Effect of Two Cryopreservation Methods on Seminal Quality and Pregnancy Rate in Alpacas Induced to Ovulation with Seminal Plasma

The objective was to evaluate the effect of two methods of freezing on seminal quality and effect of seminal plasma as an inducer of ovulation in pregnancy percentages in inseminated alpacas. The semen collection was made post copula. After the collection of the ejaculates, motility and volume were assessed. The ejaculates with volume (≥1 ml) and motility (≥ 60%) were mixed (pool), 80 % was used (3 samples/pool) and 20 % (2 samples/pool) during the experiment. Then and diluted in Tris-base with 20% egg yolk. The samples were cooled 1.5 hours at 5 °C. At that temperature it was combined with the basic dilutor plus glycerol, obtaining a final concentration of 5% glycerol, then they were packed in 0.5 mL (13 x 106 spem/straw) straws to be frozen by horizontal and vertical methods. The seminal (semen + vaginal fluid) quality analysis was performed fresh and after cooling and thawing. Insemination was performed in two groups with thawed semen from two straws of the vertical method, the first group 20 females induced to ovulate with GnRH analog, the second group 20 females induced to ovulate with seminal plasma. When comparing, the results obtained of motility, viability, HOST and acrosomal integrity of fresh sperm, after the freezing process, decreased (p<0.05) compared to fresh and refrigerated samples. On the other hand, when comparing the freezing methods, the sperm values frozen by the vertical method were higher than those obtained by the horizontal method, with (p<0.05) in motility and HOST without (p>0.05) in acrosomal integrity and viability. The vertical semen freezing method can replace the horizontal method to obtain pregnancy.

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Vitrification Using Soy Lecithin and Sucrose: A New Way to Store the Sperm for the Preservation of Canine Reproductive Function

Animals ◽

10.3390/ani10040653 ◽

2020 ◽

Vol 10 (4) ◽

pp. 653

Author(s):

Maja Zakošek Pipan ◽

Margret L. Casal ◽

Nataša Šterbenc ◽

Irma Virant Klun ◽

Janko Mrkun

Keyword(s):

Semen Quality ◽

Reproductive Function ◽

Egg Yolk ◽

Final Concentration ◽

Freezing Process ◽

Soy Lecithin ◽

Group A ◽

Short And Long Term

A challenge in freezing semen for short and long-term availability is avoiding damage to intact spermatozoa caused by the freezing process. Vitrification protocols provide better results through less manipulation of semen and shorter freezing time compared to slow freezing techniques. Our research was aimed at improving vitrification methods for canine semen. Semen quality was determined in 20 ejaculates after collection. Each ejaculate was divided into eight aliquots, each with a different extender. The control extender contained TRIS, citric acid, fructose, and antibiotics. Soy lecithin and sucrose were added to the control extender at different concentrations to make up the test extenders and final concentration of 50 × 106 spermatozoa/mL. From each group, a 33 µL (1.65 × 106 spermatozoa) suspension of spermatozoa was dropped directly into liquid nitrogen and devitrified at least one week later and evaluated as before. Soy lecithin at 1% and 0.25 M sucrose added to the base vitrification media effectively preserved all sperm qualities. Our results demonstrate the effectiveness of our methods. Vitrification media containing sucrose and soy lecithin cause a minimal decline in quality of canine semen after devitrification. Furthermore, extenders used in our research did not contain egg yolk, which was replaced by soy lecithin, thus allowing for ease of shipping to other countries with strict requirements.

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Effect of different levels of egg-yolk on freezability of Jakhrana buck semen

Indian Journal of Animal Research ◽

10.18805/ijar.11326 ◽

2016 ◽

Author(s):

Narendra Kumar ◽

B. Rai ◽

Chetna Gangwar ◽

S. A. Lone ◽

Anshuman Kumar ◽

...

Keyword(s):

