Effects of fulvic acids on goat sperm

Zygote ◽  
2018 ◽  
Vol 26 (3) ◽  
pp. 220-223
Author(s):  
Yu Xiao ◽  
Zhengmu Wu ◽  
Min Wang
Keyword(s):  
Superoxide Dismutase ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Fulvic Acids ◽  
Quality Parameters ◽  
Control Group ◽  
Progressive Motility ◽  
Acrosome Integrity

SummaryThe effects of adding fulvic acids (FAs) to semen extenders on the quality parameters of frozen–thawed goat buck spermatozoa remain undetermined. Buck semen samples collected from six mature goat bucks once a week were diluted with Tris–egg yolk-based extenders. The diluted semen samples were supplemented with FAs (0.2, 0.4 and 0.6%, w/w), cryopreserved, and evaluated for sperm-quality parameters. Addition of FAs to the extender increased progressive motility, acrosome integrity, membrane integrity, and superoxide dismutase and catalase activities and decreased percentage abnormality and sperm malondialdehyde level compared with the control group. However, excessive FA addition (>0.4%, w/w) to semen extenders did not improve the efficiency. The results indicated that FAs could be a promising cryoprotectant for goat buck sperm.

scholarly journals Effects of Taurine on Sperm Quality during Room Temperature Storage in Hu Sheep

Animals ◽  
2021 ◽  
Vol 11 (9) ◽  
pp. 2725
Author(s):  
Liuming Zhang ◽  
Yanhu Wang ◽  
Tariq Sohail ◽  
Yan Kang ◽  
Haoyuan Niu ◽  
...  
Keyword(s):  
Room Temperature ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Control Group ◽  
Negative Effects ◽  
Positive Effects ◽  
Antioxidant Parameters ◽  
Acrosome Integrity ◽  
Hu Sheep

The present study aimed to investigate whether the presence of Tau protected Hu sheep sperm from ROS stress during storage at room temperature. The semen was diluted with extender (Tris-based) at room temperature, supplemented with different concentrations of Tau (0, 10, 20, 40, 80, or 100 mM), and stored at 15 °C. Sperm quality parameters (sperm progressive motility, kinetic parameters, plasma membrane integrity rate, acrosome integrity rate, and MMP) and antioxidant parameters (ROS, MDA, SOD, CAT, and T-AOC) were evaluated during the preservation of semen. The addition of Tau, especially at a concentration of 20 mM, exerted positive effects on sperm quality parameters and antioxidant parameters compared to the sperm without Tau treatment (control group). The addition of Tau, especially at a concentration of 100 mM, exerted negative effects on sperm quality parameters and antioxidant parameters compared to the control group. Interestingly, the results indicated that the sperm acrosome integrity rate did not change during storage time. In conclusion, the addition of Tau to sperm preserved at room temperature can enhance the antioxidant ability of sperm, reduce the LPO on the 5th day, and improve the quality of semen preserved at room temperature. These results implied that Tau had potential to enhance Hu sheep sperm reproductive performance.

scholarly journals Butaphosphan and Cyanocobalamin Supplementation in Semen Extender on Chilled Boar Sperm Quality and Life Span

2020 ◽  
Vol 7 ◽  
Author(s):  
J. Suwimonteerabutr ◽  
S. Chumsri ◽  
P. Tummaruk ◽  
Morakot Nuntapaitoon
Keyword(s):  
Plasma Membrane ◽  
Life Span ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Control Group ◽  
Plasma Membrane Integrity ◽  
Progressive Motility ◽  
Boar Sperm ◽  
Acrosome Integrity ◽  
Semen Extender

