Cryopreservation of Dog Semen in a Tris Extender with 1% or 2% Soya Bean Lecithin as a Replacement of Egg Yolk

Egg yolk is usually included in extenders used for preservation of dog semen. Lecithin is an interesting animal‐protein free alternative to egg yolk for semen preservation. The aim of our study was to evaluate soya bean lecithin for cryopreservation of dog semen. Five ejaculate replicates were divided in three equal parts, centrifuged and each pellet diluted with one of the three Tris‐based extenders containing 20% egg yolk, 1% soya bean lecithin or 2% soya bean lecithin. Extended semen was loaded in 0.5‐ml straws, cooled and diluted a second time and frozen in liquid nitrogen vapours. Sperm motility parameters (CASA), acrosome integrity (FITC‐PNA/PI) and sperm membrane integrity (C‐FDA) were evaluated 5 min post‐thaw and after 2 and 4 h of incubation. Total motility was significantly better in the egg yolk extender than in any of the lecithin‐based extender and was better in the 1% lecithin extender than in the 2% lecithin extender. Sperm membrane integrity was significantly better in the egg yolk extender than in any of the lecithin‐based extenders but did not differ significantly between the 1% and 2% lecithin extenders. Acrosome integrity was significantly better in the egg yolk extender than in the 2% lecithin extender but did not differ between the egg yolk extender and the 1% lecithin extender or between the two lecithin extenders. In conclusion, egg yolk was superior to lecithin in our study. The extender with 1% lecithin preserved sperm motility better than the extender with 2% lecithin.     

The Effect of Removal of Seminal Plasma, Egg Yolk Level and Season on Sperm Freezability of Canary Buck (Capra hircus)

Goat semen is different from that of other domestic species in its limited tolerance to the inclusion of egg yolk in the freezing medium, and this tolerance depends on the presence of enzymes in the seminal plasma that react with egg yolk, producing toxic compounds to the spermatozoa. Moreover, the goat is a seasonal breeder that shows variations in semen quality throughout the year, and those variations may affect semen freezability; hence in freezing protocols, for instance, removal of seminal plasma (washing) yields varying results. This work was designed to study this problem in Canary goats: semen from six males was collected in spring, autumn or winter, washed or non-washed, diluted in a freezing extender with 1.5, 6 or 12% egg yolk, frozen, and thawed after 2 days, 2 or 6 months of cryopreservation. The effect of egg yolk concentration in the freezing extender was far more important than the effect of washing or season on sperm cryosurvival. The quality of frozen-thawed semen tended to improve as egg yolk concentration increased regardless of the effects of season, washing or period of cryopreservation. Washing produced a positive effect on frozen-thawed semen collected during spring or autumn, but the difference decreased as the concentration of yolk increased. However, washing produced a negative effect on frozen-thawed semen collected during winter, diluted with either 6 or 12% egg yolk. There was no apparent seasonal effect on gross measures of sperm production but the seasonal effect was ever present and was reinforced by freezing.     

Artificial insemination with canine spermatozoa frozen in a skim milk/glucose-based extender

Due to the recent outbreak of avian influenza, transportation of frozen canine semen with egg yolk has been sharply restricted. Thus, there is urgent need to develop a novel egg yolk-free extender for freezing canine spermatozoa. In the present study, the effect of using skim milk/glucose (SG)-based extender without egg yolk on the motility and fertilizing capacity of canine spermatozoa frozen-thawed in the presence of glycerol was examined. There was a tendency for the proportion of motile spermatozoa exposed to SG-based extender for 3 h to be higher than that exposed for 1 h, but the difference was not significant. The motility and other viability parameters of canine spermatozoa after thawing were similar to those obtained with an egg yolk-based extender. When spermatozoa frozen with SG-based extender containing glycerol after 3 h exposure were transcervically inseminated into 2 recipient bitches, a total of 6 pups were obtained. These results suggest that a simple extender composed of skim milk, glucose and glycerol is useful for cryopreservation of canine spermatozoa, which may contribute to improved exchange of genetic material and efficient production of companion and working dogs, such as guide dogs for the blind.     

