Comparison of two processing systems for bull semen with regard to post-thaw motility and nonreturn rates

Two semen processing systems based on either skim milk-yolk extender or Biladyl® were compared with regard to post-thaw motility and nonreturn rates. The skim milk-yolk extender contained penicillin and streptomycin, while Biladyl® consisted of tylosin, gentamycin, spectinomycin and lincomycin. The dilution of semen involved a 2-step procedure for both extenders. The second dilution with the skim milk-yolk extender took place at 5 °C, while the Biladyl® extender was processed at room temperature.In this study 197 ejaculates from 45 Norwegian Cattle bulls were used. The ejaculates were split-sampled and diluted with the 2 extenders. A total of 41,445 first inseminations was recorded, 21,035 with semen diluted with skim milk-yolk and 20,410 with Biladyl®. The 60-d nonreturn rates were 73.1 and 71.9% for skim milk-yolk and Biladyl®, respectively. The results for skim milk-yolk were significantly better than for Biladyl® (P < 0.01). No difference in post-thaw motility for these extenders was found.     

Caprine blastocyst development after in vitro fertilization with spermatozoa frozen in different extenders

The feasibility of using frozen-thawed semen in caprine IVF outside the breeding season was investigated. Electroejaculated spermatozoa from a Nubian buck were washed twice and then frozen in skim milk- or in egg yolk-based extenders. Goat oocytes were matured and inseminated by frozen-thawed spermatozoa selected by swim-up. In vitro fertilization was performed in a modified defined medium (mDM), altered experimentally, for 24 h. Embryos were cultured in 50 microL of c-SOF + NEA for 9 d. The percentages of oocytes exposed to heparin-capacitated spermatozoa, (previously cryopreserved in skim milk-based extender) that cleaved, reached morula, blastocyst and expanded blastocyst stages were 82.8, 57.1, 35.7 and 30.0%, respectively. Without heparin treatment the rates for cleavage, morula, blastocyst and expanded blastocyst stages were 44.3, 31.4, 18.6 and 8.6%, respectively. Therefore, heparin treatment was included in sperm capacitation. Use of spermatozoa with BSA in the IVF medium yielded no cleavage. Although extenders containing 8 to 20% egg yolk enabled good sperm motility after cryopreservation, in vitro fertilizing ability was compromised under our conditions. By contrast, semen commercially processed in season in an egg yolk-based diluent remained effective for IVF. The highest proportion of blastocysts resulted from the use of spermatozoa diluted in a skim milk extender, heparin capacitation, and insemination in medium containing lamb serum.     

Effect of milk- and TRIS-based extenders on the fertility of sheep inseminated vaginally once or twice with liquid semen

We studied the influence of two different extenders, a milk-based versus a TRIS-based extender, using a split-sample technique, on fertility after single and double vaginal inseminations in natural estrous in Norwegian Crossbred ewes. Semen from 21 Norwegian Crossbred rams, all aged approximately 0.5 years, was used for AI of totally 561 Norwegian Crossbred ewes housed at 37 different farms. The farmers performed the inseminations themselves. The ewes were allocated to four parallel groups based on the two extenders and single or double inseminations (2 x 2). The farmers were recommended to inseminate the ewes between 12 and 24 h after detection of natural standing estrous. Vaginal insemination with cooled liquid semen diluted in the milk-based extender resulted in a statistically significant (P<0.01) better fertility of about 10% units both as 25-day NR (non return rate)-and lambing rates, compared with semen diluted in the TRIS-based extender. Double inseminations gave significantly higher (P=0.03) fertility results for both extenders expressed as 25-day NR results, but was not quite statistically significant when expressed as lambing rates (P=0.06) compared with single insemination. The overall 25-day NR results for the milk-based extender (66.4%) after single inseminations is in accordance with both the national results (67.1%) based on vaginal inseminations of 11,377 ewes, as well as with the results from a previous study in the same region achieving a 25-day NR results of 63.3%. In conclusion, liquid ram semen diluted in a milk-based extender and vaginally inseminated once in natural heat, with a semen dose of 150 x 10(6) spermatozoa, gave acceptable fertility results and is to be recommended as the method of choice in Norway.     

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.     

