Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility

Objective   We evaluated combinations of two commercial semen extenders and three concentrations of glycerol to determine the combination that yielded the highest post-thaw sperm motility.
Design   A randomised 2 × 3 block design was used.
Procedure   Semen was collected from four stallions (6 collections per stallion). The sample was diluted with either a dried skim-milk glucose extender (EZ Mixin Original Formula) or a chemically defined, milk-free diluent (INRA 96), and each was used in combination with 2%, 3% or 4% glycerol in standard commercial freezing medium. Sperm motility was assessed by microscopy in fresh and post-thaw semen.
Results   There was a significant difference between the two extenders in the motility of spermatozoa after cryopreservation (48.9% for INRA 96; 38.6% for EZ Mixin OF; P < 0.0001). Glycerol at 4% in freezing medium yielded the highest post-thaw motility, significantly better than 2% ( P < 0.05). Three of four stallions had significantly higher post-thaw motility using INRA 96 relative to EZ Mixin OF ( P < 0.01), and two of four stallions had significantly higher post-thaw motility using 4% glycerol ( P < 0.05). The combination of INRA 96 and 4% glycerol in freezing medium gave the highest average post-thaw motility of 51.5%.
Conclusion   In this study, INRA 96 combined with 4% glycerol yielded an average recovery of progressively motile sperm consistently above the 35% target.     

Review of neurological diseases of ruminant livestock in Australia. VI: postnatal bovine, and ovine and caprine, neurogenetic disorders

This paper, the last in a series of reviews of neurological diseases of ruminants in Australia, discusses a range of neurogenetic disorders of cattle, sheep and goats, including necrotising encephalomyelopathy, glycogen storage disorders, cerebellar abiotrophy and β-mannosidosis. They are categorised by the species and breeds in which they occur.     

In vitro evaluation of frozen-thawed stallion semen: a review

The article reviews methods used for in vitro evaluation of sperm, with particular emphasis on frozen-thawed stallion sperm. The techniques, limitations of the methods and correlations with fertility results are discussed. Very few studies have tried to find correlation between fertility of frozen stallion semen and laboratory tests. It is difficult and expensive to inseminate an adequate number of mares to achieve statistically significant differences. Significant, but low correlations have been demonstrated between the foaling rate and subjective motility of sperm incubated for 2 h and 4 h at 37 degrees C and hypoosmotic swelling test after 0 and 3 h of incubation. Significant correlations have been reported between the pregnancy rate and viability of propidium iodide-stained sperm assessed by flow cytometry as well as for glass wool and Sephadex filtration tests. No correlations have been detected between fertility and motility immediately after thawing. In spite of that, motility estimation by light microscope is the most commonly used method to evaluate frozen-thawed stallion sperm. Computer assisted automatic sperm analyzers have replaced light microscopy in research projects, but so far nobody has been able to demonstrate a correlation between fertility of frozen stallion semen and any of the motility parameters obtained by these instruments.     

海藻糖、二甲乙酰胺对公猪精液冷冻保存效果的研究

采用5% 二甲乙酰胺(DMA)(V/V)完全替代甘油,比较乳糖、海藻糖对精液冷冻保存效果的影响。结果表明,海藻糖显著提高了冷冻——解冻后精子成活力(49.32%±1.52%)与顶体完整性 (47.33%±1.16%)(P<0.05)。然后利用海藻糖替代乳糖,评价不同浓度的DMA对公猪精液冷冻保存的影响。结果表明,当DMA添加量为4%时,解冻后精子活率、成活力、顶体完整率分别为(45.17±0.56)%、(50.33±0.67)%、(48.30±1.44)%,均显著高于3% DMA、6% DMA添加组(P<0.05),精子活率显著高于5% DMA添加组(P<0.05),但精子成活力、顶体完整性与其差异不显著(P>0.05)。因此,当利用海藻糖作为冷冻保存基础稀释液,DMA最适添加量为4%。     

Cryopreservation of ram semen: Manual versus programmable freezing and different lengths of equilibration

