The advantages of LDL (Low Density Lipoproteins) in the cryopreservation of canine semen

A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze–thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at −196 °C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively.Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p < 0.05).In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p < 0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test).In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24 h; 200 × 10⁶ spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%).In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze–thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone.Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.     

Quail Egg Yolk: A Novel Cryoprotectant for the Freeze Preservation of Poitou Jackass Sperm

For many years, attempts have been made to establish a sperm bank for the Poitou jackass population which is threatened with extinction. Unfortunately, no cryopreservation technique has ever been described for spermatozoa of this species. In an attempt to find a suitable technique, we studied the relative effectiveness of chicken egg yolk and quail egg yolk in preserving the motility and characteristics of movement of Poitou jackass spermatozoa during the freezing–thawing process. Semen was diluted to 60 × 10⁶sperm/ml in a preservation medium containing 4% (v/v) glycerol with 0, 2, 5, 10, 15, or 20% (v/v) of chicken or quail egg yolk. The chemical composition of these two eggs was compared. Effects were assessed using an automated analyzer which measured curvilinear velocity (VCL), straight line velocity (VSL), and the velocity of the average path. Linearity was defined as VSL/VCL × 100. The amplitude of the lateral head displacement was also measured. It was found that after the freeze–thaw process, quail egg yolk improved the percentages of motile and progressively undulating spermatozoa and the movement characteristics compared with chicken egg yolk. The optimal concentration of quail egg yolk was 10%. The general composition of the two types of egg yolk were similar, but quail egg yolk contained significantly more phosphatidylcholine, less phosphatidylethanolamine, and a smaller ratio of polyunsaturated to saturated fatty acids than chicken egg yolk. The improvement of motility for frozen–thawed Poitou jackass spermatozoa using frozen–thawed quail egg yolk compared to chicken egg yolk may be due to the differences in composition of the two yolks.     

A comparison of the protective action of chicken and quail egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa

Egg yolk-based diluents provide adequate cryoprotection for the sperm of several mammalian species. Traditionally, chicken egg yolk has been used as additive for the freeze preservation of spermatozoa because of its wide availability. Variations in the chemical composition of the egg yolk of different avian species appear to influence the protection afforded during cooling, freezing, and thawing. The aim of the present study was to assess the use of quail egg yolk as a novel additive for the epididymal spermatozoa of a threatened wild ruminant species—the Spanish ibex—and to compare its efficacy with chicken egg yolk. Epididymal spermatozoa were diluted using a Tris–citric acid–glucose medium (TCG) composed of 3.8% Tris (w v⁻¹), 2.2% citric acid (w v⁻¹), 0.6% glucose (w v⁻¹), 5% glycerol (v v⁻¹), and 6% egg yolk (v v⁻¹). Sperm masses from the right epididymes were diluted with TCG-6% chicken egg yolk medium, while those from the left were diluted with TCG-6% quail egg yolk. The thawed spermatozoa preserved with TCG-6% quail egg yolk extender exhibited lower motility (P < 0.001), membrane integrity (P < 0.001), and viability (P < 0.01) than those diluted with the TCG-6% chicken egg yolk extender. The fertility of spermatozoa frozen in TCG-6% chicken egg yolk tended to be higher than in those frozen with TCG-6% quail egg yolk (63.3% vs 36.4%, P = 0.19). These results show that quail egg yolk offers no advantages over chicken egg yolk in the cryopreservation of Spanish ibex epididymal spermatozoa.     

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing–thawing

Egg low-density lipoprotein (LDL) was added at concentrations of 7–10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen–thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 μm/s; VCL, curvilinear velocity: 50.2 μm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 μm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P < 0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P < 0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen–thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.     