The objective of the present study was to determine the effect of butaphosphan and cyanocobalamin supplementation in semen extender on chilled boar sperm quality and life span. A total of 35 ejaculates of boar semen were included. The semen was diluted with Beltsville thawing solution extender supplemented with different concentrations of butaphosphan and cyanocobalamin [0 (control), 0.1, 0.2, 0.3, 0.4, and 0.5%] in the diluted semen. The semen samples were evaluated using a computer-assisted sperm analysis system to determine sperm motility and sperm kinetic parameters (i.e., the curvilinear velocity, VCL; straight line velocity, VSL; average path velocity, VAP; linearity, LIN; straightness, STR; amplitude of lateral head, ALH; wobble, WOB; and beat cross frequency, BCF). Additionally, sperm viability, acrosome integrity, mitochondrial activity, and plasma membrane integrity were evaluated after 4 (day 0), 72 (day 3), 120 (day 5), and 168 (day 7) h of storage using SYBR-14–ethidium homodimer-1 (EthD-1), EthD-1, JC-1, and the short hypo-osmotic swelling test, respectively. The analyses were carried out by using the general linear mixed model (MIXED) procedure of SAS. The statistical models for each data set included group, day after storage, and interaction between group and day after storage. The boar was included as a random effect. On day 0 after storage, progressive motility, VCL, VSL, VAP, and plasma membrane integrity of boar sperm in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the 0.4 and 0.5% groups (P < 0.05). On day 3 after storage, total motility and progressive motility, VCL, VSL, VAP, LIN, WOB, BCF, and plasma membrane integrity in 0.3% of butaphosphan and cyanocobalamin supplementation were significantly greater than those in the control group (P < 0.05). The total motility and progressive motility, VAP, and WOB in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 5 after storage (P < 0.05). No effects of butaphosphan and cyanocobalamin supplementation on acrosome integrity and mitochondria activity were found on days 3, 5, and 7 after storage. However, the motility and progressive motility and the values for all sperm kinetic parameters except ALH in 0.3% of butaphosphan and cyanocobalamin supplementation were greater than those in the control group on day 7 after storage (P < 0.05). In conclusion, 0.3% of butaphosphan and cyanocobalamin supplementation in semen extender improved sperm motility, sperm activity, morphology, and life span in chilled boar sperm.

scholarly journals Influence of Seasons and Extenders on Quality and Freezability of Gir Bull Semen under Middle Gujarat Climate

2018 ◽  
Vol 13 (03) ◽  
Author(s):  
D. V. Chaudhari ◽  
J. A. Patel ◽  
K. K. Hadiya ◽  
A. J. Dhami
Keyword(s):  
Plasma Membrane ◽  
Sperm Motility ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Winter Season ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Summer Season ◽  
Progressive Motility ◽  
Bull Semen

            The study was conducted to evaluate the seasonal influence (peak winter and summer) and the efficacy of three extenders (egg yolk based TFYG extender and egg yolk free soya bean based commercial extenders Optixcell and Andromed) on quality and freezability of Gir bull semen in Middle Gujarat. Semen ejaculates (6/bull/season, total36) revealed mean ejaculate volume 6.49±0.30 ml, sperm concentration1212.36±58.10 million/ml, progressive motility 74.17±0.78 %, live sperm 81.39±0.80 %, abnormal sperm 7.36±0.31 %, and sperm with intact plasma membrane 81.31±0.98 % and intact acrosome 94.81±0.24 %. Only the progressive sperm motility was significantly (P<0.05) higher(76.39±0.97 % vs. 71.94±1.00 %) with lesser sperm abnormality(6.17±0.37 % vs. 8.56±0.30 %) during winter than in summer. Semen samples split diluted with TFYG, Optixcell and Andromed extenders recorded the overall mean values of progressive sperm motility, livability, abnormality, plasma membrane integrity and acrosomal integrity during winter season as 77.87±0.51, 77.50±0.45, 5.56±0.20,76.02±0.81 and 94.35±0.29 on dilution; 72.41±0.51, 70.50±0.64, 5.96±0.26, 71.20±0.79 and 93.09±0.32 at pre-freeze stage; 41.30 ±0.94, 50.28±1.03, 9.15±0.31, 29.89±0.40 and 90.65±0.40 at post-thaw stage, respectively. The respective values in summer season were 72.13±0.60, 75.50±0.60, 7.48±0.25, 75.61 ±0.55 and 94.09±0.30 on dilution; 65.46±0.66, 69.41±1.05, 8.89±0.28, 69.70±0.66 and 92.63 ±0.33 at pre-freeze stage; 31.48±0.52, 45.09±0.85, 13.48±0.33, 26.85±0.71 and91.26±0.38 at post-thaw stage.  The overall mean sperm post-thaw motility/longevity at 0, 30, 60 and 120 min of incubation at 37°C was 41.20±1.51, 35.19±1.47, 28.80±1.75 and 17.50±1.47 % during winter season and 31.57±0.89, 26.20±0.77, 20.37±0.83 and 13.80±0.77% in summer season, respectively. The initial quality as well as freezability of semen in terms of motile, live, normal and HOS reactive sperm including post thaw longevity were better in winter season than in summer season. Further, the values of all the five semen quality parameters studied were comparatively better in Optixcell than TFYG and Andromed extenders with significant differences only in sperm progressive motility in both the seasons.The season x extender interaction was not significant for any of the sperm quality parameters studied.