The aim of our study was to examine effects of the length of semen equilibration as well as two freezing techniques on ram sperm post-thaw quality. The ejaculates of Wallachian sheep rams (n = 12) were collected by an electro-ejaculation, equilibrated in a Triladyl® (0, 2, 4, 6, and 8 h) containing glycerol and egg yolk and frozen by programmable freezing (PF) or manual freezing (MF). After thawing, sperm samples were subjected to the motility (computer-assisted sperm analysis [CASA]), viability (SYBR-14/PI), and fertilizing ability (FA) (in vitro penetration/fertilization test on bovine oocytes) assays. It was found that the equilibration of 6 h (E-6) ensured higher post-thaw sperm motility and progressive movement compared with other lengths tested, irrespective of a freezing technique. The E-6 sperm viability did not differ between PF and MF but was lower (P < 0.05) than control. Sperm FA (E-6) was similar in PF (60.44%) and MF (62%) but slightly lower than in fresh (72.8%). Our data demonstrate that the use of MF was comparable with PF, which can be applied in the field conditions without need in a piece of cost-expensive equipment, which can greatly benefit the gene bank of animal genetic resources.     

犬精液液态保存稀释液及温度筛选试验

探讨了8种稀释液在5℃、10℃、15℃和20℃下保存犬精液的效果。结果表明:15℃为犬精液液态保存的较适温度,3号液为优等液,其中3号液在15℃精子的存活时间为105±10h,与对照液在15℃的保存效果差异极显著(p<0.01)。2、4、5号液是良等液,在15℃精子的存活时间是64±8h,与对照液在15℃的保存效果差异显著(p<0.05)。中等液为6号液,在15℃、20℃保存时与对照液没有统计学上的差异(p<0.05),在其他2种温度保存时不及对照液。7、8号液为差等液,在15℃保存时与对照液差异不显著(p<0.05),在其他3种温度时不及对照液。     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

Cryopreservation of Ghagus chicken semen: Effect of cryoprotectants,diluents and thawing temperature

The present study evaluated the effects of cryoprotectants, semen diluents and thawing temperature during Ghagus chicken semen cryopreservation. Four different experiments were conducted; Experiment 1—semen was cryopreserved using 6% dimethylacetamide (DMA) and 2% dimethylsulphoxide (DMSO) in Sasaki diluent (SD) and Lake and Ravie diluent (LR), Experiment 2 and 3—semen was cryopreserved using 8% ethylene glycol (EG) in SD, LRD and Red Fowl Extender (RFE), Experiment 4—semen was cryopreserved using 6% dimethylformamide (DMF) in SD, LR and Beltsville poultry semen extender (BPSE). Semen was cryopreserved in 0.5 ml French straws. Thawing was done at 5°C for 100 s in ice water in Experiments 1, 2 and 4, whereas in Experiment 3 thawing was done at 37°C for 30 s. The post-thaw sperm motility, viable sperm and acrosome-intact sperm were significantly (p < .05) lower in cryopreserved samples in all the experiments. No fertile eggs were obtained from cryopreserved samples in Experiments 1 and 2, except for 8% EG RFE treatment where the fertility was 0.83%. In Experiments 3 and 4, highest fertility was obtained in LR treatment 48.12 and 30.89%, respectively. In conclusion, using cryoprotectant EG (8%) and thawing at 37°C for 30 s, and DMF(6%) resulted in acceptable level of fertility in Ghagus chicken. Though the diluents influenced post-thaw in vitro semen parameters, the fertility was not affected. In addition, results indicated that thawing temperature may be a critical stage in the cryopreservation protocol.     

Comparative aspects of plant tannins on digestive physiology,nutrition and microbial community changes in sheep and goats: A review

Comparative aspects of plant tannins on digestive physiology, nutrition and microbial community in sheep and goats are discussed in the context of differences due to feed intake, digestibility, utilization of nutrients and microbial community. The purpose of this review was to present an overview of the potential benefits of tannin‐containing diets for sheep and goats and specie differences in their response to tannins. It is well established that moderate level of tannins in the diet (3%–4% tannins DM) can precipitate with soluble proteins and increase protein supply to the sheep, but comparative aspects of tannin‐containing diets in sheep and goats on animal performance, digestive physiology, rumen microbial changes and potential benefits to sustainable animal production by those compounds have received little attention. In addition, developing plant‐based tannin‐containing diets for control of rumen microbiota and rumen fermentation (e.g., methane gas) would be expected to have a greater impact on the ruminant health, productivity and emission of greenhouse gasses. The positive impacts of the plant tannin compounds mainly depend on their influence on the gut microbiome diversity and ability to generate fermentation end products (short‐chain fatty acids) that have diverse biological roles. Diets which contain optimal levels of tannins have potential benefits for sustainability of small ruminant production systems. However, there is a need for an improved understanding of the utilization of tannin‐containing forages to improve their management. This implies investigations of animal responses to tannin‐containing forages or browse species and, in particular, a better understanding of the interactions that can arise between sheep and goats on digestion, DMD, rumen fermentation and microbial community changes. This knowledge could help to improve current feeding systems in terms of efficiency of feed use and environmental impacts (reduce methane gas production) and thus contribute to the development of a sustainable sheep and goat production.     

