Cryopreservation of collared peccaries (Tayassu tajacu) semen using a powdered coconut water (ACP-116c) based extender plus various concentrations of egg yolk and glycerol

The objective was to determine the effectiveness of a powdered coconut water-based extender (ACP-116c), plus various concentrations of egg-yolk and glycerol, as an alternative for cryopreservation of collared peccary semen. Twelve ejaculates were obtained from captive adult males by electroejaculation, and evaluated for sperm motility, kinetic rating, viability, morphology, and functional membrane integrity. The ejaculates were apportioned into aliquots that were diluted in Tris plus 10% egg yolk and 3% glycerol, or in ACP-116c plus 10 or 20% egg yolk and 1.5 or 3% glycerol. Samples were frozen in liquid nitrogen and, after 1 mo, thawed at 37 °C for 1 min. After thawing, samples were evaluated as reported for fresh semen, and also for sperm membrane integrity (fluorescent probes) and kinematic parameters (computerized analysis). Results were presented as means ± SEM. Freezing and thawing decreased sperm characteristics relative to fresh semen. Overall, ACP-116c plus 20% egg yolk and 3% glycerol provided better (P < 0.05) sperm motility and kinetic rating (48 ± 6.1% and 2.8 ± 0.2, respectively) after thawing than Tris extender (30.4 ± 5.7% and 2.4 ± 0.2). However, there were no differences (P > 0.05) among treatments with regard to the other sperm characteristics. Based on computerized motion analysis, total (26.5 ± 5.9%) and progressive (8.1 ± 2.2%) motility were best preserved (P < 0.05) with the above-mentioned treatment. In conclusion, a coconut water-based extender, ACP-116c, plus 20% egg yolk and 3% glycerol, was effective for cryopreservation of semen from collared peccaries.     

Cryopreservation of buffalo (Bubalus bubalis) semen in Bioxcell extender

This study was designed to compare commercially available extender Bioxcell^® with tris-citric egg yolk extender for post thaw quality and in vivo fertility of buffalo semen. For comparison of post thaw semen quality: semen was collected from five adult Nili-Ravi buffalo (Bubalus bubalis) bulls of similar age group with artificial vagina (at 42 °C) for three weeks (replicates). Qualifying ejaculates having motility >60% from each buffalo bull were divided in two aliquots and diluted (at 37 °C having 50 × 10⁶ spermatozoa/ml) in tris-citric egg yolk or Bioxcell^® extender. Diluted semen was cooled to 4 °C in 2 hours, equilibrated for 4 hours and filled in 0.5 ml straws. Semen straws were kept over liquid nitrogen vapors (5 cm) for 10 minutes. Straws were then plunged and stored in liquid nitrogen (−196 °C). After 24 hours of storage, semen straws were thawed at 37 °C for 30 seconds to assess sperm motility, viability, plasma membrane integrity, normal apical ridge, and abnormalities (head, mid piece, and tail). For comparison of in vivo fertility: semen from two buffalo bulls of known fertility was cryopreserved in tris-citric egg yolk and Bioxcell^® as described earlier, and used for inseminations under field conditions. Post-thaw percentage of sperm motility (45.3 ± 1.1, 45.0 ± 1.4), viability (66.2 ± 1.1, 64.4 ± 1.3) plasma membrane integrity (60.4 ± 1.2, 59.2 ± 1.4) and normal apical ridge (82.9 ± 0.5, 80.7 ± 0.5) did not differ (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. Similarly, sperm abnormalities of head (1.20 ± 0.1, 1.20 ± 0.1), mid piece (0.67 ± 0.1, 0.87 ± 0.1) and tail (11.7 ± 0.2, 11.6 ± 0.3) remained similar (P > 0.05) in tris-citric egg yolk and Bioxcell^® extender, respectively. In vivo fertility rates of buffalo semen cryopreserved in tris-citric egg yolk and Bioxcell^® also remained similar (44% vs. 47%). It is concluded that commercially available Bioxcell^® may be used for the cryopreservation of buffalo semen with an equal efficiency to tris-citric egg yolk extender.     