Soya-lecithin in extender improves the freezability and fertility of buffalo (Bubalus bubalis) bull spermatozoa

Egg yolk is routinely used as a cryoprotectant in semen extenders. However, it may contain cryoprotective antagonists, and there are hygienic risks associated with its use. Proteins of plant origin, like soya-lecithin, lack these hazards. The aim of this study was to use soya-lecithin as a cryoprotectant in extender and to investigate its effects on in vitro quality and in vivo fertility of buffalo semen. Semen from three buffalo bulls was frozen in tris-citric extender containing 5.0%, 10% or 15% soya-lecithin or 20% egg yolk. Sperm motility, plasma membrane integrity and viability were assessed post-dilution, pre-freezing and post-thaw. In Post-dilution and pre-freezing, the values for motility, plasma membrane integrity and viability remained higher (p ≤ 0.05) in extenders containing 10% soya-lecithin and control compared with extender containing 5% and 15% soya-lecithin. However, motility, plasma membrane integrity and viability were higher (p < 0.05) in extender containing 10% soya-lecithin compared with control and extenders containing 5% and 15% soya-lecithin. Semen from two buffalo bulls was frozen in tris-citric extender containing either 10% soya-lecithin or 20% egg yolk. Higher (p < 0.05) fertility rate was recorded in buffaloes inseminated with semen containing 10% soya-lecithin (56%) compared with 20% egg yolk (41.5%). The results suggest that 10% soya-lecithin in extender improves the freezability and fertility of buffalo bull spermatozoa and can be used as an alternate to egg yolk in cryopreservation of buffalo semen.     

A comparison of duck and chicken egg yolk for cryopreservation of stallion sperm

OBJECTIVE: Duck and chicken egg yolk were compared for their protective effects against cold shock during the cryopreservation of stallion sperm in a lactose-EDTA-glycerol cryodiluent. DESIGN: A completely randomised design was used. Procedure Ejaculates from five stallions (n = 14 ejaculates) were split and diluted to either 20 or 200 x 10(6) sperm/mL in a lactose-EDTA extender containing either duck or chicken egg yolk. The extended semen was then frozen in liquid nitrogen. The percentage of sperm total motility and forward progressive motility were assessed before freezing and at 0 and 1 hr after thawing. Morphology data were also collected at 0 and 1 hr post thaw. RESULTS: Total and forward progressive motility were higher when the sperm were frozen in the presence of duck rather than chicken egg yolk. Furthermore, the total and forward progressive motility and percentage of morphologically normal sperm were higher when frozen at a concentration of 200 than 20 x 10(6)/mL. CONCLUSION: The results of this study demonstrate that the motility parameters of stallion sperm are improved when the semen is frozen in lactose EDTA extender supplemented with duck egg yolk rather than chicken egg yolk. Moreover, sperm motility and the percentage of morphologically normal sperm were higher after freezing at a concentration of 200 x 10(6)/ml rather than 20 x 10(6)/ml.     

大豆卵磷脂对马精液冷冻保存的效果研究

为探索适宜浓度的大豆卵磷脂(soybean lecithin,SL)代替卵黄(egg yolk,EY)对马精液冷冻保存的效果,本试验分别以5%卵黄(V/V)和10%、20%、30%大豆卵磷脂(m/V)作为精液的冷冻保护剂冷冻解冻马精液,解冻后分别对精子细胞的运动参数、精子细胞膜的完整性、精子细胞脂质氧化物丙二醛的值和线粒体膜完整性进行检测。结果发现,精液冷冻解冻后,5%卵黄和10%、20%、30%大豆卵磷脂对精子的总运动精子数无显著影响(P>0.05),但30%大豆卵磷脂具有最大的原地摆动精子数和最小的直线前进精子数(P<0.05),其他运动参数差异不显著(P>0.05);20%大豆卵磷脂代替卵黄后具有最高的质膜完整性,同时产生最少的脂质氧化物丙二醛;线粒体膜电位经流式细胞仪检测时,30%大豆卵磷脂活精子高膜电位数最高,但与20%大豆卵磷脂之间差异不显著(P>0.05)。试验结果表明,20%大豆卵磷脂能够代替卵黄作为冷冻保护剂用于马精液的冷冻保存。     

Comparison of Different Glycerol and Egg Yolk Concentrations Added to Tris‐based Extender for the Collared Peccaries (Tayassu tajacu) Semen Freezing