Characterization and Evaluation of Semen in Growing Awassi Ram Lambs

This study was conducted to determine when semen can be collected and to characterize and evaluate the semen collected from growing Awassi ram lambs. Semen was collected regularly once a week for 20 months, starting at 11 months of age, from 14 Awassi ram lambs of milk and meat lines that accepted the artificial vagina. After each collection, the semen was evaluated in terms of its appearance, ejaculate volume, progressive motility, spermatozoa concentration and density. There were significant effects (p<0.01) for the age and weight of the lambs on ejaculate volume, progressive motility and spermatozoal concentration, while the types of birth and production line had no significant effects on these characteristics. Ejaculate volume and spermatozoal concentration increased significantly (p<0.01) with age, despite monthly variations. Progressive motility was similar throughout the year. Average values for ejaculate volume, progressive motility and sperm concentration were 1.2±0.5 ml, 75±10% and (4.0±1.6)×10⁹ sperm/ml, respectively. The highest positive and significant correlations were found between the semen characteristics (r = 0.29–0.68). On the other hand, a negative and significant (p<0.01) correlation (r = –0.66) was found between the spermiodensimeter readings and spermatozoal concentration, and the relationship could be represented by a linear equation Y = 7.85 – 0.07X ±0.37, where Y = expected concentration of sperm (units of 10⁹ sperm/ml) and X = spermiodensimeter reading. However the modest correlation coefficient indicates that the accuracy and precision of the resulting predictions will not be high. It was concluded that semen can be collected with a good quality from growing Awassi ram lambs at 11 months of age.     

Cryopreservation of Black Bengal buck semen: Effects of diluents and freezing on sperm motility and morphology

Semen from Black Bengal bucks was collected to establish a cooling protocol (to −196°C) for buck semen preservation, and to study the effect of freezing on sperm motility and morphology. Semen was diluted with diluents (Triladyl & Tris) and cryoprotectants, filled into straws, sealed, cooled (to 5°C) and equilibrated. After dilution, motility ranged from 75.00% to 76.67% and from 73.33% to 80.00% in Triladyl and Tris diluents, respectively. Motility of sperm after cooling to 5°C in Triladyl and Tris diluents ranged from 65.00% to 66.67% and from 63.33% to 70.00%, respectively. After equilibration in straws, the semen was subjected to a freezing protocol in a computer-controlled biofreezer CL-3000 (cooling at 10°C per minute, from 5°C to −80°C) and plunged into liquid nitrogen. Sperm motility of re-thawed semen varied from 38.33% to 43.33% and from 6.00% to−6.67% in Triladyl and Tris diluents, respectively. Sperm morphology of re-thawed semen was studied and head damage or cryoinjury was found in 2–3% of sperm in Triladyl diluents and 3–6% in Tris diluents. Whether the differences of sperm motility and head damage reflect fertility after artificial insemination is yet unknown and needs to be studied further.     

Diseases caused by Pythium insidiosum in sheep and goats: a review

Pythiosis is characterized most commonly by ulcerative dermatitis, mainly in the limbs of sheep and occasionally of goats. In sheep, Pythium insidiosum is also responsible for necrotizing rhinitis characterized by marked enlargement and deformity of the nasal region, severe respiratory difficulty, and bloody nasal discharge. A third form of pythiosis in sheep affects the digestive tract, involving ulceration of the esophagus, forestomachs, and abomasum. Pythiosis in sheep and goats has been reported only in Brazil where it occurs mainly in the semiarid region of the country, when animals congregate and stay for longer periods of time within or around water reservoirs. However, it has been reported as well in areas of humid environments, such as the Pantanal of Mato Grosso and in the Brazilian Cerrado. The diagnosis of the different presentations of pythiosis is based on gross and microscopic findings, coupled with detection of the agent by immunohistochemical, molecular, and/or culture-based methods.     