Sperm Motility ◽

Egg Yolk ◽

Osmotic Swelling ◽

Semen Collection ◽

Sperm Abnormality ◽

Before And After ◽

Different Levels ◽

Artificial Vagina ◽

Intensive System ◽

Acrosomal Integrity

The present study was designed to determine the effect of different levels of egg-yolk on freezability of Jakhrana buck semen. Six healthy Jakhrana bucks (BW=30 ± 2kg, age=12 ± 0.5 month) were used for semen collection. These bucks were maintained under semi-intensive system at Jakhrana Unit of C.I.R.G. Makhdoom, Mathura. A total of 48 ejaculates (6 bucks × 8 replicates) were collected twice a week using artificial vagina. Each ejaculate was divided into 4 groups (G1, G2, G3 and G4). The G1, G2, G3 and G4 were extended with Tris-egg yolk-citric acid- fructose-glycerol (TEYCFG) extenders containing 5, 10, 15 and 20% egg yolk level, respectively. Each ejaculate was evaluated for sperm motility, viability, abnormality, and hypo-osmotic swelling (HOS) response and acrosome integrity before and after freezing. At pre-freeze stage no significant (P>0.05) difference in sperm motility and viability was found among all groups. Sperm abnormality was significantly (P<0.05) higher in G4 as compared to other groups (G1, G2, G3). The HOS response and acrosomal integrity was significantly (P<0.05) higher in G1, G2 and G3 as compared to G4. However, no significant (P>0.05) difference was observed in HOS response and acrosomal integrity among G1, G2 and G3. At post thaw stage, sperm motility, viability and HOS response was significantly (P<0.05) higher in G1 and G2 as compared to G3 and G4. Sperm abnormality was significantly (P<0.05) lower in G2 as compared to other groups. The acrosomal integrity was significantly (P<0.01) higher in G1 and G2 as compared to G3 and G4. It is concluded that 10% egg yolk in Tris based extender may be the best for successful cryopreservation of Jakhrana buck semen.

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Detection of osteopontin transcript in seminal plasma and its association with post-freeze-thaw quality of cryopreserved spermatozoa in mithun (Bos frontalis)

Indian Journal of Animal Research ◽

10.18805/ijar.v0iof.4553 ◽

2016 ◽

Author(s):

K. K. Baruah ◽

A. Dhali ◽

B. Bora ◽

A. Mech ◽

M. Mondal

Keyword(s):

Seminal Plasma ◽

Egg Yolk ◽

Sperm Viability ◽

Rt Pcr ◽

Freeze Thaw ◽

Sperm Membrane ◽

The Relationship ◽

Acrosomal Integrity

The study aimed to detect osteopontin (OPN) transcript in seminal plasma and to assess the relationship between the presence of the transcript and post-freeze-thaw quality of cryopreserved spermatozoa in mithun (Bos frontalis). Semen samples were collected from five adult bulls through rectal massage method and cryopreserved in tris-egg yolk-glycerol extender. OPN transcript was detected by RT-PCR in the RNA purified from seminal plasma. OPN transcript was found to be present consistently in three animals (OPN+) and absent in two animals (OPN-). Although, sperm viability and acrosomal integrity were found similar at different stages of cryopreservation in both the groups, tail abnormality after final dilution and, head and tail abnormalities after equilibration were found significantly (P less than 0.05) lesser in the OPN+ compared to OPN- animals. The results indicated that OPN is probably important in stabilizing sperm membrane that resulted in better resistance of sperm to cryopreservation process in OPN+ animals.

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118 CRYOPRESERVATION OF CONVENTIONAL AND SEX-SORTED BULL SPERM USING A DIRECTIONAL FREEZING METHOD

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab118 ◽

2007 ◽

Vol 19 (1) ◽

pp. 176 ◽

Cited By ~ 7

Author(s):

H. Hayakawa ◽

T. Yamazaki ◽

M. Oshi ◽

M. Hoshino ◽

O. Dochi ◽

...

Keyword(s):