Effect of low density lipoprotein on the quality of cryopreserved dog semen

Egg yolk is included in extenders for semen cryopreservation due to its protective effect against cold shock, which is attributed to the presence of low density lipoprotein (LDL). This study evaluates how semen quality is affected by using LDL as a replacement for egg yolk in extenders for cooled and frozen dog semen. In Experiment 1, semen was extended in TRIS–glucose at 5 °C, in four treatments: 20% egg yolk (T1); 6% (T2); 8% (T3); and 10% LDL (T4). Sperm motility and membrane integrity after 24, 48, 72 and 96 h and the 50% conservation rate of motile spermatozoa (50 M) were evaluated. The 50 M was less for T1 than for the other treatments (P < 0.01), but T2–T4 did not differ (P > 0.05). In Experiment 2, glycerol at 10% was included in the freezing extender, in treatments similar to those from Experiment 1. Sperm motility and membrane integrity did not differ for T2, T3 and T4 at any period in Experiment 1 and after thawing in Experiment 2 (P > 0.05), but were greater for all LDL treatments than for T1 (P < 0.01), in both experiments. Thus, LDL can replace egg yolk in the composition of the TRIS–glucose extender for cooled or frozen dog semen.     

The advantages of low-density lipoproteins in the cryopreservation of bull semen

Egg low-density lipoprotein (LDL) was added at concentrations (w/v) of 7%, 8% or 9% to the extenders used to freeze bull semen and its effects on seminal parameters and anti-oxidant activities of frozen–thawed sperm were assessed. Analysis of data showed that sperm exposed to 8% LDL exhibited the greatest percentages of sperm motility, acrosome integrity and membrane integrity, compared to the control which differed from the treatment groups by replacing LDL with 20% egg yolk (P < 0.05). No difference was observed for membrane integrity between 8% and 9% LDL groups (P > 0.05). The extender supplemented with LDL did not exhibit improvement in SOD levels. However, 8% LDL group favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison to other groups (7%, 9% LDL and the control) (P < 0.05). No difference was observed for CAT activity between 9% LDL and the control group. In conclusion, sperm cryopreserved in the extender containing 8% LDL in place of egg yolk exhibited the greatest percentages of post-thaw sperm motility, acrosome integrity and membrane integrity, in comparison with the control, and favored the highest anti-oxidant activities of CAT, GSH-Px and GSH in comparison with other groups. The replacement of egg yolk by LDL in the composition of extenders was beneficial for bull sperm cryopreservation.     

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.     

Effect of egg yolk, cryoprotectant, and various sugars on semen cryopreservation in endangered Cuvier's gazelle (Gazella cuvieri)

Cryopreservation of spermatozoa from endangered species is a valuable tool for genetic management. Previous studies showed the feasibility of cryopreservation of spermatozoa from various endangered gazelles but have also revealed difficulties with available protocols for semen freezing in Cuvier's gazelle (Gazella cuvieri). Experiments were carried out to investigate the effect of (a) 5% or 20% egg yolk or 4% or 6% glycerol, and (b) addition of sugars (glucose, fructose, lactose and raffinose) on cryopreservation using a Tes-Tris-based diluent (TEST). A diluent containing 13.5% raffinose, 5% or 20% egg yolk, and 6% glycerol (REYG) was also evaluated. Semen was obtained by electroejaculation from 22 G. cuvieri males. Diluted samples were loaded into 0.25 ml straws, cooled to 5 °C over 1.5 h (−0.16 °C/min), equilibrated at that temperature for 2 h, frozen in nitrogen vapours for 10 min and plunged into liquid nitrogen. Subsamples were assessed for motility and acrosome integrity upon collection, after refrigeration–equilibration, after freezing and thawing, and 2 h after thawing. Use of TEST with 20% egg yolk or with 4% glycerol led to worse motility preservation, whereas TEST with 5% egg yolk and 6% glycerol led to better results. Addition of fructose, lactose or raffinose to TEST resulted in similar or worse preservation of motility than inclusion of glucose. On the other hand, use of a raffinose-based medium with 20% egg yolk and 6% glycerol (REYG) afforded better preservation of motility than use of TEST. With REYG, 20% egg yolk was better than 5% egg yolk for motility preservation. Differences were noted between males in their responses to cryopreservation when using different egg yolk or glycerol concentrations. Moreover, spermatozoa from most males exhibited better cryopreservation with REYG although some were better cryopreserved in TEST. The raffinose-based diluent thus represents an improvement over previous results but more work is needed to better characterize cryopreservation conditions for future routine banking of Cuvier's gazelle spermatozoa.     