Effect of supplementing vitamin E analogues on post-thaw semen parameters and fertility in chicken

1. The effect of supplementing water-soluble vitamin E analogues 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (trolox) and butylated hydroxy toluene (BHT) was studied in two separate experiments.

2. In the first experiment, trolox was supplemented at 0.2 mM, 0.4 mM and 0.8 mM concentrations along with N-methylacetamide (MA; 12% final concentration) and semen was cryopreserved in 0.5 ml French straws. Different semen parameters and fertility were assessed from post-thaw samples.

3. Sperm motility, live sperm, and mitochondrial activity were significantly lower (P < 0.05) in cryopreserved semen. Lipid peroxidation (LPO) was significantly higher (P < 0.05) in cryopreserved semen that was reduced by trolox supplementation. The treatment containing trolox at 0.2 mM concentration produced significantly higher (P < 0.05) fertility compared to unsupplemented cryopreservation treatment.

4. In the second experiment, BHT was supplemented at 0.25 mM, 0.5 mM, and 1 mM concentrations along with MA during semen cryopreservation.

5. Sperm motility, live sperm and MTT dye reduction test were significantly lower (P < 0.05) in cryopreserved semen. These parameters declined with increasing BHT concentration. Abnormal sperm was significantly higher (P < 0.05) in the BHT supplemented treatments. The sperm chromatin dispersion (SCD) test was significantly higher (P < 0.05) in cryopreserved samples and was highest in samples supplemented with 0.5 mM and 1 mM BHT. The percentage fertility was significantly lower (P < 0.05) in cryopreserved semen and BHT supplementation did not improve fertility.

6. In conclusion, trolox supplementation at 0.2 mM concentration during semen cryopreservation improved fertility, whereas BHT supplementation resulted in a decline in post-thaw semen parameters.     

Effect of supplementation with the reduced form of coenzyme Q10 on semen quality and antioxidant status in dogs with poor semen quality: Three case studies

Oxidative stress owing to an imbalance between reactive oxygen species and antioxidants, such as coenzyme Q10 (CoQ10), is a major contributor to male infertility. We investigated the effects of the reduced form of CoQ10 (ubiquinol) supplementation on semen quality in dogs with poor semen quality. Three dogs received 100 mg of ubiquinol orally once daily for 12 weeks. Semen quality, serum testosterone, and seminal plasma superoxide dismutase (SOD) activity were examined at 2-week intervals from 2 weeks before ubiquinol supplementation to 4 weeks after the treatment. Ubiquinol improved sperm motility, reduced morphologically abnormal sperm, and increased seminal plasma SOD activity; however, it had no effect on testosterone level, semen volume, and sperm number. Ubiquinol supplementation could be used as a non-endocrine therapy for infertile dogs.     

Use of nutraceuticals in the stallion: Effects on semen quality and preservation

Nutritional supplements are widely used in the equine industry with the aim of improving horse health, sports or reproductive performances. Over the years, a number of studies have focused on investigating the effects of several dietary compounds on the quality and preservation of stallion semen. This paper reviews the literature available on the use of nutritional supplementation for the improvement of reproductive performance and semen quality in equine species, critically appraising the benefits and negative effects of several compounds found in complementary feeds such as PUFAs from different sources, vitamins and antioxidants, carnitine and botanical extracts. Different nutraceuticals have been highlighted to improve stallion fertility by providing optimal levels of antioxidants, with the most promising results obtained by the combination of PUFAs and antioxidants that resulted to be essential for the maintenance of normal reproductive functions and the reduction of cryodamage in cooled and frozen equine semen.     