Comparison between glycerol and ethylene glycol for dog semen cryopreservation

The aim of this study was to compare the effect of ethylene glycol versus glycerol for dog semen freezing, on post-thaw longevity, motility and motility parameters, and on plasma membrane functional integrity. Semen was diluted in two steps with an egg yolk TRIS extender containing a final concentration of either 5% glycerol or 5% ethylene glycol, and frozen in 0.5 ml straws, with 100 x 10(6) spermatozoa/ml, over nitrogen vapours. Semen motility was evaluated both under a light microscope and with a Computer Assisted Motility Analyser System, immediately after thawing and then hourly till 4h of incubation. Sperm membrane functional integrity was assessed with the hypoosmotic swelling test (60 mOsm fructose solution) applied at thawing and then hourly, for 4 h, on incubated samples. Motility (light microscope) and total and progressive motility (analyser) were significantly higher in ethylene glycol frozen samples at thawing (P < 0.01); from hour 1 onwards the effect of the cryoprotectant became not significant. Semen frozen with ethylene glycol showed higher path velocity and higher straight line velocity till 3 h after thawing; however, ethylene glycol semen samples also showed higher curvilinear velocity and higher lateral head displacement, which may indicate a capacitation-like condition affecting sperm membranes and possibly reducing post-thaw longevity. Functional integrity of plasma membrane was similar in glycerol and ethylene glycol samples till 3 h after thawing, then ethylene glycol samples showed a higher decline. The strong though short-lived positive effect of ethylene glycol is worth being evaluated further.     

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.     

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.     

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.     

Trehalose enhanced the freezability of Poodle dog sperm collected by an artificial vagina (AV)

In an attempt to develop a suitable freezing method for Poodle dog sperm, an experiment was conducted to investigate semen collection methods of digital stimulation and an artificial vagina (AV), using Tris and trehalose-egg yolk extender, on the characteristics and cryopreservation of sperm. Two dogs (dogs A and B) were subjected to semen collection by digital stimulation and AV. The volume, sperm concentration, sperm motility index (SMI) and acrosome status of ejaculates were determined immediately after collection. The remainder was frozen as pellets in Tris and trehalose-egg yolk extender. Sperm motility index was evaluated after thawing and during a thermal resistance test, and acrosome integrity was also assessed. No significant differences regarding sperm concentrations, SMI and acrosome integrity were observed between semen collected by AV and digital stimulation. However, when dog sperm were collected by an AV and frozen in trehalose-egg yolk extender, the motility index of frozen-thawed sperm was significantly improved compared to sperm frozen in Tris-egg yolk extender which were collected by digital stimulation. In conclusion, semen collected by an AV and frozen in trehalose-egg yolk extender was effective in enhancing the freezability of Poodle dog sperm.     

Influence of the thawing rate on the cryopreservation of semen from collared peccaries (Tayassu tajacu) using Tris-based extenders

The objective was to evaluate the influence of the thawing rate on the quality of frozen-thawed (cryopreserved in Tris-based extenders) semen obtained from collared peccaries (Tayassu tajacu). Semen from 13 sexually mature collared peccaries males were collected by electroejaculation, and evaluated for motility, vigor, sperm viability, membrane integrity, and sperm morphology. Semen was divided in two equal portions: the first was diluted in Tris-fructose and the other in Tris-glucose, with egg yolk (20%) and glycerol (3%) added to each portion. Extended semen was frozen in liquid nitrogen and thawed using two thawing protocols (37 °C for 1 min or 55 °C for 7 s, followed by an additional 30 s at 37 °C). There were no significant differences between the two extenders after extension, chilling, or glycerol addition. After thawing at 37 °C, there were 37.9 ± 4.2% and 28.5 ± 5.1% motile spermatozoa for samples extended in Tris-fructose and Tris-glucose, respectively, with 33.8 ± 3.7% and 28.2 ± 3.5% motile spermatozoa after thawing at 55 °C (no significant differences). Furthermore, there were no significant interactions between extenders and thawing protocols for any semen end point. In conclusion, semen from collared peccaries was successfully cryopreserved in Tris-based extenders and thawed with two protocols (37 °C for 1 min or 55 °C for 7 s).     

四种渗透性防冻剂在猕猴精子低温冷冻保存中对精子功能状态的影响

以冷冻精子的复苏运动度、荧光染料Hoechst 3 3 2 5 8检测的细胞膜完整率、异硫氰酸荧光素标记的花生凝集素 (FITC PNA)检测的顶体完整率作为精子功能状态的指标 ,对甘油、二甲亚砜、乙二醇和丙二醇 4种常用渗透性防冻剂在猕猴精子冷冻保存过程中的作用进行了比较。结果表明 :冷冻保存精子的复苏运动度 ,甘油 ( 4 7 3± 5 7% )和乙二醇 ( 4 4 8± 6 7% ) >二甲亚砜 ( 2 2 9± 0 9% ) >丙二醇 ( 0± 0 % ) ;细胞膜完整率 ,甘油 ( 5 4 8± 3 2 % )和乙二醇 ( 5 4 0± 6 7% ) >二甲亚砜 ( 3 7 5± 7 0 % ) >丙二醇 ( 2 8 3± 6 5 % ) ;顶体完整率 ,甘油 ( 82 2± 2 4 % )和乙二醇 ( 82 4± 2 4 % ) >二甲亚砜 ( 6 8 7± 5 7% )和丙二醇 ( 72 3±3 5 % ) (P <0 0 5 )。结果提示 :二甲亚砜和丙二醇 ,尤其是丙二醇并不适合猕猴精子的冷冻保存 ;而乙二醇具有和甘油相似的保护作用 ,是一种极具潜力的猕猴精子冷冻保存的渗透性防冻剂。     