This study aimed to evaluate various concentrations of egg yolk (5, 10, or 20%) in combination with different concentrations of glycerol (3% or 6%) added to a Tris‐based extender on the post‐thaw characteristics of sperm obtained from Tayassu tajacu. For this purpose, semen from 10 sexually male mature collared peccaries was collected by electroejaculation and evaluated for sperm motility, vigour, viability, morphology and functional membrane integrity. The ejaculates were initially extended in Tris‐fructose plus egg yolk (5%, 10% or 20%). After cooling, the semen was added to Tris‐egg yolk plus glycerol (6% or 12%), resulting in a final concentration of 3% or 6% glycerol of the extender. Straws were frozen using liquid nitrogen and thawed in a water bath at 37°C for 30 s. The frozen–thawed semen was evaluated as reported for fresh semen. After thawing, a significant decrease was verified for sperm motility and vigour, for all the samples in comparison with fresh semen. However, no differences were evidenced among treatments for any sperm characteristics evaluated (p > 0.05), except for the combination between 10% egg yolk and 6% glycerol, which provided the worst preservation of functional membrane integrity (p < 0.05). The interactions between higher concentrations of egg yolk (20%) and glycerol (6%) and also between lower concentrations of the same substances (5% egg yolk and 3% glycerol) added to the Tris‐based extender negatively affected the preservation of the normal sperm morphology after thawing (p < 0.05). In conclusion, the use of Tris‐based extender added to 10% or 20% egg yolk plus 3% glycerol is recommended for effective sperm cryopreservation in collared peccaries.     

Seminal parameters and field fertility of cryopreserved donkey jack semen after insemination of horse mares

Reasons for performing study: As mule production is often concentrated in remote areas of the world, a simplified semen cryopreservation protocol is required. Aim: To compare the seminal parameters of cryopreserved donkey semen in lactose‐EDTA and lactose‐yolk extenders and the fertility rates on horse mares. Methods: Trial 1: Sperm total and progressive motility, vigour (scale 0–5), morphology (major and minor defects) and plasma membrane integrity (HOST) were evaluated in 25 ejaculates from 5 donkey jacks immediately after collection (raw), after chilling to 5°C (chilled) and after freezing/thawing. The semen was mixed with skimmed‐milk extender, centrifuged, and then re‐suspended in lactose‐EDTA or lactose‐yolk extender. Semen was loaded into 0.5 ml straws and chilled to 5°C for 1 h, after which samples were either evaluated (chilled semen) or placed above liquid nitrogen for 20 min prior to immersion. Seminal parameters were evaluated by ANOVA and Tukey's test. Trial 2: Cryopreserved semen from 3 males was used to inseminate 53 mares at 60 oestrous cycles randomly assigned to lactose‐yolk (n = 30 cycles) or lactose‐EDTA (n = 30 cycles) extenders. Pregnancy diagnosis was performed 15 and 25 days post ovulation. The pregnancy rates were compared using Chi‐squared tests. Results: Trial 1: No significant differences were evident in any seminal parameters between extenders after either chilling or cryopreservation. Total and progressive motility were significantly (P<0.05) lower in cryopreserved semen than raw and chilled semen for both extenders. Trial 2: Pregnancy rates did not significantly differ between extenders (lactose‐EDTA extender 53.33 and 43.33%; lactose‐yolk 50.0 and 46.66% for Days 15 and 25 post ovulation, respectively). Conclusions: Cryopreservation of donkey semen using the simplified lactose‐yolk extender resulted in similar seminal parameters and fertility rates when compared to lactose‐EDTA extender. Potential relevance: Lactose‐yolk extender may be advocated as a simple, easy to prepare extender, for use in geographically isolated enterprises producing mules throughout the world.     

‘Soya Milk Tris‐based Phytoextender Reduces Apoptosis in Cryopreserved Buffalo (Bubalus bubalis) Spermatozoa’