Supplementation of salvianic acid A to boar semen extender to improve seminal quality and antioxidant capacity

The purpose of this test was to investigate the effect of salvianic acid A (SAA, CAS No. 76822‐21‐4) on the quality of boar semen during liquid storage at 17°C. The effects of different concentrations of SAA on semen quality and antioxidant capacity were analyzed. Boar semen was diluted with Beltsville Thawing Solution (BTS) containing different concentrations (0, 15, 30, 45, 60, 75 μM of SAA). During the storage period, sperm activity was measured every 24 hr, and plasma membrane integrity, acrosome integrity, total antioxidant capacity (T‐AOC), malondialdehyde (MDA) content, and catalase (CAT) activity were measured at 0, 1, 3, and 5 days. The results from our study suggest that different concentrations of SAA have different effects on semen preservation. Semen samples supplemented with SAA showed reduced effects of oxidative stress on sperm compared to the control samples. Supplementation of 30 μM of SAA significantly improved sperm motility, plasma membrane integrity, acrosome integrity, and antioxidant capacity. However, the addition of SAA to the extender was scarcely beneficial to the improvement of results of artificial insemination with boar semen after liquid preservation. Further studies are necessary in order to demonstrate that SAA has good effects on the liquid preservation of semen.     

Semen collection by urethral catheterization and electro-ejaculation with different voltages,and the effect of holding temperature and cooling rate before cryopreservation on semen quality in the Japanese macaque (Macaca fuscata)

In the Japanese macaque, semen has been collected by electro-ejaculation (EE), using the higher voltage stimuli compared to other species including genus Macaca. Semen coagulates immediately after ejaculation, which makes difficult to produce high-quality semen for artificial insemination. Recently, semen collection using urethral catheterization (UC) has been reported in carnivore and this technique may allow semen collection without coagulation in a less invasive manner. Further, the temporal preservation temperature and cooling rate of semen during cryopreservation affect post thawing sperm quality. In this study, to improve semen quality and quantity, as well as the animal welfare, semen collection was performed by EE with high (5–15 V) or low (3–6 V) voltage, UC and a combination of the two (EE-UC). It has been suggested that a high voltage is necessary for semen collection, but 10 V stimulation was effective enough and 15 V is for additional sperm collection. Also, liquid semen was collected by EE-UC and this could increase the total number of sperm. Further, to improve the post thawing sperm motility, semen was kept at four temperatures (4, 15, 25 and 37°C) for 60 min, and processed with two cooling procedures (slow cooling before second dilution and fast cooling after second dilution). Holding semen at 25°C and fast cooling after the second dilution maintained progressive motile sperm rate. The present results will contribute to the improvement of semen collection and animal welfare of Japanese macaques.     

A preliminary study on the effect of dietary supplementation with cod liver oil on the polyunsaturated fatty acid composition of boar semen

Eight mature Norwegian Landrace boars, of proven fertility and in routine semen production for AI, were fed individually with the same basic diet for 9 weeks. One group of 4 animals served as the control, the remaining 4 boars received a daily supplement of 75 ml cod liver oil (CLO-group). Fifteen consecutive semen samples were collected from each boar. The fatty acid composition of the semen was determined, and the content of the 15 most numerous fatty acids with a chain length longer than 12 carbon atoms was followed over time. In both groups, the proportion of 16:1n-7 decreased significantly, while 16:0 and 22:6n-3 (DHA) increased. By the end of the experiment, DHA had tended to increase and 22:5n-6 to decrease to a greater extent in the CLO-group. A significant difference between the groups was seen for onen-6 PUFA (22:4n-6), which remained unchanged in the control group but decreased in the CLO-group. No change was seen in docosapentaenoic acid (22:5n-3) and eicosapentaenoic acid (20:5n-3) was not found in any sample. These results indicate that CLO supplementation affects the fatty acid composition of boar semen. There were no significant differences in the non-return rates (4–25 days) between the two groups before, during or after the experiment.Abbreviations AA arachidonic acid - AI artificial insemination - area% each fatty acid percentage of the total area of the peaks from gas chromatographic analysis of methylated fatty acids - CLO cod liver oil - DHA docosahexaneoic acid - DPA docosapentaenoic acid - EPA eicosapentaenoic acid - LA linoleic acid - LNA linolenic acid - NR non-return rate - PUFA polyunsaturated fatty acid - T _c the temperature marking the beginning of the phase transition Fatty acid nomenclature: Example, 22:6n-3: 22=number of carbon atoms, 6=number of double bonds,n-3=position of the first double bond counted from the terminate (n or methyl end of the fatty acid chain     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.