Lipid and protein oxidation levels in spermatozoa and seminal plasma of Asian Elephants (Elephas maximus) and their relationship with semen parameters

Peroxidation damage to spermatozoa and seminal plasma has an important role in sperm quality. Thus, the objective of this study was to determine the levels of lipid and protein oxidation in spermatozoa and seminal plasma of Asian elephants (Elephas maximus) with varying percentage of progressive motility. Lipid and protein oxidation was measured by the thiobarbituric acid‐reactive species (TBARS) assay and the 2, 4‐dinitrophenylhydrazine (DNPH) carbonyl groups assay, respectively. Fresh semen samples were collected from Asian elephants and classified according to the percentage of motile spermatozoa into good (>60%) and poor (≤20%) motility. Results revealed that seminal plasma malondialdehyde (MDA) and seminal plasma protein carbonyls (PCs) were significantly higher in poor motility than in good motility (p < .05). The MDA and PC levels in seminal plasma were negatively correlated with the percentages of progressive motility (p < .05). In addition, the negative correlation between sperm concentration and seminal plasma MDA level was investigated (p < .05). The sperm viability was also negatively correlated with sperm PC level (p < .05). This study indicated that lipid and protein oxidation has deleterious effect on semen quality of Asian elephants.     

Comparison of density gradient and single layer centrifugation of stallion spermatozoa: Yield,motility and survival

Reasons for performing study: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. Objective: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. Methods: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4°C overnight, were also available for testing. Results: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18–20% when cold‐stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. Potential relevance: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.     

Biochemical observations on beagle dog semen.

Semen samples were collected at weekly intervals for six weeks from eight sexually mature beagles previously shown to produce normal ejaculates. Seminal plasma and sperm fractions were separated by centrifugation and the sodium, potassium, alanine and aspartate aminotransferases, acid and alkaline phosphatase concentrations in the two fractions determined. Regression analysis of the mean weekly values obtained from physical and biochemical examination of the ejaculates showed that sodium ion concentration was highest in seminal plasma. The highest levels of aminotransferases were found in sperm fractions. Those enzymes may be indices of abnormal or damaged spermatozoa. Acid and alkaline phosphatase activity was 100 times greater in seminal plasma than in sperm fractions. Phosphatase concentrations are likely to be dependent on prostate activity. Measurement of acid phosphatase in canine semen therefore may be a useful index of prostate function. The motility of the semen samples was independent of the potassium concentration in seminal plasma. However, there was some evidence of a correlation between sperm motility and the enzyme and sodium content of seminal plasma.     

Influence of different anaesthetic protocols over the sperm quality on the fresh,chilled (4°C) and frozen‐thawed epididymal sperm samples in domestic dogs

This study assessed the influence of three different anaesthetic protocols on semen quality obtained from the epididymis. Sixty male dogs undergoing to routine sterilization were assigned to three anaesthetic protocols: thiopental group (TG, n = 20), propofol group (PG, n = 20) and ketamine–dexmedetomidine group (KDG, n = 20). Immediately after orchidectomy, the cauda epididymides and vas deferent ducts were isolated and then a retrograde flushing was performed to collect spermatozoa. In experiment 1, after the initial evaluation of the semen (sperm concentration, sperm motility and the percentages of live spermatozoa, abnormal spermatozoa and acrosome membrane integrity), semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 48 hr, and the sperm motility was assessed at 6, 24 and 48 hr. In experiment 2, semen samples were diluted in Tris‐glucose‐egg yolk extender and chilled for 24 hr, and then samples were frozen in two extenders with different glycerol concentrations, to reach a final concentration of 50–100 × 10⁶ spermatozoa ml^?1, 20% egg yolk, 0.5% Equex and 4% and 5% glycerol, respectively. Mean values of total sperm concentration, sperm viability and the percentages of intact acrosome and abnormal spermatozoa were not significantly different between experimental groups, and therefore, the anaesthetic protocols assessed did not affect sperm parameters mentioned above. However, our study confirmed a detrimental effect of the use of thiopental (TG) over the total sperm motility (p < 0.05) and progressive sperm motility (p < 0.05) of the fresh and chilled epididymal sperm samples. The anaesthetic protocols including the application of propofol or ketamine–dexmedetomidine can be used to recover sperm in domestic canids without significant changes in sperm quality compared when semen is collected routinely and these techniques could be applicable to endangered wild canids.     