Use of semen from a bull heterozygous for the 1 29 translocation in an IVF program

In cattle, a translocation of the Robertsonian type between the largest and smallest chromosome leads to a reduction in fertility. This is substantiated by reduced nonreturn rates in daughter groups of bulls carrying the 1 29 translocation and in the heterozygous bulls themselves. This reduction in fertility is thought to be due to the early death of embryos with unbalanced karyotypes. The influence of semen from a bull known to be heterozygous for the 1 29 translocation on the outcome of a bovine IVF program was investigated. There was a significant difference (P<0.005) in terms of cleavage rate (59.8 vs 71.1%) and blastocyst rate (12.0 vs 20.0%) between the carrier and control bull, respectively. There was no difference in blastocyst quality as measured by cell number. The results observed in vitro are consistent with the field fertility records of the 2 bulls in terms of nonreturn rates (59.2 vs 70.6%, for the carrier and control bull, respectively).     

Effect of solid storage on caprine semen conservation at 5 degrees C

In this work, we investigated the effect of storage in solid-phase extender on buck semen conserved at 5 degrees C. Furthermore, we studied the effect of addition of cysteine to the extender and the washing of seminal plasma on sperm survival. In Experiment 1, milk-based extender (M) was used as a control to study the effect of solid media storage (G) and cysteine supplementation (C), and the combination of both (GC), on in vitro sperm survival of washed and non-washed semen, conserved up to 72 h at 5 degrees C. Motility, acrosome integrity (NAR) and hypo-osmotic swelling tests (HOST) were evaluated to assess in vitro sperm survival. In Experiment 2, an artificial insemination (AI) field trial was performed to compare G versus M. Solid media (G) maintained motility of spermatozoa during storage higher than any other extender (67% G versus 62% GC; 61% M and 59% C; P<0.05), but there was no difference in NAR or HOST between extenders (P>0.05). No improvement in sperm viability was obtained by addition of cysteine to the media. Washing of semen improved motility (65% versus 60%; P<0.05), NAR (70% versus 64%; P<0.05) and HOST (37% versus 28%; P<0.05). No significant differences in fertility were obtained between G and M extenders (47% versus 41%; P>0.05). In conclusion, washing of semen and dilution in gelatin-supplemented milk extender (solid storage) appears to be a successful method for goat semen storage at 5 degrees C.     

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.     

Cryopreservation of goat spermatozoa: comparison of two freezing extenders based on post-thaw sperm quality and fertility rates after artificial insemination

TRIS-glucose or skim milk extenders are most commonly used for cryopreserving goat sperm. The aim of this study was to compare the ability of two extenders based on TRIS and skimmed milk buffer to maintain sperm viability after cryopreservation. Goat semen samples (n=110) were frozen with TRIS and with milk extender and thaw. Sperm motion parameters, morphology and acrosomal integrity were assessed in fresh and frozen-thawed samples by Sperm Class Analyzer (SCA) and Diff-Quik and Spermac staining techniques. Pregnancy rates were obtained after cervical insemination with frozen semen doses. The cryopreservation process had a significant effect on acrosome and kinematic parameters. TRIS extender provided more effective preservation of total motility, velocity parameters and amplitude of lateral head displacement after freezing. The percentage of acrosome intact spermatozoa was significantly higher in samples diluted with milk extender. In the insemination doses, mean values of velocity parameters and lateral head displacement were higher in doses processed in TRIS. Spermatozoa frozen in milk extender was mathematically greater than for those frozen with TRIS extenders, though no significant difference exists. We conclude that post-thaw kinematic parameters and acrosome integrity assessed after 1h of incubation was acceptable in both extenders which indicated the feasibility of cryopreserving goat spermatozoa. TRIS extender results in better in vitro performance compared to milk, though these improvements were not reflected in fertility results. Semen doses cryopreserved in milk extender provided greater pregnancy rates after intra-cervical insemination compared to those in TRIS extender (52.4% versus 42.9%).     

Relationship between embryo development in vitro and 56-day nonreturn rates of cows inseminated with frozen-thawed semen from dairy bulls