scholarly journals Short communication: The percentage of egg yolk in the freezing media affects mouflon (Ovis musimon) epididymal sperm cryosurvival

2018 ◽  
Vol 16 (3) ◽  
pp. e04SC04 ◽  
Author(s):  
Lucía Martínez-Fresneda ◽  
Milagros C. Esteso ◽  
Adolfo Toledano-Díaz ◽  
Cristina Castaño ◽  
Rosario Velázquez ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Epididymal Sperm ◽  
Morphological Abnormalities ◽  
Head Displacement ◽  
Straight Line ◽  
Acrosome Integrity ◽  
Ovis Musimon

Sperm cryopreservation protocols are not well defined in many wild species such as the mouflon. The aim was to study the effect of different concentrations of EY on mouflon epididymal sperm cryosurvival. Samples were collected by the flushing method and cryopreserved by the conventional freezing technique in straws using a TEST (TES, Tris, glucose) 5% v/v glycerol medium containing either 6% v/v (n=16) or 12% v/v (n=13) clarified EY. The membrane integrity, acrosome integrity, motility, and morphological abnormalities were assessed in fresh and frozen/thawed samples. Fresh sperm quality parameters did not differ between groups except for the acrosome integrity that was lower in the TEST-6% EY than in the TEST-12% EY group (88.9 ± 2.1% vs 94.7 ± 0.8%). Membrane integrity (31.6 ± 4.6% vs 11.6 ± 4.5%), total motility (32.8 ± 4.6% vs 17.2 ± 5.6%), progressive motility (13.3 ± 2.7% vs 6.1 ± 2.9) were higher in frozen-thawed sperm with TEST-6% EY than with TEST-12% EY (p<0.05). Other motility parameters such as curvilinear velocity, straight-line velocity, average path velocity and amplitude of lateral head displacement were also higher (p<0.05) in frozen-thawed sperm with TEST-6% EY. Frozen-thawed acrosome integrity (85.1 ± 3.3% vs 91.9 ± 2.3) and morphological abnormalities (34.0 ± 3.7% vs 29.1 ± 3.6%) did not differ between extenders. In conclusion, high EY concentration had detrimental effects on post-thaw quality parameters, therefore the use of TEST based extender containing 6% EY is recommended for the cryopreservation of mouflon epididymal sperm.

scholarly journals Finding an Effective Freezing Protocol for Turkey Semen: Benefits of Ficoll as Non-Permeant Cryoprotectant and 1:4 as Dilution Rate

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 421 ◽  
Author(s):  
Michele Di Iorio ◽  
Giusy Rusco ◽  
Roberta Iampietro ◽  
Maria Antonietta Colonna ◽  
Luisa Zaniboni ◽  
...  
Keyword(s):  
Dilution Rate ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Quality Parameters ◽  
Dna Integrity ◽  
Osmotic Resistance ◽  
Progressive Motility ◽  
Nitrogen Vapor ◽  
Combined Use ◽  
Cryopreservation Protocol