The objective of this study was to investigate the effect of newly developed soya milk Tris (SMT)‐based phytoextender as an alternative to egg yolk Tris (EYT) extender used for cryopreservation of buffalo (Bubalus bubalis) spermatozoa on apoptosis. Fresh buffalo semen (control without dilution) was cryopreserved in conventional EYT (20% egg yolk v/v in Tris) and SMT (25% soya milk v/v in Tris) extender and used for the assessment of expression of apoptotic proteins. Proteins extracted from a total number of nine ejaculates from three individual buffalo bulls chosen at random were separated using SDS–PAGE followed by immunoblotting against caspase‐8, caspase‐9, caspase‐3, poly(ADP‐ribose)polymerase (PARP), cytochrome c and apoptosis inducing factor (AIF). In addition, fluorescence microscopy was used for the detection of mitochondrial membrane potential (JC‐1 assay) and apoptotic cells (annexin V‐FITC/PI assay). The results obtained clearly indicate the significant (p < 0.05) reduction in the expression of caspase‐3 (27 kDa), caspase‐8 (53 kDa), caspase‐9 (50 kDa) precursor and cytochrome c (17 kDa) in semen cryopreserved in SMT extender in comparison with EYT extender. A non‐significant (p > 0.05) reduction in expression of PARP‐DNA‐binding subunit (24 kDa) was observed in SMT extender. No expression of AIF was found in cryopreserved semen samples. A significant (p < 0.05) increase in the mean percentage of cells having high mitochondrial membrane potential and a non‐significant (p > 0.05) decrease in late apoptotic cells (AN+/PI+) was observed in SMT extender when compared to EYT extender. The results demonstrated that cryopreservation of buffalo semen in SMT‐based phytoextender can replace the traditional egg yolk extenders as it reduces the expression of apoptotic proteins maintaining high mitochondrial membrane potential and gives better protection to sperms in terms of its non‐animal origin.     

Inactivation of bovine herpesvirus 1 in semen using a hyperimmune egg yolk semen extender

Hyperimmune egg yolk semen extender was used for the inactivation of bovine herpesvirus (BHV-1) in experimentally infected bovine semen. As much as 5 x 10(4) TCID50/ml of virus was inactivated in semen as assayed by tissue culture. Moreover the hyperimmune egg yolk semen extender did not produce any adverse effect on the quality of the semen after being frozen/thawed in comparison with normal egg yolk semen extender (P > 0.05). The hyperimmune egg yolk semen extender is considered an important tool for containing the spread of BHV-1 from infected semen.     

Alternatives in Donkey semen cryopreservation: Mare vs. Jenny Colostrum

The aim of this study was to test and compare two new components in extenders for freezing donkey semen: mare colostrum and jenny colostrum. Colostrum was obtained from four mares and four jennies right after the foal's birth. Ejaculates were collected from five fertile donkeys. Sperm samples were pooled, diluted and cryopreserved in three different experimental extender groups: lactose supplemented with egg yolk extender (20%) as the control group, lactose supplemented with jenny colostrum extender (20%), and lactose supplemented with mare colostrum extender (20%). After thawing, we evaluated the sperm motility by means of computer‐assisted analysis, viability by SYBR‐14 and propidium iodide (PI), membrane functional by HOS test and acrosome integrity by isothiocyanate conjugated with peanut agglutinin (FITC‐PNA) and PI. The results demonstrated that lactose–jenny colostrum extender displayed significantly higher values (p < .05) in nearly all parameters evaluated – Total Motility, Viability, HOS test, VCL, VSL, VAP, LIN, STR and WOB –, compared with mare colostrum and egg yolk extenders after thawing. In conclusion, the extender containing jenny colostrum used for donkey semen cryopreservation improved the donkey sperm quality after the freezing–thawing process.     

Fertility after artificial insemination using a soybean-based semen extender in sheep

The present study aimed to compare the fertility of ewes intrauterinally inseminated with frozen-thawed semen using a soybean-based semen extender (AndroMed) with those of ewes intrauterinally inseminated with frozen-thawed semen using a Tris-based extender containing either egg yolk or BSA. Suffolk ewes (n=104) were treated with an intravaginal sponge containing 40 mg fluoroprogesterone acetate (FGA) for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin to induce estrus and ovulation during the non-breeding season (July, 2007). Intrauterine insemination was carried out 40-46 h after removal of the FGA sponge (n=90), regardless of the incidence of estrus. The pregnancy rates were not significantly different among the semen extenders containing egg yolk (64.5%) or BSA (58.6%) and AndroMed extender (56.7%). The lambing rates (64.5, 55.2 and 56.7% for the semen extenders containing egg yolk, BSA and AndroMed, respectively) and prolificacy (1.59 to 1.75) were also not significantly different. The present results indicate that an egg yolk-containing semen extender can be replaced with the non-animal derived extender AndroMed, which could be used for intrauterine insemination using frozen-thawed ram semen without reducing fertility.     