Effect of semen collection in extender solution on the characteristics of goat spermatozoa

The objective of this experiment was to investigate whether the motility parameters and acrosome integrity of goat ejaculated spermatozoa are affected by collecting semen into tubes containing an extender, and thereby determine the significance of reducing contact between seminal plasma and the sperm membrane at ejaculation. Semen were collected from three goats into tubes containing 0, 1 or 10 ml extender, or collected into tubes containing 10 ml extender supplemented with 0.1, 1 or 5% BSA. Sperm motion parameters were evaluated immediately after collection, after washing, and during a 3-h thermal resistance test. Acrosome integrity was assessed using FITC-PNA staining. Semen collection into tubes containing 10 ml extender produced higher sperm motility, progressive motility, and acrosome integrity than that using a smaller volume of extender. Furthermore, collection into 5% BSA-containing extender exhibited higher sperm characteristics and maintained high sperm motility and progressive motility throughout incubation. In conclusion, semen collection into tubes with a large volume of extender, especially extender containing higher concentrations of BSA, improved the quality of ejaculated spermatozoa, strongly suggesting that the in vitro functional characteristics of the spermatozoa were abruptly modified by flash sperm contact with accessory sex gland fluid at ejaculation.     

Metformin blocks mitochondrial membrane potential and inhibits sperm motility in fresh and refrigerated boar spermatozoa

Metformin is clinically used to treat diabetes. Given its role‐impacting metabolism, metformin has been also added to semen cryopreservation media showing specie‐dependent effects. We aimed to investigate metformin effects in both fresh (38.5°C for 2, 24 hr) and refrigerated (17°C for 10 days) boar spermatozoa. Metformin (2 hr) does not affect fresh sperm viability, membrane lipid organization nor acrosome integrity. However, metformin (24 hr) blocks sperm ΔΨm and significantly reduces % motile spermatozoa (65%), % progressive spermatozoa (50%), % rapid (100%), velocities VCL (69%), VSL (86%), VAP (78%) and motility coefficients. Metformin‐including extender does not modify sperm viability, membrane lipid organization or acrosome integrity. Furthermore, it significantly reduces high ΔΨ‐population spermatozoa at refrigeration day 4. Metformin also significantly reduces sperm motility during refrigeration. Summarizing, metformin inhibits both boar sperm ΔΨ and motility in any sperm condition studied: fresh and refrigerated. These findings dissuade metformin as an additive to improve boar sperm quality.     

Influence of Macrophages on the Rooster Spermatozoa Quality

The goal of this study was to evaluate the occurrence of macrophages in rooster semen and to investigate their impact on the spermatozoa quality. Ross 308 breeder males (n = 30) with no evidence of genital tract infections were used to determine the concentration of macrophages using fluorescently conjugated acetylated low‐density lipoprotein (AcLDL). Subsequently, the roosters were divided into two groups on the basis of semen macrophage concentration, and semen quality was compared in two heterospermic samples. We applied computer‐assisted semen analysis (CASA) system to determine motility parameters. Fluorescence microscopy and flow cytometry were used to evaluate occurrence of apoptotic and dead spermatozoa. Spermatozoa fertility potential was examined after intravaginal artificial insemination of hens. Eighteen roosters (control group) contained 0.2–3% of macrophages within spermatozoa population and ten roosters (macrophage group) had 10–15% of macrophages. Males from macrophage group had lower (p < 0.05) motility parameters (total and progressive movement, velocity curved line) and increased concentration of dead spermatozoa detected by flow cytometry and fluorescence microscopy (p < 0.001 and p ? 0.05, respectively). Differences (p < 0.05) between fluorescent microscopy and flow cytometry in results on spermatozoa apoptosis and viability were observed. No significant difference was found between groups in fertility of spermatozoa. In conclusion, the higher presence of macrophages in rooster semen may have a negative effect on some parameters of rooster spermatozoa evaluated in vitro. Furthermore, our study suggests that flow cytometry allows more precise examination of spermatozoa viability and apoptosis in a very short time compared with the fluorescent microscopy.     