Cryoprotectant effect of trehalose and low-density lipoprotein in extenders for frozen ram semen

This study tested trehalose and low-density lipoprotein (LDL) as cryoprotectants in extenders for frozen ram semen. In the first experiment, the extenders were Tris, with 20% egg yolk (E1-1); E1-1 with 5% glycerol (E1-2); E1-1 with 100 mM trehalose (E1-3); and E1-1 with 100 mM trehalose and 5% glycerol (E1-4). Sperm motility and membrane integrity of the E1-2, E1-3 and E1-4 extenders were greater than for E1-1 (P < 0.05), but acrosome integrity following cryopreservation did not differ. In the second experiment, the extenders were Tris, with 20% egg yolk and 100 mM trehalose (E2-1); Tris with 8% LDL and 5% glycerol (E2-2); Tris with 8% LDL and 100 mM trehalose (E2-3); and Tris with 8% LDL, 100 mM trehalose and 5% glycerol (E2-4). Sperm membrane integrity was lowest for the E2-1 extender (P < 0.05), but similar for extenders including LDL. Sperm motility post-thawing was highest for E2-2 and E2-3 extenders (P < 0.05), but acrosome integrity did not differ. Thus, extenders including trehalose and LDL as cryoprotectants recorded a post-thawing ram sperm quality similar to that achieved when using conventional cryoprotectants.     

Recovery and cryopreservation of epididymal sperm from agouti (Dasiprocta aguti) using powdered coconut water (ACP-109c) and Tris extenders

The objective was to compare the use of powdered coconut water (ACP-109c; ACP Biotecnologia, Fortaleza, CE, Brazil) and Tris extenders for recovery and cryopreservation of epididymal sperm from agouti. The caudae epididymus and proximal ductus deferens from 10 sexually mature agoutis were subjected to retrograde washing using ACP-109c (ACP Biotecnologia) or Tris. Epididymal sperm were evaluated for motility, vigor, sperm viability, membrane integrity, and morphology. Samples were centrifuged, and extended in the same diluents plus egg yolk (20%) and glycerol (6%), frozen in liquid nitrogen, and subsequently thawed at 37°C for 1 min, followed by re-evaluation of sperm characteristics. The two extenders were similarly efficient for epididymal recovery, with regard to the number and quality of sperm recovered. However, for both extenders, sperm quality decreased (P < 0.05) after centrifugation and dilution. After sperm cryopreservation and thawing, there were (mean ± SEM) 26.5 ± 2.6% motile sperm with 2.6 ± 0.2 vigor in the ACP-109c (ACP Biotecnologia) group, which was significantly better than 9.7 ± 2.6% motile sperm with 1.2 ± 0.3 vigor in Tris. In conclusion, agouti epididymal sperm were successfully recovered using either ACP-109c (ACP Biotecnologia) or Tris extenders; however, ACP-109c (ACP Biotecnologia) was a significantly better extender for processing and cryopreserving these sperm.     

The effect of zwitterion buffers on the freezability of Boer goat semen

Twenty semen samples with mass activity greater than +3 were collected from six healthy, mature Boer goat bucks. Each ejaculate was divided into four equal parts and extended at 37 degrees C in Tris, Test, Tes and Bes buffers containing egg yolk and glycerol. Semen was placed into medium size French strawsand after 2 hours of equilibration at 5 degrees C, frozen in the vapour phase and stored in liquid nitrogen for 7 days at -196 degrees C. Progressive motility, the number of live spermatozoa and glutamic oxaloacetic transaminase (GOT) release were studied after the initial extension, after equilibration and after 15 minutes and 7 days of freezing of semen. Semen samples when extended with Tris yolk glycerol showed significantly (P<0.01) higher progressive motility and live spermatozoa than when extended with the other zwitterion buffer-based extenders. The change of extenders did not influence the release of GOT at various stages of freezing of semen (P>0.05).     