Frozen-thawed bull semen with > 50% post-thaw motility from 40 batches (21 bulls, 2 consecutive ejaculates per batch) was used for fertilization (IVF) and embryo development in vitro to assess the relationship between field and laboratory fertility using a retrospective approach. Each frozen batch was tested in 3 or 4 replicates with 30 oocytes per replicate. Field fertility, quantified as the 56-d nonreturn rate and based on 89 to 441 artificial inseminations per frozen batch, ranged between 46.2 and 74.8%. The cleavage and blastocyst rates after IVF varied from 29.0 to 81.9% and from 1.8 to 32.0%, respectively, with significant differences among frozen batches. Rates of cleavage and blastocyst formation were significantly related to the nonreturn rate (r = 0.59, P < 0.001; r = 0.35, P < 0.05, respectively). The interaction between cleavage and blastocyst rate was 0.69 (P < 0.001). Significant variations (P < 0.05) among frozen semen batches within 15 bulls with >/= 2 different semen batches were found for the nonreturn rate (13.3%) of 2 bulls, for cleavage rates (26.7%) in 4 bulls and for blastocyst rates (20.0%) in 3 bulls. Significant differences (P < 0.05) among replicates within the 40 frozen semen batches were only found in 3 batches (7.5%) for the cleavage rate and in 7 batches (17.5%) for blastocyst rate. Overall, bull and frozen semen batch were the greatest sources of variation in the cleavage rate (30.6 and 29.4%, respectively), while testing date was the greatest source of variation in the blastocyst development rate (21.7%). The results indicated that in vitro fertilization and, to a lesser extent, culture to the blastocyst stage could be useful in estimating the potential fertilizing ability of frozen-thawed semen from dairy bulls.     

Freezability of spermatozoa from Finn and Dorset rams in multiple semen extenders

Ten semen extenders were tested in two experiments for cryopreservation of semen collected from four Finn and four Dorset rams. Two ejaculates of semen were combined from each ram for testing each extender treatment. The extenders consisted of a series of commonly used egg yolk-TRIS media with and without sodium and triethanolamine lauryl sulfate (STLS), a similar extender with 3-N-morpholino propane sulfonic acid (MOPS), and milk and whey extenders. In Experiment 1, extender treatments were replicated with three sets of collections from the eight rams, and in Experiment 2 with two sets. The egg yolk-TRIS-glycerol-STLS (EY(1)TSTLS) extender was significantly superior to other extenders except whole milk in protecting the sperm during freezing and thawing. In Experiment 1, a 20% egg yolk-TRIS-glycerol-STLS extender preserved 71% of the progressively motile Finn sperm (post-thaw divided by pre-freeze percentage of motile sperm), and 76% of the Dorset sperm. In Experiment 2, the corresponding values for the same EY(1)TSTLS extender used with Finn and Dorset sperm were 86 and 64%, respectively. Without STLS the egg yolk extenders were significantly less effective in protecting cryopreserved ram sperm. This egg yolk-TRIS extender, containing STLS and glycerol, may hold promise for freezing ram sperm that could be used successfully for intracervical insemination.     

Fertility results using bovine semen cryopreserved with extenders based on egg yolk and soy bean extract

Semen extenders containing components such as egg yolk and skim milk are difficult to standardize and they introduce the risk of microbial contamination. A well-defined extender not originating from animal tissues would present a valuable contribution to the AI industry. We evaluated the fertility of bovine semen cryopreserved with 3 different extenders: 1) TRIS-Standard, prepared at 2 local AI laboratories, containing 20% (v/v) pasteurized egg yolk, 2) TRIS-Concentrate, prepared by adding 20% (v/v) pasteurized egg yolk and 1:5 (v/v) nonpyrogenic water, and 3) Biociphos Plus, a soybean extract containing extender, prepared by adding 1:5 nonpyrogenic water. Ejaculates of 4 Holstein bulls were split into 3 aliquots and cryopreserved with the 3 extenders. Prior to this study, the semen dose-response curve for each of the 4 bulls was developed in a field trial by freezing the semen and randomly distributing the straws throughout the Netherlands for insemination. Optimal semen doses were thus established to detect the effect of extenders on fertility, evaluated by 56-day non-return rate (NR56), and by the estimated conception rate and the calving rate, given a conception. We used the multiphasic model developed by Grossman et al. (7). A total of 22,246 first and second inseminations were recorded. The NR56 ranged among bulls from 67.0 to 70.1% for Tris-Standard, from 67.5 to 69.9% for Tris-Concentrate and from 60.2 to 66.7% for Biociphos Plus. No significant differences in NR56 were detected between Tris-Standard and Tris-Concentrate (P=0.54), whereas Biociphos Plus resulted in a significantly lower NR56 than Tris-Standard and Tris-Concentrate (P<0.05). Estimated conception rate was 72.1, 73.6 and 69.6% and estimated calving rate, given a conception was 80.6, 78.3 and 77.1 for Tris-Standard, Tris-Concentrate and Biociphos Plus, respectively. These results indicate that 1) semen extended with a custom made TRIS-Concentrate can be succesfully used in the field resulting in comparable fertility with Tris-Standard; 2) semen extended with Biociphos Plus results in a significant reduction in the NR56; 3) extender source may affect both conception rate and calving rate, given a conception, i.e., extrinsic and intrinsic sperm factors (4).     