Single and double layer centrifugation improve the quality of cryopreserved bovine sperm from poor quality ejaculates

Background: Density gradient centrifugation was reported as a technique of semen preparation in assisted reproductive techniques in humans and animals. This technique was found to be efficient in improving semen quality after harmful techniques such as cryopreservation. Recently a modified technique, single layer centrifugation,was proposed as a technique providing a large amount of high quality spermatozoa, and this treatment was performed before conservation. Single layer centrifugation has been studied prevalently in stallions and in boars,but limited data were available for bulls. Occasionally bulls are known to experience a transient reduction in semen quality, thus techniques that allow improvement in semen quality could be applied in this context. The aim of this study was the evaluation of single layer and double layer centrifugation by the use of iodixanol, compared with conventional centrifugation and non-centrifuged semen, on the sperm characteristics during the cryopreservation process in bulls with normal and poor semen quality.Results: Single layer centrifugation and double layer centrifugation both significantly increased the percentage of normal spermatozoa and decreased the percentage of non-sperm cells in poor quality samples, while both were ineffective in those of normal quality. Sperm characteristics in poor quality samples increased after single layer centrifugation and double layer centrifugation, reaching values similar to those recorded in normal samples, and this trend is maintained after equilibration and after cryopreservation. On the other hand, SLC and DLC resulted in a consistent reduction in the spermatozoa recovered, and this resulted in a reduction of the absolute amount of spermatozoa cryopreserved in the normal samples, without a clear improvement in sperm characteristics in this type of sample.Conclusions: These data suggested that both SLC and DLC could be performed in practice, but their application should be limited to the cases in which the quality of the spermatozoa recovered is more important than the total amount of spermatozoa.     

Assessment of Different Sperm Quality Parameters to Predict in vitro Fertility of Bulls

Frozen‐thawed semen from six bulls with high (> 60%) and low (20–35%) in vitro fertility was used for studying the predictive value of simple sperm quality tests with respect to in vitro fertilization (IVF) outcome as assessed by pronucleus (PN) formation ability. Sperm quality parameters, such as sperm concentration, motility, progressive motility, live‐dead sperm ratio, morphology, membrane integrity, mitochondrial activity and acrosomal status were analysed using both conventional and automatic techniques at three time points during the IVF process, namely after sperm thawing, Percoll differential gradient centrifugation and IVF. Associations between the sperm quality parameters before and after IVF, and PN formation ability were assessed by using linear regression analyses. The percentages of motility, progressive motility and normal morphology determined after sperm thawing, and the percentage of live spermatozoa assessed after Percoll preparation by using nigrosin‐eosin (N‐E) staining showed a good correlation with PN formation ability, but the regression parameters were borderline not significant. These parameters formed the most reliable basis for predicting IVF outcome. After IVF, the percentage of live spermatozoa determined by using N‐E staining was the only sperm quality parameter showing a significant association with the PN formation ability of a given bull. This sperm quality test can be used as a non‐invasive method to estimate the PN formation ability of oocytes which are further cultured to assess embryonic development.     

First successful frozen semen of the maned wolf (Chrysocyon brachyurus)

This study aimed to describe successful cryopreservation of sperm from maned wolves (Chrysocyon brachyurus). Three ejaculates from 2 maned wolves were collected by digital manipulation of the penis and evaluated subjectively, centrifuged and frozen in BotuCrio^® (Botupharma, Botucatu, Brazil) or Tris–yolk egg extender. Spermatozoa were thawed at 37ºC/30s or 70ºC/4s and evaluated for kinetics, morphology, plasma and acrosome membrane integrity, mitochondrial potential, hydrogen peroxide, superoxide anion and lipid peroxidation. From 5 thawed samples, two had sperm total motility >55% (56.0% and 64.0%) and progressive motility ~35% (35% and 40%), both frozen with Tris–yolk egg. Plasma and acrosome membrane integrity decreased and percentage of sperm defects increased post-thawing. We concluded that is possible to freeze spermatozoa from maned wolves using semen collection and processing methods applied for domestic dogs.     