Liquid Nitrogen ◽

Interface Velocity ◽

Egg Yolk ◽

Sperm Viability ◽

Freezing Method ◽

Progressive Motility ◽

Nitrogen Vapor ◽

Bull Sperm ◽

Directional Freezing ◽

Acrosomal Integrity

The objective of this study was to find an optimal parameter (liquid-ice interface velocity; velocity) of the directional freezing method (Arav et al. 2002 Reprod. Nutr. Dev. 42, 583–586) for conventionally processed bull sperm stored in 0.5-mL straws. We also evaluated sex-sorted bull sperm frozen using this method. Experiment 1: Each single ejaculate from two Holstein bulls was split and processed in egg yolk citrate extender (2 steps, 6% glycerol final; EYC) or egg yolk Tris extender (1 step, 7% glycerol final; EYT1). After cooling and loading in 0.5-mL plastic straws, each batch of semen was frozen using a directional 0.5-mL straw freezer (MTG 550; IMT, Ltd., Ness-Ziona, Israel) in 6 different velocities (v2.0, v2.2, v2.4, v2.6, v2.8, v3.0; mm s-1). Ice seeding time was 45 s. Static freezing in liquid nitrogen vapor was used as the control. Sperm parameters were measured using computer-assisted semen analysis (CASA) at 0, 1, and 2 h after thawing. The thawed samples were also evaluated for sperm viability and acrosomal integrity by triple staining. The experiment was repeated 3 times. Data were analyzed by ANOVA. No significant differences in general and progressive motility were observed in each extender regardless of the freezing method. However, sperm viability and acrosomal integrity of sperm frozen in EYC were highest at v3.0 (73.2% and 84.7%, respectively) and were generally higher after MTG freezing (63.4 to 73.2% and 80.6 to 84.7%, respectively) than in the control (52.6% and 73.1%, respectively; P < 0.05 or 0.01) except for v2.4 (54.5% and 70.8%). Sperm viability (74.6 to 77.9% vs. 63.2%) and acrosomal integrity (84.8 to 88.9% vs. 79.3%) in EYT after MTG freezing were also higher than in the control (P < 0.01). Experiment 2: Two single ejaculates from a Holstein bull were flow cytometrically sex-sorted (Schenk et al. 1999 Theriogenology 52, 1375–1391) for X sperm. Each ejaculate was assigned to processing in EYC or egg yolk Tris extender (2 steps, 6% glycerol final; EYT2). Processed sperm was frozen using MTG 550 (velocity set at v3.0) or in liquid nitrogen vapor as control and analyzed as in Experiment 1. In the control, general (40.2% vs. 21.1%; P < 0.01) and progressive (16.3% vs. 4.3%; P < 0.05) motility in EYC were higher than in EYT2, respectively, at 0 h. In MTG freezing, motility (at 0 h, 42.0% vs. 28.3%; P < 0.05), viability (60.5% vs. 40.8%; P < 0.01), and acrosomal integrity (86.9% vs. 71.0%; P < 0.05) in EYC were higher than in EYT2, respectively. But in experiment 2, there were no differences in motility, progressive motility, viability, and acrosomal integrity between MTG freezing and control. The above results suggest that MTG freezing improves membrane and acrosomal quality of bull sperm frozen in 0.5-mL straws. Faster interface velocity (3.0 mm s-1) seems optimal for the given condition. Egg yolk citrate extender seems to be beneficial for MTG freezing of sex-sorted bull sperm, but further studies using ejaculates from different bulls are required to confirm the apparent benefit.

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Seminal plasma proteins protect flow-sorted ram spermatozoa from freeze - thaw damage

Reproduction Fertility and Development ◽

10.1071/rd08238 ◽

2009 ◽

Vol 21 (4) ◽

pp. 571 ◽

Cited By ~ 23

Author(s):

T. Leahy ◽

J. I. Marti ◽

G. Evans ◽

W. M. C. Maxwell

Keyword(s):

Seminal Plasma ◽

High Speed ◽

Sperm Quality ◽

Membrane Integrity ◽

Weight Fraction ◽

Protein Supplementation ◽

Freezing Process ◽

Spermatozoa Motility ◽

Functional Integrity

Seminal plasma improves the functional integrity of compromised ram spermatozoa but has been reported to be toxic to sorted spermatozoa. The present study attempted to clarify this paradoxical effect and improve the functional integrity of spermatozoa following sorting and cryopreservation. The in vitro function of sorted spermatozoa (motility characteristics and membrane integrity) was examined after supplementation with differing concentrations and protein fractions of seminal plasma at various stages of the sorting and freezing process. For all experiments, spermatozoa (two males, n = four ejaculates per male) were processed through a high-speed flow cytometer before cryopreservation, thawing and incubation for 6 h (37°C). Supplementation of crude seminal plasma (CP), its low molecular weight fraction (LP; <10 kDa) or protein-rich fraction (SPP; >10 kDa), immediately before freezing improved the functional integrity of sorted spermatozoa compared with no supplementation (control), whereas supplementation after thawing had no effect for CP and LP. The protective effect of seminal plasma was not altered by increasing the amount of protein supplementation. No toxic effect of CP, SPP or LP was evident even when supplemented at high protein concentrations. It is concluded that seminal plasma protein, if added to ram spermatozoa after sorting and before freezing, can improve post-thaw sperm quality and consequently the efficiency of sorting. This effect is most likely related to protection of the spermatozoa during freeze–thawing.

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Plasma Lipid Peroxidation as a Marker for Seminal Oxidative Stress in Stallion

Journal of Agricultural Science ◽

10.5539/jas.v11n6p401 ◽

2019 ◽

Vol 11 (6) ◽

pp. 401

Author(s):

Patricia Wolkmer ◽

Andressa M. G. Stumm ◽

Luiz F. K. Borges ◽

Eduarda P. T. Ferreira ◽

Bruna Favaretto ◽

...