Mink semen studies: I. Liquid preservation and prospect of freezing spermatozoa

Using 22 males, 41 semen samples were collected from the vagina of mink by means of a plastic tubing attached to a 1 ml syringe. Subsamples of vaginal semen were diluted in 4 different extenders, viz., tris (tris, citric acid, glycine, fructose, glycerol and egg yolk), PVP (tris extender with polyvinyl pyrrolidone and caproic acid), milk (boiled and filtered milk with glycerol) and sodium citrate. The extended semen samples were stored at 23, 5 and −196°C for varying periods and evaluated for % motile spermatozoa. In the tris extender storage for 3 days at 5°C or for 2 days at 23°C reduced the number of spermatozoa by more than 50%. When milk was used as the extender, the motility decreased from an initial value of 68% to 10% after 5 days of 5°C and to 8% after 4 days at 23°C. The PVP extender was not suitable for storage at any temperature. After being frozen at −196°C for 2 hr, the motility ranged from 3–10% in the tris extender and was zero in milk and PVP extenders. Prolonged storage for 7 days in tris extender reduced the motility to 1–7%.     

Effects of the 7 21 Robertsonian translocation on fertilization rates and preimplantation development of bovine oocytes in vitro

A study was conducted to investigate the effect of the 7/21 Robertsonian translocation on fertilization and subsequent development of bovine oocytes matured in vitro. Semen from Japanese Black bulls, 2 with a normal karyotype (Bulls A and B) and 2 that were heterozygous for the 7/21 translocation (Bulls C and D), was used in this study. In vitro matured bovine oocytes were inseminated with frozen-thawed sperm capacitated with heparin. After insemination, oocytes were cultured at 38.5 degrees C on a monolayer of cumulus cells in TCM-199 supplemented with 5% superovulated cow serum and 0.5 mM sodium pyruvate in an atmosphere of 2% CO2 in air. Cleavage rate was evaluated at 54 h after insemination, and development of embryos to the blastocyst stage was observed 7 to 10 d post insemination. There was no difference in the fertilization rate among the 4 bulls. Although the cleavage rate of oocytes inseminated with semen from Bull C (heterozygote) was lower (P < 0.05) than that obtained with semen from Bull B (normal), the blastocyst formation rate did not differ among the 4 bulls. These results indicate that the 7/21 Robertsonian translocation had no effect on the fertilization and blastocyst formation rates of bovine in vitro-matured oocytes.     

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.     

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.     

Effect of reducing sperm concentration during IVF on the ability to distinguish between bulls of high and low field fertility: work in progress

The objectives of this study were to evaluate the effect of sperm dose and sire on the fertilization rate, cleavage rate and blastocyst yield following insemination in vitro, to examine the relationship between these parameters and field fertility in cattle, and to examine the relationship between blastocyst quality and sire used in IVF. Frozen semen from four bulls with 150-day nonreturn rates ranging from 57 to 78% was used. In Experiment 1, oocytes were inseminated with sperm from one of the four bulls at concentrations ranging from 0.016 to 0.5 x 10(6)sperm/ml. A proportion of presumptive zygotes were fixed at 17 h post-insemination (hpi), while the remainder was transferred to in vitro culture (IVC) in droplets of synthetic oviduct fluid (SOF). Cleavage at 48 hpi and the percentage of oocytes reaching the blastocyst stage by Day 8 were recorded. In Experiment 2, to assess blastocyst quality, after insemination with semen from one of the four bulls, presumptive zygotes were cultured in SOF until Day 7. Blastocysts for each bull were removed and vitrified/warmed and survival was recorded at 24, 48 and 72 h after warming. Regardless of bull used, a concentration of 0.125 x 10(6)sperm/ml or above resulted in higher blastocyst yields than any lower concentration used. Fertilization and cleavage rates were also higher at higher sperm concentrations. The best predictor of field fertility was fertilization rate at a concentration of 0.5 x 10(6)sperm/ml (r=0.94, P<0.0001). There was also a significant correlation between cleavage rate at a concentration of 0.5 x 10(6)sperm/ml and nonreturn rate (r=0.90, P<0.0001). In Experiment 2, blastocysts derived from one bull, HTA, were of superior quality as measured by survival 24h after thawing, although these differences were less significant at the subsequent time points measured. In conclusion, these data show that differences between the field fertility of bulls can be determined at sperm concentrations routinely used in IVF. Lowering the sperm concentration does not increase the likelihood of optimizing the differences in fertility or cleavage rate between bulls of different field fertility. We have also demonstrated that the bull can have a significant effect on the quality of blastocysts produced using IVF techniques.     