Antagonist effect of DMSO on the cryoprotection ability of glycerol during cryopreservation of buffalo sperm

The objective of the present study was to investigate the synergistic effect of DMSO and glycerol added at various temperatures on the post-thaw quality of buffalo sperm. Pooled ejaculates from four Nili-Ravi buffalo bulls were divided into 18 aliquots and extended (1:10) in Tris-citric acid extender differing in glycerol:DMSO ratios (0:0, 0:1.5, 0:3; 3:0, 3:1.5, 3:3; and 6:0, 6:1.5, 6:3, respectively; %, v:v) either at 37 or 4 degrees C. Semen was packaged in 0.5 mL French straws and frozen in a programmable cell freezer. Thawing was performed at 37 degrees C for 50s. Post-thaw motion characteristics, plasma membrane integrity and acrosome morphology of buffalo sperm were determined using computer-assisted semen analyzer (CASA), hypoosmotic swelling (HOS) assay and phase-contrast microscopy, respectively. Glycerol (6%) in extender yielded better post-thaw sperm motility, velocities (straight-line and average path), plasma membrane integrity, and normal acrosomes (P<0.05). Post-thaw sperm motility and plasma membrane integrity declined in the presence of DMSO (P<0.01). The addition of glycerol (6%) at 37 degrees C yielded better post-thaw sperm motility, plasma membrane integrity and velocities than addition at 4 degrees C (P<0.05). In conclusion, glycerol is still an essential cryoprotectant for buffalo sperm. The addition of DMSO antagonized the cryoprotection ability of glycerol and reduced the post-thaw quality of buffalo sperm. Furthermore, 6% glycerol added at 37 degrees C, provided better cryoprotection to the motility apparatus and plasma membrane integrity of buffalo sperm.     

Effect of basic factors of extender composition on post-thawing quality of brown bear electroejaculated spermatozoa

The improvement of freezing extenders is critical when defining sperm cryopreservation protocols for wild species, in order to create germplasm banks. The aim of this study was to evaluate the effect of additives (Equex Paste and EDTA) supplementation, egg-yolk (10 and 20%) and glycerol (4 and 8%) concentrations and extender osmolality (300 and 320 mOsm/kg) on the post-thawing quality of brown bear semen. Semen was obtained from 20 adult males by electroejaculation, and centrifugated individually (600 × g for 6 min). The pellets were diluted 1:1 in the corresponding extender TTF (TES-Tris-Fructose with the aforementioned variants) and cooled to 5 °C. Then, it was diluted down to 100 × 10⁶ spz/mL, loaded in 0.25 mL straws and frozen at −20°C/min. After thawing (in water at 65 °C for 6s), the semen samples were assessed for motility (CASA), viability (SYBR-14 with propidium iodide), acrosomal status (PNA-FITC with propidium iodide) and mitochondrial activity (JC-1). Extender supplementation with additives rendered significantly higher results for these sperm parameters. Comparing the two percentages of egg yolk, 20% egg yolk showed the highest motility results, percentages of viable spermatozoa and viable spermatozoa with intact acrosome. No differences were detected among samples frozen using 4 or 8% glycerol. For extender osmolality, 300 mOsm/kg showed higher values of VAP, VCL, VSL, and ALH than 320 mOsm/kg. Based on the best performance of sperm motility, viability and acrosome status, we conclude that the most suitable extender to cryopreserve brown bear spermatozoa was TTF adjusted to 300 mOsm/kg, supplemented with 20% egg yolk, 4-8% glycerol, and the additives 1% Equex paste and 2% EDTA.     

A procedure for Poitou jackass sperm cryopreservation

We have tried to establish sperm banking for the endangered Poitou donkeys. No successful cryopreservation technique had been described for spermatozoa of this species; our preliminary work indicated that a particular medium and procedure may be effective for cryopreservation of Poitou jackass spermatozoa as evaluated by sperm motility, membrane integrity and pregnancy rate after AI with frozen-thawed semen. We found that glutamine at 80 mM and 10% (v/v) quail egg yolk in a basal medium containing 4% (v/v) glycerol (T2-94 medium) improved the post-thaw total and progressive motility and velocity assessed with the automated analyzer ATS-M. The T2-94 medium also preserved the sperm nuclear, acrosom, and plasma membrane integrity as assessed with the acridine orange method, fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) lectin procedure, and hypo-osmotic swelling test, respectively. Semen frozen-thawed in T2-94 medium as used to artificially inseminate. 13 Poitou jennies from the beginning of estrus to ovulation during 4 cycles at a rate of one AI per day. Heigh pregnancies and 3 foals were obtained, but only when the glycerol was removed from sperm before AI. We conclude that the cryopreservation of Poitou jackass semen for sperm banking may succeed by using the T2-94 medium and removing the glycerol post-thaw, but before AI.     