Effects of supplementation of iodixanol to semen extender on quality and fertilization ability of frozen–thawed Thai native bull sperm

This study investigates the effects of iodixanol supplementation in varied concentrations to Tris egg yolk (TEY) extender on the quality and fertilization ability of frozen–thawed sperm of Thai native bulls. Each ejaculate was divided into four different groups, as follows: sperm were treated with TEY extender (control group) and TEY extender supplemented with three different concentrations of iodixanol (1.25%, 2.50% and 5.00%). Semen straws were frozen in liquid nitrogen vapor. After thawing, sperm motility characteristics, viability, plasma membrane integrity and acrosome integrity were determined. Also, frozen–thawed spermatozoa from all groups were used for in vitro fertilization and artificial insemination (AI) in natural estrus Thai native cows. The results showed that the post‐thaw quality of the 2.50% iodixanol group was superior to the other iodixanol groups (P < 0.05). However, iodixanol had no beneficial effect on post‐thaw sperm in vitro fertilization ability and pregnancy rate after AI (P > 0.05). It can be concluded that the supplementation of 2.50% iodixanol extender significantly improves the progressive motility, viability, plasma membrane integrity and acrosome integrity of cryopreserved semen from Thai native bulls, but it has no beneficial effect on in vitro fertilization ability and pregnancy rate after AI.     

Characterization and cooled storage of semen from corn snakes (Elaphe guttata).

The phylogenetic order Squamata has many representatives that could benefit from the use of semen preservation as a tool for assisting conservation. To date, few studies have been made evaluating the potential for collecting and preserving semen from snakes. The objectives of this study were to characterize semen parameters of the corn snake (Elaphe guttata), including appearance, volume, concentration, sperm motility, and sperm morphology, and to determine the longevity of corn snake sperm motility stored at 4 degrees C. Single semen samples were collected from 22 adult corn snakes. The appearance of the corn snake semen was generally cloudy, and the color was white to tan. Corn snake spermatozoa initially exhibited a median motility of 92.5%. Corn snakes were found to produce small-volume ejaculates (median 0.01 ml). However, the overall concentration of the snake ejaculate was high (chi = 852 x 10(6) +/- 585 x 10(6) spermatozoa/ml). Morphologically, a mean of 75.7 +/- 9.3% of the sperm cells in an ejaculate were normal. Snake ejaculate with a white appearance had significantly higher sperm concentrations (chi = 1,859 x 10(6) +/- 1,008 x 106 sperm cells/ml; F = 15.74, P = 0.001) than tan ejaculates (chi = 601 x 10(6) +/- 439 x 106 sperm cells/ml). Sperm motility decreased significantly in samples that were stored at 4 degrees C for greater than 48 hr in a refrigerator or Equitainer I. This is the first study to characterize semen volume, appearance, and concentration; sperm motility; and sperm morphology in captive corn snakes. The information derived from this study can be used to develop a model for a collection, cooled storage, and shipping program for semen from endangered or threatened captive and wild snakes.     

Effect of caffeine on metabolic activity of ejaculated and epididymal spermatozoa of buffalo (Bubalus bubalis)

Split fractions of 25 ejaculated semen samples and spermatozoa from 5 caudae epididymides were used to study the effect of different levels of caffeine on the motility and fructolytic activity. During the first hour of incubation at 37°C, addition of caffeine to unwashed ejaculated buffalo sperm significantly increased the percentage of motility and amount of fructose utilized. In presence of 2–8 mM caffeine, sperm maintained their initial motility at least 2 hours at 37°C. Maximal stimulation of fructolytic activity was obtained with 2 mM whereas the minimal stimulation was found with higher concentration of 8–10 mM caffeine. Compared to ejaculated spermatozoa, epididymal sperm appeared to be more influenced by caffeine. Fructolytic activity was stimulated at least 1.5 times in the presence of 2 mM caffeine. Nearly during all incubation periods, the amounts of fructose utilized by caffeine-treated sperm were greater than in control samples. Data on epididymal sperm motility demonstrated that caffeine (2–10 mM) significantly stimulated and maintained the initial motility at least 3 hours at 37°C .     