Comparison of an extender containing defined milk protein fractions with a skim milk-based extender for storage of equine semen at 5 degrees C

A problem of semen extenders based on milk or egg yolk is the fact that these biological products consist of a variety of substances. Extenders containing only components with clearly protective effects on spermatozoa would thus be an advantage. In this study, we have compared the effects of an extender containing defined caseinates and whey proteins only (EquiPro, defined milk protein extender) with skim milk extender on equine spermatozoa during cooled storage. The defined milk protein extender was used with and without the antioxidant N-acetyl cysteine (NAC). In a second experiment, semen was diluted with PBS or defined milk protein extender and was either stored directly or 90% of seminal plasma was removed by centrifugation and replaced by defined milk protein extender before storage. In both experiments, eight stallions were available for semen collections. Motility, velocity and membrane integrity of spermatozoa were determined by CASA immediately after semen processing and after 24, 48 and 72 h of storage at 5 degrees C. Total motility after 24 h of storage was lowest in semen diluted with PBS (p<0.05 versus all extenders). At 48 and 72 h, motility of spermatozoa in defined milk protein extender was significantly (p<0.05) higher than in PBS or skim milk extender. Velocity of spermatozoa after storage was highest in defined milk protein extender. Membrane integrity after storage was significantly (p<0.05) lower in semen diluted with PBS than in semen diluted with both extenders. Addition of NAC was without effect on the examined parameters. Centrifugation further increased the percentage of motile and membrane-intact spermatozoa in the defined milk protein extender (p<0.05). Velocity of spermatozoa in this extender was not negatively affected by centrifugation.     

Heterologous in vitro fertilization and embryo production for assessment of jaguar (Panthera onca Linnaeus, 1758) frozen-thawed semen in different extenders

Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 10⁶ spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.     

Effect of native phosphocaseinate on the in vitro preservation of fresh semen

The fertilization capacity of goat sperm stored in milk extenders is approximately 12-24h. Long-term storage of goat sperm (up to 3 days) is desirable as it would confer greater flexibility to breeding farms. The aim of this study was to evaluate in vitro motility parameters of buck spermatozoa for up to 7 days of storage using skim milk or chemically defined extender supplemented with native phosphocaseinate (NPPC). Four experiments were conducted to determine optimum temperature (4 or 15 degrees C) and storage conditions (aerobic versus anaerobic), the effect of seminal plasma on sperm survival, the optimal concentration of NPPC and the effect of beta lactoglobulin (BL). Both skim milk and NPPC were found to be more efficient for preserving goat sperm at 4 degrees C than at 15 degrees C (P<0.01). Furthermore, when sperm was stored at 4 degrees C, no detrimental effects of seminal plasma were observed. Our results showed that motility parameters can be maintained with success until Day 4. However, NPPC-based extenders extend the in vitro survival to 7 days of storage. The optimal concentration of NPPC for the preservation of sperm cells for 4 days of storage was 81g/l and for 7 days of storage was 81 and 54g/l. No effect of the supplementation of the NPPC extender with BL was found.     

In vitro fertilization of in vitro matured canine oocytes using frozen-thawed dog semen

Experiments were conducted to evaluate in vitro fertilization (IVF) of in vitro matured (IVM) bitch oocytes using dog spermatozoa frozen in three different extenders. Sperm-rich fraction from eight ejaculates of five dogs was frozen in each one of three egg yolk Tris extenders with additional: (A) 1.4 g citric acid and 0.8 g glucose; (B) 0.7 g citric acid and 3.5 g glucose; or (C) 1.4 g citric acid and 0.8 g fructose (all with 5% glycerol in 100 mL milliQ water). Thawed sperm were co-incubated with IVM bitch oocytes for 6 h. Oocytes were fixed and evaluated under an epifluorescence microscope; penetrated oocytes were defined as those having sperm heads in the perivitelline space or in the oocyte cytoplasm. Higher penetration rates (P < 0.05) were obtained in oocytes cultured with spermatozoa frozen in extenders B and C than those frozen in extender A (33.1, 34.2 and 26.4%, respectively).