Effects of Soy Lecithin Extender on Dog Sperm Cryopreservation

Semen cryopreservation is an essential biotechnology in canine reproduction and during the cryopreservation process commonly egg yolk are used. The discrepancy in the egg yolk composition and the potential risk of disease dissemination are obstacles for semen exportation and use. Therefore, studies aiming to substitute egg yolk are extremely important. In this context, soy lecithin contains a low-density lipoprotein fraction, is an interesting alternative. Thus, the objective of this study was to compare extenders based on soy lecithin (several concentrations and forms) with egg yolk during the cryopreservation process of dog sperm. For this purpose, we used twelve dogs. Semen was evaluated at different time points (after refrigeration, glycerolization, and thawing), by motility analysis (CASA) and functional tests (e.g., membrane integrity—eosin/nigrosin, acrosome integrity—fast green/Bengal rose, mitochondrial activity—3’3 diaminobenzidine, Chromatin susceptibility to acid-induced denaturation—SCSA, and susceptibility to oxidative stress—thiobarbituric acid reactive substances). The results indicated that egg yolk and lower concentrations of lecithin had similar effects on mitochondrial activity and motility. Thus, soy lecithin is a potentially viable alternative to egg yolk for the cryopreservation of dog semen.     

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.     

Fertility of stallion semen processed, frozen and thawed by a new procedure

The fertility of frozen-thawed and fresh semen from three stallions was compared in a trial using a randomized block design and 90 mares for 108 cycles. Semen was collected every third day, diluted to 50 x 10(6) sperm/ml with a citrate-based centrifugation medium, and centrifuged. The cells were resuspended at 700 x 10(6) progressively motile sperm/1.0 ml of added lactose-EDTA-egg yolk extender containing 4% glycerol, packaged by placing 0.55 ml into polypropylene straws, and frozen. Semen was thawed by immersion in 75 degrees C water for 10 sec. All of the 43 ejaculates collected were frozen, but 21 were discarded because progressive sperm motility was <35% immediately after thawing or <40% after 30 min of incubation at 37 degrees C. semen from the same stallions was collected daily for inseminations with fresh semen. Semen containing 200 x 10(6) progressively motile sperm was added to 10 ml of heated skimmilk extender. Mares were inseminated daily starting on the third day of estrus or when a >/=4-cm follicle was detected, whichever came later, and continuing through the end of estrus or for nine days. Based on palpation per rectum on day 50 postovulation, the pregnancy rates from inseminations during one estrus were 50, 56 and 61% with frozen semen and 67, 67 and 61% with fresh semen (P>0.05) from the three stallions, respectively. Thus, mean pregnancy rate with frozen semen was 86% of the rate attained with fresh semen.     

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

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

Impact of Eurycoma longifolia extract on DNA integrity,lipid peroxidation,and functional parameters in chilled and cryopreserved bull sperm

This study aims to assess the effect of Eurycoma longifolia aqueous extract on chilled and cryopreserved quality of bull sperm. Semen samples were obtained from four Simmental–Brangus. Each sample was divided into two fractions: the first fraction was used for chilling the semen, and the second fraction was used for the freezing process. Both fractions were extended with Tris–egg yolk extender supplemented with 0.0, 0.25, 0.5, 1.0, 2.5, 5.0, and 7.5 mg/ml Eurycoma longifolia aqueous extract. The diluted chilled fraction was chilled at 5 °C for 6 days, whereas the frozen–thawed fraction was frozen in liquid nitrogen. Data revealed that 1 mg/ml E. longifolia aqueous extract yielded significantly (p < .05) higher sperm motility, morphology, viability, and sperm membrane integrity compared with the control group and other treated groups in chilled semen evaluation. For cryopreserved sperm, a significant difference (p < .05) in sperm motility, viability, sperm membrane integrity, DNA integrity, and lipid peroxidation was observed between 5 mg/ml E. longifolia aqueous extract and other treated and control groups. However, no significant difference in the percentage of sperm exhibiting normal sperm morphology was observed among the groups. In conclusion, the addition of 0.25 and 1 mg/ml E. langifolia extract to chilled semen and 5 mg/ml E. longifolia aqueous extract to cryopreserved sperm into Tris–egg yolk extender helps in maintaining superior quality of bull spermatozoa during chilling and freezing.