The present study aimed to find an effective cryopreservation protocol for turkey semen through the combined use of dimethylsulfoxide (DMSO) and three non-permeant cryoprotectants (NP-CPAs), sucrose, trehalose, and Ficoll 70. In addition, the action of two dilution rates (1:2 and 1:4) were also investigated. Semen was processed according to two final dilution rates and the following treatments: Tselutin extender (TE)/DMSO (control), TE/DMSO + sucrose or trehalose 50, 100, 200, or 400 mM, and TE/DMSO + Ficoll 0.5, 0.75, 1, or 1.5 mM. In total 26 different combinations treatments were achieved. The diluted semen was filled up into straws and frozen on liquid nitrogen vapor. The post-thawing sperm quality was assessed by analyzing motility, membrane integrity, osmotic resistance, and DNA integrity. The results obtained revealed a significant effect of NP-CPA concentration on total and progressive motility, on most of the kinetic parameters, on membrane integrity and DNA integrity, while the post-thaw quality was less affected by dilution rate. The highest post-thaw quality for all sperm quality parameters assessed except curvilinear velocity (VCL) and DNA integrity were found in semen frozen with 1 mM Ficoll/1:4 (p < 0.05). Our findings provide an important contribution for the identification of a reference procedure for turkey semen cryopreservation, in order to create the first national avian semen cryobank.

scholarly journals The Antibacterial and Antioxidant Roles of Buckwheat Honey (BH) in Liquid Preservation of Boar Semen

2021 ◽  
Vol 2021 ◽  
pp. 1-9
Author(s):  
Qun Lan ◽  
Yingyu Xie ◽  
Jiahua Pan ◽  
Qiaohui Chen ◽  
Tianfang Xiao ◽  
...  
Keyword(s):  
Sperm Quality ◽  
Antioxidant Properties ◽  
Membrane Integrity ◽  
Storage Period ◽  
Antibiotic Use ◽  
Quality Parameters ◽  
Progressive Motility ◽  
Liquid Storage ◽  
Boar Semen

In the present study, we hypothesized that buckwheat honey (BH) should be regarded as a potential alternative to antibacterial and antioxidant agent in liquid storage of boar semen. To this end, boar semen was firstly studied for in vitro dose tolerability to BH by measuring sperm progressive motility. The optimum progressive motility of boar spermatozoa was observed in extender with 0.5% and 0.6% BH addition. Afterward, sperm quality parameters, bacterial profile and composition, total antioxidant (T-AOC), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels of control, BH supplementation, antibiotics supplementation, and incorporated supplementation were compared during liquid storage period, to further investigate antibacterial and antioxidant properties of BH. The results showed that BH supplementation significantly improved sperm motility, acrosome integrity, plasma membrane integrity, inhibited opportunistic bacterial growth, and altered microbial compositions at the end of preservation. Additionally, T-AOC, SOD, and CAT levels were significantly higher in the BH supplementation group than those in the control and antibiotic supplementation group, whereas MDA level exhibited opposite change pattern. Importantly, BH addition to the extender was able to exert a synergistic effect in combination of antibiotic use. Our findings suggested that the appropriate concentrations (0.5% and 0.6%) of BH were added to the extender could act antibacterial and antioxidant roles in liquid preservation of boar semen.

Effects of low-density lipoproteins extracted from different avian yolks on boar spermatozoa quality following freezing–thawing

Zygote ◽  
2012 ◽  
Vol 22 (2) ◽  
pp. 175-181 ◽  
Author(s):  
Peng Wang ◽  
Yan-Feng Wang ◽  
Chun-Wei Wang ◽  
Shu-Hai Bu ◽  
Jian-Hong Hu ◽  
...  
Keyword(s):  
Semen Quality ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Low Density Lipoproteins ◽  
Low Density ◽  
Boar Sperm ◽  
Boar Semen ◽  
Avian Egg

SummaryLow-density lipoproteins (LDL) is known to protect boar sperm during freezing–thawing, but little information is known about the effects of LDL extracted from different avian egg yolks on post-thaw boar semen quality. The purpose of this study was to compare and analyze the effects of LDL at various concentrations and different species on boar sperm quality after freezing–thawing. LDL extracted from the yolk of hen egg, duck egg, quail egg, pigeon egg or ostrich egg was added to the extender at the concentrations of 0.06, 0.07, 0.08, 0.09 and 0.1 g/ml, respectively, and their effects on frozen–thawed boar sperm quality were assessed. According to all measured parameters, the results showed that sperm motility, acrosome integrity and plasma membrane integrity were 43.20%, 52.57% and 48.13%, respectively, after being frozen–thawed with 0.09 g/ml LDL extracted from pigeon egg yolk. All these quality parameters were higher than that of other groups (P < 0.05). In conclusion, our results confirmed that LDL extracted from pigeon egg yolk had the best cryoprotective effects on frozen–thawed boar sperm among all of the groups supplemented with LDL from five kinds of avian egg in extender. The optimum concentration of LDL extracted from pigeon egg in boar semen freezing extender was 0.09 g/ml.