Antioxidant effects of clove bud (Syzygium aromaticum) extract used with different extenders on ram spermatozoa during cryopreservation

Clove bud (Syzygium aromaticum) extract was added at concentrations of 0, 35, 75, and 115 μg/ml to ovine semen extenders in order to investigate the antioxidant activities of clove bud extract and its effects on semen quality parameters after cryopreservation of ram spermatozoa. The basic extender was composed of Tris, egg yolk, and glycerol. Two other extenders were prepared by substitution of egg yolk with either LDL or egg yolk + SDS. The DPPH inhibition test was employed to assess the antioxidant activity of clove bud extract. Results showed that, compared to vitamin E, clove bud extract had a higher antioxidant activity. Better sperm motility and movement characteristics (P < 0.05) were observed in the semen diluted with medium containing egg yolk + SDS than in that containing egg yolk and LDL. Progressive motility and movement characteristics of the sperm were significantly improved (P < 0.05) by adding 35 and/or 75 μg/ml of clove bud extract to semen extenders. Sperm viability and plasma membrane integrity were also higher (P < 0.05) in the semen exposed to medium containing egg yolk + SDS and 75 μg of clove buds extract after cryopreservation processes. Higher levels of clove bud extract, however, had adverse effects on all the sperm quality parameters and significantly reduced (P < 0.05) the motility, movement parameters, viability, and plasma membrane integrity of ovine sperm. It was concluded that the clove bud extract had an antioxidant potential that makes it useful for addition to semen extenders and that the best results are obtained with a maximum clove bud extract of 75 μg/ml. Moreover, the combination of egg yolk and a detergent was found to improve sperm quality after the cooling and freeze–thawing processes.     

Cryoprotective and contraceptive properties of egg yolk as an additive in rooster sperm diluents

The addition of chicken egg yolk to semen extenders is thought to reduce the fertilizing potential of rooster spermatozoa - but not (or at least not as much) that of other avian species. The aim of the present study was to determine whether quail egg yolk, a novel extender additive, provides advantages over chicken egg yolk in the cryopreservation of rooster spermatozoa. Experiments were also performed to determine whether the harmful effect of egg yolk occurs during cryopreservation or during fertilization after artificial insemination. Heterospermic rooster semen samples were divided into aliquots and cooled in a polyvinylpyrrolidone-based medium containing 15% chicken egg yolk, 15% quail egg yolk or no egg yolk at all. The viability of spermatozoa of cooled samples (5 °C) without egg yolk were less viable (P < 0.01) than those of samples containing either type of egg yolk. The same aliquots were then cryopreserved for 15 days. Thawed spermatozoa preserved without egg yolk showed lower motility (P < 0.001) and viability (P < 0.001) than those in samples diluted with either type of egg yolk extender. No eggs were fertilized when hens were inseminated with semen that had been diluted with chicken egg yolk. The fertilization rate was only slightly higher when sperm diluted with quail egg yolk was used (1.5%). The best results were obtained when no egg yolk was used (13.8%). These results show that the addition of egg yolk of either type protects rooster sperm cells against cold shock and during freezing and thawing, but exerts a contraceptive effect in the genital tract of the hen.     

Effect of hen's egg yolk on capacitation and acrosome reaction of diluted canine spermatozoa

