milk extender after 12 or 24 hours at 20 degrees C

The effects of extender and storage at 20 degrees C on equine spermatozoa were evaluated in two experiments using embryo recovery as the end point. In both experiments, inseminations were every other day, starting on Day 2 or 3 of estrus or after a 35-mm follicle was detected, with 250 x 10(6) progressively motile cells (based on initial evaluation). In Experiment 1, semen from two stallions was used to compare the motility and fertility of spermatozoa maintained in a) heated skim milk extender at 37 degrees C with insemination in <1 h; b) E-Z Mixin extender at 37 degrees C with insemination in <1 h; and c) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 12 h at 20 degrees C. The percentage of motile spermatozoa was 34% after 12 h compared to 55% at 0 h (P < 0.05). However, the percentage of mares from which an embryo was recovered 6.5 d after ovulation was 62, 56, and 50% for Treatments A, B, and C (P > 0.05). In Experiment 2, semen from three stallions was used to compare the motility and fertility of spermatozoa in a) E-Z Mixin extender at 37 degrees C with insemination in <1 h or b) E-Z Mixin extender at 37 degrees C with cooling to 20 degrees C and insemination after storage for 24 h at 20 degrees C. The percentage of motile spermatozoa was 17% after 24 h compared to 54% at 0 h (P < 0.05). There was no difference between treatments (P > 0.05) in the percentage of mares from which an embryo was recovered 6.0 d after ovulation (68 vs 62%) or among stallions. Thus, stallion semen extended in E-Z Mixin was held at 20 degrees C for 24 h without a marked decline in fertility.     

Effect of milk fractions on survival of equine spermatozoa

Milk-based semen diluents are known to be practical and effective in protecting equine spermatozoa during storage. Due to complex composition of milk, the components which are beneficial or harmful to spermatozoa are unknown. To address these unknowns the effect of various milk fractions on motility of stallion spermatozoa was evaluated. The fractions tested were native phosphocaseinate (NPPC), beta-casein, whey protein concentrate (WPC), alpha-lactalbumin, beta-lactoglobulin, microfiltrate, and ultrafiltrate. The standard reference diluents were INRA 82, commercial skim milk, and Hank's salts solution supplemented with Hepes, glucose, lactose (HGLL) supplemented with BSA. After 48 and 96 h storage at 4 or 15 degrees C some milk fractions (ultrafiltrate, microfiltrate, and alpha-lactalbumin fraction) decreased spermatozoal survival. Others (beta-lactoglobulin (BL) and native phosphocaseinate) were protective. Native phosphocaseinate (NPPC) at milk concentration afforted better protection than did the standard reference diluents. The optimal concentration of beta-lactoglobulin afforted significantly better protection than did BSA. The protection afforded by native phophocaseinate was not synergistic with beta-lactoglobulin. This implies a similar mechanism of protective action of these two components. Semen diluted in HGLL supplemented with NPPC (HGLL-NPPC) or in INRA 82 and stored 24 h at 15 degrees C or 4 degrees C, respectively, produced no difference of spermatozoal motility. However, fertility of semen stored in HGLL-NPPC (60%) was higher (p < 0.05) than that stored in INRA 82 (36%).