Melatonin supplementation improved cryopreserved Thai swamp buffalo semen

The production of reactive oxygen species (ROS) during cryopreservation process impairs the sperm characteristics and fertilizing ability. However, melatonin, an antioxidant, could protect spermatozoa against this cell damage during cryopreservation. Therefore, we attempted to evaluate whether the melatonin supplementing in the semen extender could improve the sperm quality of swamp buffalo during cryopreservation. The semen collected from six swamp buffalo bulls were diluted with tris-citrate egg yolk extender supplementing with 0, 0.1, 0.5, 1.0, 2.0 and 3.0 mM of melatonin. The parameters of sperm viability and motility were evaluated using computer-assisted semen analyser (CASA) after cryopreservation on days 1, 7, 15 and 30. The group supplemented with 1.0 mM melatonin exhibited the higher viability after cryopreservation on days 1, 7, 15 and 30 with 58.346 ± 2.1^a, 57.586 ± 2.0^a, 55.082 ± 1.8^a and 55.714 ± 1.8^a, respectively, and showed the best results of motility parameters. However, higher concentration of melatonin at 3.0 mM impaired all the parameters. In conclusion, the addition of melatonin at 1 mM to semen extender could exert the best protection against sperm damage in swamp buffalo bull during cryopreservation.     

鸵鸟卵黄低密度脂蛋白对猪精子冻后品质的影响

在家畜精液冷冻中,卵黄被广泛应用,且其中的低密度脂蛋白(LDL)对精子起主要保护作用。本研究利用含6%、7%、8%和9%鸵鸟卵黄LDL配制的稀释液制作猪细管冷冻精液,分析鸵鸟卵黄LDL对冷冻-解冻后猪精子质量参数的影响。结果表明:在含不同浓度鸵鸟卵黄LDL的稀释液中,8%LDL的稀释液冷冻效果最好,冻后精子活率平均可达52.13%,显著高于其他组(P<0.05);精子顶体完整率平均为58.33%,质膜完整率为72.38%,与其他处理组相比差异显著(P<0.05)。但与鸡蛋卵黄LDL和鸽子蛋卵黄LDL处理组相比,鸵鸟卵黄LDL处理组冷冻-解冻后猪精子质量参数相对较低。本研究表明,虽然鸵鸟卵黄LDL在冷冻过程中对猪精子具有一定的保护作用,但相对于鸽子蛋和鸡蛋卵黄LDL效果并不理想。     

Optimization of Ram Semen Cryopreservation Using a Chemically Defined Soybean Lecithin‐Based Extender

The purpose of the present study was to investigate the effects of a chemically defined soybean lecithin‐based semen extender as a substitute for egg yolk‐based extenders in ram semen cryopreservation. In this study, 28 ejaculates were collected from four Zandi rams in the breeding season and then pooled together. The pooled semen was divided into six equal aliquots and diluted with six different extenders: (i) Tris‐based extender (TE) containing 0.5% (w/v) soybean lecithin (SL0.5), (ii) TE containing 1% (w/v) soybean lecithin (SL1), (iii) TE containing 1.5% (w/v) soybean lecithin (SL1.5), (iv) TE containing 2% (w/v) soybean lecithin (SL2), (v) TE containing 2.5% (w/v) soybean lecithin (SL2.5) and (vi) TE containing 20% (v/v) egg yolk (EYT). After thawing, sperm motility and motion parameters, plasma membrane and acrosome integrity, apoptosis status and mitochondrial activity were evaluated. The results shown that total and progressive motility (54.43 ± 1.33% and 25.43 ± 0.96%, respectively) were significantly higher in SL1.5 when compared to other semen extenders. Sperm motion parameters (VAP, VSL, VCL, ALH and STR) were significantly higher in SL1.5 compared to other extender, with the exception of SL1 extender. Plasma membrane integrity (48.86 ± 1.38%) was significantly higher in SL1.5 when compared to other semen extenders. Also, percentage of spermatozoa with intact acrosome in SL1.5 (85.35 ± 2.19%) extender was significantly higher than that in SL0.5, SL2.5 and EYT extenders. The results showed that the proportion of live post‐thawed sperm was significantly increased in SL1.5 extender compared to SL0.5, SL2 and EYT extenders. In addition, SL1, SL1.5 and SL2.5 extenders resulted in significantly lower percentage of early‐apoptotic sperm than that in EYT extender. There were no significant differences in different semen extenders for percentage of post‐thawed necrotic and late‐apoptotic spermatozoa. Also, the results indicated that there are slight differences for percentage of live spermatozoa with active mitochondria between extenders. In conclusion, SL1.5 extender was better than other extenders in most in vitro evaluated sperm parameters.     