The aim of this study was to investigate the influence of progesterone, cholesterol and calcium (Ca²⁺) in an egg-yolk-containing extender on capacitation and acrosome reactions (AR) of diluted canine spermatozoa during 4 days of cooled-storage. For this purpose, we first investigated the effect of supplementation of a Tris–citrate–fructose buffer (TCF) with progesterone in a final concentration of 0.1, 0.2 and 1.0 μg progesterone/ml TCF-diluted semen. We then compared the effects of TCF and the same buffer-containing 20% egg yolk (TCF–EY). In egg yolks and the TCF–EY, progesterone was measured by enzyme immunoassay, cholesterol by enzymatic colorimetry and Ca²⁺ by flame atomic absorption spectrophotometry. For both experiments, ejaculates from eight dogs were used. For the comparison of diluents, one ejaculate was divided and one half diluted with TCF, the other with TCF–EY. One half of each TCF- and TCF–EY-diluted sample was evaluated immediately (D1), the other after storage for 4 days at +4 °C (D4). In diluted semen, motility and viability were measured by a computer assisted sperm analyzer (CASA; Sperm Vision, Minitüb, Germany), capacitation and AR were evaluated with a modified chlortetracycline assay (CTC) and the AR additionally by flow cytometry. Results: Supplementation of progesterone revealed, that between D1 and D4, total and progressive motility decreased with all progesterone concentrations, while viability as well as percentage of capacitated and acrosome reacted spermatozoa stayed constant. Progesterone-, cholesterol- and Ca²⁺ concentrations in egg yolks were 524.8 ± 131.4 ng/g, 13.9 ± 2.03 mg/g and 1.27 ± 0.17 mg/g, respectively. In the TCF–EY-diluent, the respective values were 210.9 ng/g, 2.52 mg/g and 1.1 mg/g. In TCF–semen, at D1, motility and viability were significantly higher than in TCF–EY-samples (p < 0.05), however at D4, no significant differences were detectable. Further, in TCF–semen, percentages of spermatozoa with intact membranes decreased significantly (p < 0.05) and capacitated spermatozoa increased (p < 0.05), which was not seen in TCF–EY-samples. In all samples, low percentages of AR were detected and after 4 days, the highest value of AR in TCF–EY-samples was 5.3% on average, as detected by flow cytometry. We therefore conclude that progesterone from egg yolk in routine extenders does not substantially influence semen longevity or AR of canine semen during cold-storage for 4 days. In contrary, egg yolk seems to prevent a significant increase in capacitated spermatozoa.     

Freezability of rat epididymal sperm induced by raffinose in modified Krebs-Ringer bicarbonate (mKRB) based extender solution

The objective of this study was to develop an ideal freezing extender and method for rat epididymal sperm cryopreservation. Epididymal sperm collected from 30 Wistar males was frozen, and experiments were conducted to study its post-thaw characteristics when freezing with raffinose-free buffer or various concentrations of raffinose and egg yolk dissolved in distilled and deionised water, PBS, or modified Krebs–Ringer bicarbonate (mKRB)-based extender. Different concentrations of glycerol, Equex STM, or sodium dodecyl sulfate (SDS) dissolved in either PBS or mKRB containing egg yolk were also tested. Based on the data from these experiments, further experiments tested how different sugars such as raffinose, trehalose, lactose, fructose, and glucose dissolved in mKRB with Equex STM, SDS and egg yolk supplementation affected the post-thaw characteristics of cryopreserved sperm. Cryosurvival of frozen-thawed sperm were judged by microscopic assessment of the sperm motility index (SMI), and acrosome integrity was measured using FITC-PNA staining. Thawed sperm were subjected to 3 h of a thermal resistance test. Beneficial effects on the post-thaw survival of sperm were obtained when 0.1 M raffinose in mKRB was used with 0.75% Equex STM, 0.05% SDS, and 20% egg yolk. Sperm cryopreserved with this treatment exhibited a higher motility index and maintained greater SMI and acrosome integrity throughout incubation when compared to sperm frozen in various concentrations of other cryoprotectants and trehalose, lactose, fructose, glucose. In conclusion, cryopreservation in an extender solution of raffinose dissolved in mKRB containing Equex STM, SDS and egg yolk greatly enhances the freezability of rat epididymal sperm.     

Effect of sperm coating on the survival and penetrating ability of in vitro stored bovine spermatozoa

The aim of this study was to examine the effect of sperm coating on the survival and penetrating ability of in vitro stored diluted spermatozoa. Bovine semen was collected by means of an artificial vagina connected with a tube containing 5 ml of the commercial Triladyl diluent supplemented with 20% egg yolk and 6.7% glycerol (EYTG). Both EYTG and seminal plasma were removed by centrifugation and the spermatozoa were stored under different in vitro storage conditions. In the first and second experiment, "control" and "coated" spermatozoa were stored in Hepes-TALP (pH 6 and 7) at room temperature. After 4 days of storage, the progressive motility, membrane integrity, mitochondrial membrane potential or DNA integrity of the spermatozoa were evaluated before and after Percoll centrifugation. The in vitro penetration rate of the spermatozoa was examined only after Percoll centrifugation. A significantly (P<0.05) positive influence of sperm coating was observed on the tested sperm characteristics and penetration rate of spermatozoa when they were stored in Hepes-TALP at pH 7, but not at pH 6. In the last experiment, the influence of the storage medium Hepes-TALP (pH 7) or EYTG was investigated on motility, membrane integrity, mitochondrial membrane potential and in vitro penetration potential of "coated" spermatozoa stored at room temperature or at 4 degrees C during 4, 5 and 6 days. After 6 days of storage, a significantly (P<0.05) higher percentage of motile and membrane intact spermatozoa with high mitochondrial membrane potential was obtained in EYTG at both temperatures leading to a significantly higher in vitro penetration rate. These results indicate that sperm coating could preserve sperm characteristics and penetrating capacity of fresh bovine spermatozoa stored in egg yolk containing diluent for up to 6 days.     