scholarly journals Ram semen preserved at 0°C with soybean lecithin Tris-based extender substituted for egg yolk

2021 ◽  
Vol 34 (2) ◽  
pp. 192-197
Author(s):  
Jian-qing Zhao ◽  
Guo-liang Xiao ◽  
Wen-liang Zhu ◽  
Di Fang ◽  
Na Li ◽  
...  
Keyword(s):  
Soybean Lecithin ◽  
Egg Yolk ◽  
Control Group ◽  
Progressive Motility ◽  
Significant Difference ◽  
Acrosome Integrity ◽  
Normal Offspring ◽  
And Control ◽  
Ram Sperm ◽  
Artificial Vagina

Objective: The present study evaluated the preservation of ram semen at 0°C using soybean lecithin with a Tris-fructose extender.Methods: Semen was collected by artificial vagina ejaculation from six rams with proven fertility. High quality ejaculates were diluted by soybean lecithin (0.25%, 0.5%, 0.75%, 1.0%, 1.25%) using Tris-fructose extender and control (Tris-fructose egg yolk extender), respectively. The ejaculates were diluted to a concentration of 5×10<sup>8</sup> sperm/mL, followed by cooling to 0°C in 90 min and maintaining the temperature for 12 days. The diluted semen samples were examined and recorded for sperm progressive motility, acrosome integrity at 0, 24, 72, 144, 216, 288 h, respectively. Two hundred and twenty-three ewes were inseminated for 216 h with optimal soybean lecithin concentrated semen or control via trans-cervical insemination.Results: The results showed that there were no differences in sperm progressive motility at 0, 24, 72, and 144 h (p>0.05). After 216 h, the sperm progressive motility in the control group and 0.5% concentration groups was significantly higher when compared to 0.25% concentration (p<0.05). The 0.5% concentration group demonstrated the highest survival rate and had no difference with the control group (p>0.05). At 216 h, the sperm progressive motility of all groups was still above 50%. The acrosome integrity of all groups was decreased with prolongation of storage time, but there was no difference at each time point (p>0.05). There was no significant difference in the lambing rate and pregnancy rate between the 0.5% concentration group and the control group (p>0.05).Conclusion: These results suggest that ram sperm is capable of fertilization after preservation at 0°C with 0.5% of soybean lecithin in Tris-based extender substituted for egg yolk and produce normal offspring after insemination.

scholarly journals The Re-Addition of Seminal Plasma after Thawing Does Not Improve Buck Sperm Quality Parameters

Animals ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 3452
Author(s):  
Uchechi Linda Ohaneje ◽  
Uchebuchi Ike Osuagwuh ◽  
Manuel Alvarez-Rodríguez ◽  
Iván Yánez-Ortiz ◽  
Abigail Tabarez ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Sperm Quality ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Quality Parameters ◽  
Mitochondrial Activity ◽  
Dna Integrity ◽  
Vitro Fertilization