Comparison of two different centrifugation extenders for preservation of frozen equine semen

This study compares a commercial semen extender (control group) to ultra high temperature (UHT) skimmed milk (treatment group) used during centrifugation for subsequent cryopreservation of equine semen. Following post‐thawing of semen samples parameters measured included motility, sperm motion kinetics (using computerised assisted semen analysis) as well as acrosome and plasmatic membrane integrity (using fluorescent dyes). After collection and analysis, the sperm‐rich fraction was divided and diluted with either: control (1:1 dilution in a skimmed milk‐glucose extender) or treatment (1:1 dilution in UHT skimmed milk). The milk used in this experiment was of the same source, commercial brand, of only one lot. After dilution, samples were subjected to centrifugation at 600 g for 10 min and sperm pellets were resuspended in a freezing extender to a concentration of 200 × 10⁶ cells/ml. Aliquots were packed into 0.5 ml straws placed in a stainless steel support and kept inside the refrigerator (5°C) for 20 min. Subsequently, these straws were placed at a height of 6 cm over liquid nitrogen for 20 min in an isotherm box. No significant differences were observed in total sperm motility (42.71 vs. 38.29%), progressive sperm motility (12.29 vs. 7.86%), plasma membrane integrity (53.43 vs. 60.14%) or acrosomal membrane integrity (93.29 vs. 93.71%) with a P>0.05 calculated between the control and the treatment groups, respectively. Considering that UHT skimmed milk has a lower cost than the commercial semen extender, this could be an option used during the centrifugation protocol to decrease the expense of the equine semen cryopreservation process and increase shelf life.     

不同卵黄成分、大豆卵磷脂及β-环糊精胆固醇对马精子冷冻效果的影响

本研究旨在比较添加不同浓度的卵黄成分（全卵黄（EY）、卵黄浆蛋白（EYP）、低密度脂蛋白（LDL）及水溶性卵黄蛋白（livetin））、大豆卵磷脂（SL）及β-环糊精胆固醇（CLC）对马精子冷冻效果的影响。选择4匹成年英纯血种公马进行采精,试验1：以INRA82+3.5%的混合抗冻剂作为基础冷冻液,添加0、2%、4%和8%浓度的EY、LDL、EYP或水溶性卵黄蛋白;试验2：以4%全卵黄组为对照,添加1%、2%和4% SL;试验3：在INRA82+3.5%的混合抗冻剂+4% EY基础液中添加0、0.75、1.5和3 mg/mL浓度的CLC。结果显示,EYP、LDL及水溶性卵黄蛋白3种卵黄组分均对马精子冷冻具有显著保护作用（P<0.05）,但不同卵黄组分之间及与EY组相比保护效果无显著差异（P>0.05）;添加低浓度（1%）的SL精子冻融后活力与对照组（4% EY组）相比无显著差异（P>0.05）,但添加高浓度SL组（2%和4%）冻融后精子TM、PM及RAP值均显著低于对照组（P<0.05）;添加不同浓度的CLC解冻后精子活力与未添加组无显著差异（P>0.05）。研究结果表明,卵黄中EYP、LDL和水溶性卵黄蛋白均对精子保护有效,保护效果与EY无显著差异;1% SL可获得与卵黄相似的保护效果;在含有卵黄的基础冷冻液中添加CLC未能提高精子冻后活力。     

Influence of Cool Storage before Freezing on the Quality of Frozen–Thawed Semen Samples in Dogs