Low density lipoproteins extracted from hen egg yolk by an easy method: cryoprotective effect on frozen-thawed bull semen

Hen egg yolk is widely used as a cryoprotective agent in semen freezing extenders in order to protect the spermatozoa against cold shock. The protective action of yolk is largely presumed to be due to low density lipoproteins (LDL). In recent years, arguments concerning the presence of cryoprotective antagonists in egg yolk, have reinforced interest in the use of only the LDL extracted from egg yolk in the extenders. However, current methods of LDL purification do not support the use of LDL in commercial extenders because they offer a poor recovery rate. Consequently, we have developed an easy method to extract LDL from egg yolk. Several concentrations of purified LDL (between 2.5 and 20%, w/v) were tested in freezing extenders for bull semen, and compared with commercial extenders. Our extraction method reached 97% purity and about 67% yield, and is easily reproducible on an industrial scale. Analysis of sperm motility showed that the motility and characteristics of spermatozoa movement were improved with LDL in the extender, as compared to a commercial extender containing egg yolk. The optimum LDL concentration in the extender was 8%. In conclusion, we propose that an extender containing LDL extracted from egg yolk could be used as cryoprotective media with a better efficiency than present commercial extenders.     

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.     

Effect of low density lipoproteins in extender on freezability and fertility of buffalo (Bubalus bubalis) bull semen

This study was designed to determine whether low-density lipoporoteins (LDLs) extracted from egg yolk in extender improve the freezability and fertility of buffalo bull semen. Semen from three Nili-Ravi buffalo bulls was diluted at 37 °C with tris-citric acid extender (50 × 10⁶ motile spermatozoa mL⁻¹) containing LDLs 2.5%, 5%, 10%, and 15% extracted from egg yolk and extender containing 20% egg yolk was kept as control. Diluted semen was cooled to 4 °C in 2 h, equilibrated at 4 °C for 4 h, filled in 0.5 mL French straws, and kept on liquid nitrogen vapors for 10 min. Straws were then plunged and stored in liquid nitrogen (-196 °C). Sperm motility (visually; %), plasma membrane integrity (%; with supravital hypo-osmotic swelling test), and viability (%; with dual staining test using Trypan-blue Giemsa) were assessed at post-dilution, pre-freezing and post-thawing. At post-dilution and pre-freezing, sperm progressive motility, plasma membrane integrity and viability was similar (P > 0.05) in extender containing 10% LDLs or the control. However, at post-thaw the aforementioned parameters were higher (P < 0.05) in extender containing 10% LDLs compared with the control and other experimental extenders. The fertility rate of inseminations performed were higher (P < 0.05) with extender containing 10% LDLs than the control. It was concluded that LDLs (10%) in extender improved the freezability and fertility of buffalo bull spermatozoa.     

Freeze-dried egg yolk based extenders containing various antioxidants improve post-thawing quality and incubation resilience of goat spermatozoa

The aim of this study was to evaluate different antioxidants-supplemented freeze-dried egg yolk based extenders for the post-thawing quality and incubation resilience of goat spermatozoa. Pooled semen were diluted in a two-step dilution method to a final concentration of 1/5 (semen/extender) in control and antoxidant supplemented freeze-dried extenders (methionine, cysteamine and butylated hydroxytoluene). Semen samples were assessed for sperm motility, plasma membrane functional integrity using hypoosmotic swelling test (HOST), damaged acrosome using FITC-Pisum sativum agglutinin (PSA-FITC) and DNA integrity using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Membrane lipid peroxidation status was also analyzed using the malondialdehyde (MDA) concentration. In the study, antioxidant supplemented freeze-dried egg yolk based extenders have beneficial effect on goat sperm parameters. In addition, we achieved a higher quality in post thawed goat semen even after 6 h incubation when the extender was supplemented by 5 mM BHT or cysteamine.     

Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality

Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll^® centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR^®14-PI) and DNA fragmentation (TUNEL). After addition of a Tris–glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 °C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P < 0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.     

Natural, but not lyophilized, low density lypoproteins were an acceptable alternative to egg yolk for cryopreservation of ram semen

The objective was to evaluate the suitability of using natural or lyophilized low density lipoproteins (LDL), in lieu of whole egg yolk, in extenders for cryopreserving ram semen. Once extragonadal sperm reserves were depleted in 10 fertile Santa Inês cross rams, two ejaculates per ram were collected for cryopreservation. Nine extenders were used: Tris-16% egg yolk extender with 5% glycerol as a control (T1), and substitution of whole egg yolk with 8, 12, 16 or 20% natural LDL (T2-T5, respectively), or with 8, 12, 16, or 20% lyophilized LDL (T6-T9). Semen was diluted to 100 × 10⁶ sperm/mL, packaged into 0.25 mL straws, cooled, held at 5 °C for 3 h, and then frozen in liquid nitrogen vapor. Immediately after thawing (37 °C for 30 s), sperm total and progressive motility, and kinetic parameters were analyzed with computer assisted semen analysis (CASA). Percentage of sperm with plasma membrane functional integrity was assessed by the hypoosmotic swelling test (HOST), sperm membrane physical integrity with propidium iodide (PI), and acrosome integrity with FITC-PSA using an epifluorescent microscope. For all sperm end points, there was no difference between the control and natural LDL treatments (P > 0.05): total motility (T1: 20.9 ± 11.9 and average of T2-T5: 25.9 ± 13.6%; mean ± SD), progressive motility (T1: 6.6 ± 4.2 and average of T2-T5: 11.7 ± 7.5%), HOST⁺ (T1: 23.7 ± 6.9 and average of T2-T5: 23.2 ± 8.7 %) and PI⁻/PSA⁻ (T1: 13.8 ± 7.8 and average of T2-T5: 18.1 ± 7.8%). However, lyophilization was apparently unable to preserve the protective function of LDL; every sperm end point was significantly worse than in the control and natural LDL groups. We concluded that natural LDL was appropriate for cryopreserving ram semen, as it yielded results similar to those obtained with whole egg yolk.     

Effect of semen dilution to low-sperm number per dose on motility and functionality of cryopreserved bovine spermatozoa using low-density lipoproteins (LDL) extender: Comparison to Triladyl and Bioxcell

Artificial insemination with doses containing low-sperm numbers has been utilized to optimize the use of elite bulls. Hen egg yolk is widely used as a cryoprotective agent in semen freezing extender protecting the spermatozoa. Its action is due to the presence of low-density lipoproteins (LDL) in the hen egg yolk. The objectives of the present study were to evaluate the effects of the semen dilution to low-sperm number/dose on sperm motility and integrity of sperm plasma membrane in the cryopreservation process, using two commercial extenders (Triladyl^®, Bioxcell^®) and LDL extender prepared in our laboratory, 97% purity. Fifteen ejaculates were collected from five fertile crossbred bulls (Bos taurus × Bos indicus). After collection, sperm motility was examined by Computer-Assisted Semen Analysis (Hamilton Thorne), morphological sperm characteristics were evaluated by differential interference microscopy and the integrity of plasma membranes was determined using the hypo-osmotic swelling test. The semen was subsequently divided into three aliquots and diluted with the three extenders into 120 × 10⁶, 60 × 10⁶ and 20 × 10⁶ sperm/mL, corresponding to 30 × 10⁶, 15 × 10⁶ and 5 × 10⁶ sperm/dose, respectively. This study revealed that LDL extender was more effective in preservation of motility and integrity of the plasma membrane of spermatozoa than Bioxcell^® and Triladyl^® (p < 0.05), but no significant difference was observed between Triladyl^® and Bioxcell^®. Therefore we can conclude that LDL extender could be used instead of Triladyl^® or Bioxcell^® at low semen concentration per dose for elite bulls, it also could be envisaged for the industry of sex-stored semen.