In order to achieve a higher post-thaw buck sperm quality, an approach in the thawing protocol of cryopreserved sperm doses under in vitro capacitation conditions mimicking the in vivo female environment was studied. Therefore, functional and kinetic characteristics of buck thawed sperm from males of different ages, the season of collection, and melatonin implanted males in the non-breeding season were assessed after 3 h of incubation in an in vitro fertilization (IVF) media with 20% of buck seminal plasma (SP). Previously, fresh ejaculates were collected via artificial vagina from eight males of the Cabra Blanca de Rasquera breed during two consecutive years in breeding and non-breeding periods. Prior to semen collection in non-breeding seasons, males were split into two groups: one group was implanted with melatonin, while the other was not. In each group, semen samples were pooled, centrifuged, and diluted in an extender containing 15% powdered egg yolk and 5% glycerol before freezing. After thawing, sperm were washed and incubated in three different media: (a) control media (modified phosphate-buffered saline (PBS), (b) IVF commercial media, and (c) IVF media + 20% SP. Sperm motility was evaluated by CASA, while plasma and acrosome membrane integrity, mitochondria activity, and DNA fragmentation were analysed by flow cytometer at 0 h and after 3 h incubation. A significant reduction in motility, mitochondrial activity, plasma, and acrosome membrane integrity were observed after incubation in the presence of SP, although similar to that observed in IVF media alone. DNA integrity was not affected under in vitro capacitation conditions, regardless of SP addition. In conclusion, the addition of SP failed to improve post-thaw buck sperm quality under in vitro conditions irrespective of male age, the season of collection, and melatonin implant.

138 EFFECT OF SEMINAL PLASMA ON CHILLING AND FREEZING OF CANINE SPERMATOZOA

2007 ◽  
Vol 19 (1) ◽  
pp. 187
Author(s):  
I. Yu ◽  
Y. J. Kim ◽  
I. S. Kim ◽  
S. P. Leibo ◽  
N. Songsasen
Keyword(s):  
Pisum Sativum ◽  
Seminal Plasma ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Beneficial Effects ◽  
Progressive Motility ◽  
Healthy Dogs ◽  
Sperm Survival ◽  
Acrosome Integrity ◽  
Group 1

Seminal plasma (SP) is usually removed from semen that is to be cryopreserved. However, some reports indicate that SP has beneficial effects on spermatozoa during chilling and freezing (Barrios et al. 2000 Biol. Reprod. 63, 1531–1537; Moore et al. 2005 Theriogenology 63, 2372–2381; Vadnais et al. 2005 Anim. Reprod. Sci. 87, 121–132). The purpose of this study was to determine the effect on sperm survival of adding SP to the extender before cooling and freezing canine spermatozoa. In replicate experiments, ejaculates obtained from 4 healthy dogs (3–4 years old) of various breeds were pooled and centrifuged at 300g for 10 min at 25�C; the supernatant of seminal plasma was decanted. Spermatozoa were suspended in egg yolk-Tris (EYT) buffer. The study comprised 2 experiments: Exp. 1: Sperm were suspended in EYT extender containing 0%, 20%, 50%, 80%, or 100% SP, and were slowly cooled to 4�C for 2 h or held at 25�C as controls. Exp. 2: Sperm samples, each of which contained 1 � 108 sperm mL-1, were assigned to 5 groups to be frozen. In group 1, sperm in EYT + 20% SP were cooled to 4�C, diluted to contain final concentrations of 5% glycerol + 10% SP in EYT, and then frozen. In the 4 other groups, sperm in EYT alone were first cooled slowly to 4�C, then diluted to contain 5% glycerol plus 0%, 20%, 40%, or 50% SP in EYT, and then frozen. Spermatozoa were frozen at 25�C min in plastic straws that were suspended above liquid nitrogen and thawed in water at 38�C for 30 s. Sperm survival was assayed by determining progressive motility and integrity of plasma and acrosome membranes. Progressive motility was determined by microscopic examination at 400� magnification. Membrane integrity was assessed by use of a double fluorescent dye, and acrosome integrity by staining sperm with Pisum sativum agglutinin. The results of the first experiment showed that 20%, 50%, 80%, or 100% SP did not improve motility, membrane integrity, or acrosome integrity of spermatozoa chilled to 4�C compared to those chilled without SP (P &gt; 0.05). Survival of spermatozoa suspended in EYT + 20% SP and maintained at 25�C was significantly higher than for those that were chilled (P &lt; 0.05). The results of the second experiment showed that spermatozoa suspended in EYT + 20% SP and then diluted at 4�C to contain 5% glycerol + 10% SP exhibited the highest progressive motility and membrane integrity after being frozen and thawed (P &lt; 0.05). In summary, although seminal plasma did not affect spermatozoa that were only chilled, addition of seminal plasma did significantly improve survival of canine spermatozoa that were frozen and thawed.

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