The aim of this study was to determinate the semen quality of frozen–thawed samples that were chilled for up to 2 days before freezing. The ejaculates (n = 18) from six dogs were collected, pooled and divided into six aliquots. The first aliquot (C, control) was frozen in liquid nitrogen using a conventional protocol to reach a final concentration of 100 × 10⁶ spermatozoa/ml, 20% egg yolk and 5% glycerol. The remaining five aliquots were diluted with a chilled extender (Tris‐glucose and 20% egg yolk) and cooled at 4°C as follows: R1, the semen was cooled for 1 h; R6, the semen was cooled for 6 h; R12, the semen was cooled for 12 h; R24, the semen was cooled for 24 h and R48, the semen was cooled for 48 h. After the chilling period, a second extender was added (Tris‐glucose, 20% egg yolk, 10% glycerol and Equex at 1%) to reach a final composition similar to aliquot C, and then, the semen samples (R1, R6, R12, R24 and R48) were frozen in liquid nitrogen. The post‐thaw sperm quality was assessed in 30 straws from each experimental group. After freezing–thawing, the total sperm motility (approximately 60–70%) in the semen chilled for up to 48 h did not show any differences from the samples frozen by the conventional cryopreservation method (63.2%). No significant differences were detected in the percentages of abnormal sperm cells among the fresh semen, the control group and the frozen samples after the different cooling times. Finally, the post‐thaw percentages of damaged acrosomes showed a very uniform distribution, with mean values ranging between 7% and 10.5%. The results clearly demonstrated that cooling the semen up to 48 h before freezing did not produce a decrease in the semen quality when was compared with semen frozen by a traditional procedure.     

Fertility of ewes inseminated intrauterinally with frozen semen using extender containing bovine serum albumin

The present study was conducted to examine the fertility of ewes inseminated intrauterinally with frozen semen using semen extender containing either egg yolk or bovine serum albumin (BSA). Sixty Suffolk and cross-bred ewes were treated with controlled internal drug release (CIDR) devices during the non-breeding season (July 2006). A CIDR was inserted into the vagina for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin was administered one day before its removal. Ejaculates from a suffolk ram were diluted with a Tris-based extender containing either 15% (v/v) egg yolk or 10% (w/v) BSA, and the diluted semen was frozen in 0.25 ml straws. A fixed-time intrauterine artificial insemination (AI) was performed 43-47 h after CIDR removal, regardless of incidence of estrus. There was no significant difference in pregnancy rates at 60 days after AI between the extenders containing egg yolk (66.7%, 20/30 animals) or BSA (65.5%, 19/29 animals). Furthermore, there were no significant difference in the lambing rates (66.7% and 62.1%) and prolificacy (1.25 and 1.56) between the two semen extenders. The present study indicates that a semi-defined semen extender containing 10% BSA produces fertility after intrauterine AI that is similar to that achieved with semen extender containing egg yolk.     

Protocol Optimization for Long‐Term Liquid Storage of Goat Semen in a Chemically Defined Extender

A specific problem in the preservation of goat semen has been the detrimental effect of seminal plasma on the viability of spermatozoa in extenders containing egg yolk or milk. The use of chemically defined extenders will have obvious advantages in liquid storage of buck semen. Our previous study showed that the self‐made mZAP extender performed better than commercial extenders, and maintained a sperm motility of 34% for 9 days and a fertilizing potential for successful pregnancies for 7 days. The aim of this study was to extend the viability and fertilizing potential of liquid‐stored goat spermatozoa by optimizing procedures for semen processing and storage in the mZAP extender. Semen samples collected from five goat bucks of the Lubei White and Boer breeds were diluted with the extender, cooled and stored at 5°C. Stored semen was evaluated for sperm viability parameters, every 48 h of storage. Data from three ejaculates of different bucks were analysed for each treatment. The percentage data were arcsine‐transformed before being analysed with anova and Duncan’s multiple comparison test. While cooling at the rate of 0.1–0.25°C/min did not affect sperm viability parameters, doing so at the rate of 0.6°C/min from 30 to 15°C reduced goat sperm motility and membrane integrity. Sperm motility and membrane integrity were significantly higher in semen coated with the extender containing 20% egg yolk than in non‐coated semen. Sperm motility, membrane integrity and acrosomal intactness were significantly higher when coated semen was 21‐fold diluted than when it was 11‐ or 51‐fold diluted and when extender was renewed at 48‐h intervals than when it was not renewed during storage. When goat semen coated with the egg yolk‐containing extender was 21‐fold diluted, cooled at the rate of 0.07–0.25°C/min, stored at 5°C and the extender renewed every 48 h, a sperm motility of 48% was maintained for 13 days, and an in vitro‐fertilizing potential similar to that of fresh semen was maintained for 11 days.