Keyword(s):

Oxidative Stress ◽

Lipid Peroxidation ◽

Seminal Plasma ◽

Plasma Lipid ◽

Thiobarbituric Acid ◽

Thiobarbituric Acid Reactive Substances ◽

Semen Collection ◽

Semen Storage ◽

Plasma Lipid Peroxidation ◽

Antioxidant Enzyme Catalase

This experiment aims to evaluate the correlation between lipid peroxidation levels in serum and seminal plasma in equines. Also, it investigates the lipid peroxidation in extended semen samples and its effects and sperm motility during a 72 hr refrigeration period. Blood and semen were collected from fertile Crioulo stallions. Serum and seminal plasma lipid peroxidation levels were analyzed by thiobarbituric acid reactive substances (TBARS) immediately after semen collection. After addition of extender (hour = 0), diluted semen was refrigerated and stored at 5 °C. Semen analyses, TBARS and catalase activity were performed in extended semen at 0, 24, 48, and 72 hours. We noted that levels of plasma lipid peroxidation can be used as an indicative of seminal oxidative stress. Also, lipid peroxidation does not increase substantially during semen storage. Lipid peroxidation and the antioxidant enzyme catalase do not seem to be the major cause of loss and motility and consequently reduction in fertility in stallion semen during storage for 72 h at 5 °C.

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Anovel Immunological Technique for Identification of Human Seminal Fluid

Baghdad Science Journal ◽

10.21123/bsj.7.2.1023-1027 ◽

2010 ◽

Vol 7 (2) ◽

pp. 1023-1027

Author(s):

Baghdad Science Journal

Keyword(s):

Seminal Plasma ◽

Human Body ◽

Seminal Fluid ◽

Cross Reactivity ◽

Body Fluids ◽

Vaginal Fluid ◽

Human Semen ◽

Human Seminal Plasma ◽

Ear Wax ◽

Immunological Technique

An immunological technique was investigated for the detection of human semen in forensic analysis.This technique included a preparation of anti-human seminal plasma antibodies, by immunizing rabbits with treated human semen. The human semen was treated with an acid to prevent cross reactivity with other human body fluids. The antibody produced was tested against different animal,s seminal fluid samples (dog, goat ,sheep, cow) and human body fluids( saliva, blood , vaginal fluid, ear wax and human semen). It was found that using this developed technique was only selectively responsed with human semen . The prepered kit was evaluated and tested in Forensic laboratory- Ministry of Health. Finally, results were obtained in a comparison with the recommended techniques.

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Effects of Seminal Plasma Components on Motility and Acrosomal Integrity of Meishan Boar Spermatozoa after Cooling Treatments

Nihon Chikusan Gakkaiho ◽

10.2508/chikusan.69.720 ◽

1998 ◽

Vol 69 (8) ◽

pp. 720-727

Author(s):

Hiroshi HARAYAMA ◽

Akira IMANO ◽

Masashi MIYAKE ◽

Seishiro KATO

Keyword(s):

Seminal Plasma ◽

Boar Spermatozoa ◽

Acrosomal Integrity

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Changes in Quality of Native and Frozenthawed Semen in Relation to Two Collections Performed in a 24-hour Interval and Adition of Clarified Egg Yolk to Extender

Scientia Agriculturae Bohemica ◽

10.1515/sab-2016-0010 ◽

2016 ◽

Vol 47 (2) ◽

pp. 60-67

Author(s):

P. Folková ◽

J. Šichtař ◽

O. Šimoník ◽

A. Dokoupilová ◽

R. Rajmon

Keyword(s):

Sperm Motility ◽

Egg Yolk ◽

Semen Collection ◽

Semen Extender

Abstract The aim of the study was to evaluate the effect of repeated semen collection and the substitution of normal egg yolk with clarified egg yolk to commercially produced semen extender on qualitative parameters of frozen-thawed canine semen. Two semen collections were scheduled in a 24-hour interval and in each of six dogs, three 1st and three 2nd collections were performed. The frozen-thawed sperm samples were prepared either with clarified or normal egg yolk and motility and viability were evaluated. The effect of the sequence of semen collection was demonstrated by significant differences in motility and also in viability of sperms both in native and frozen-thawed ejaculate. The percentage of viable sperms was significantly higher in samples from the 2nd compared to the 1st collection. This trend was the same also in motility except in native ejaculate. The addition of clarified egg yolk was beneficial for higher survival of sperms immediately after thawing and also after 30 min of incubation, compared to samples with normal egg yolk. Sperm motility evaluated after thawing was higher in samples with clarified egg yolk, without an apparent connection with semen collection sequence. The decrease of values of the qualitative parameters of sperms observed in the period of 30 min of incubation was significantly slowed down when clarified egg yolk was used. This was especially obvious in samples from the 2nd collection.

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