Colloid centrifugation of boar semen

Colloid centrifugation of boar semen has been reported sporadically for at least the last two decades, beginning with density gradient centrifugation (DGC) and progressing more recently to single layer centrifugation (SLC). Single layer centrifugation through a species-specific colloid has been shown to be effective in selecting the best spermatozoa (spermatozoa with good motility and normal morphology) from boar sperm samples. The method is easier to use and less time-consuming than DGC and has been scaled-up to allow whole ejaculates from other species, e.g. stallions, to be processed in a practical manner. The SLC technique is described, and various scale-up versions are presented. The potential applications for SLC in boar semen preservation are as follows: to improve sperm quality in artificial insemination (AI) doses for 'problem' boars; to increase the shelf-life of normal stored sperm samples, either by processing the fresh semen before preparing AI doses or by processing the stored semen dose to extract the best spermatozoa; to remove pathogens (viruses, bacteria), thus improving biosecurity of semen doses and potentially reducing the use of antibiotics; to improve cryosurvival by removing dead and dying spermatozoa prior to cryopreservation; to select spermatozoa for in vitro fertilization. These applications are discussed and practical examples are provided. Finally, a few thoughts about the economic value of the technique to the boar semen industry are presented.     

Quality of Fresh,Cooled, and Frozen Semen From Stallions Supplemented with Antioxidants and Fatty Acids

This study assessed the effect of oral supplementation with the primary antioxidants and fatty acids involved in spermatogenesis (L-carnitine, selenium, vitamin E, omega-3, and omega-6) on the seminal quality in fresh, cooled, and frozen semen of stallions (n = 8), using a randomized design. The animals were divided into Group I (n = 4) and Group II (n = 4) for a 30-week experiment. The two groups alternated between nutraceutical supplementation and a placebo over the course of the experiment. Semen collections were performed in two sets: once in the middle of the experiment, before the two groups switched treatments, and once at the end. The volume, appearance, sperm concentration, spermatozoa kinetics, and membrane integrity of fresh semen were evaluated. The spermatozoa kinetics and membrane integrity of cooled (for 24, 36, and 48 hours) and frozen semen were also evaluated. No differences were observed in volume, appearance, and sperm concentration between treatment and control. However, compared with placebo, nutraceutical supplementation increased (P < .05) total motility, trajectory speed, as well as plasma and acrosomal membrane integrity in spermatozoa from fresh semen. In cooled semen, nutraceutical treatment also increased (P < .05) total motility, speed, and membrane integrity of spermatozoa compared with the control. In frozen semen, supplementation increased (P < .05) spermatozoa progressive motility and plasma membrane integrity. Our results suggest a positive, synergistic effect of the antioxidant L-carnitine and selenium on spermatozoa kinetics. Similarly, the increase in plasma and acrosomal membrane integrity could be attributed to higher concentrations of polyunsaturated fatty acids (a key cell-membrane component), combined with the prevention of excess lipid peroxidation by antioxidants. In conclusion, supplementation with nutraceuticals containing fatty acids and antioxidants improved the quality of fresh, cooled, and frozen stallion semen. Therefore, nutraceutical use should increase the success of artificial insemination with cooled and cryopreserved semen.     

Retained Functional Integrity of Bull Spermatozoa after Double Freezing and Thawing Using PureSperm® Density Gradient Centrifugation

The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88.2 +/- 6.2) and after re-freezing and thawing (83.6 +/- 6.56% NAR). However, flow cytometric assessment of the sperm membranes revealed a decline in the percentage of viable spermatozoa with intact membranes after the second freezing and thawing compared with after gradient centrifugation (76.3 +/- 6.0% vs 46.6 +/- 6.6%, p < 0.001) to levels equivalent to those obtained after the first round of freeze-thawing (53.4 +/- 11.7% viable acrosome-intact spermatozoa). Sperm movement characteristics assessed by computer-assisted analysis were unaffected in the population selected on the PureSperm gradients but declined after cooling of the selected and extended spermatozoa to 4 degrees C. There was no further change in these kinematic measurements after the cooled spermatozoa had undergone the second round of freeze-thawing. These results demonstrate that bull semen can be frozen and thawed, followed by a second freeze-thawing cycle of a population of spermatozoa selected by PureSperm, with retained motility and functional integrity. This points to the possibility of using double frozen spermatozoa in bovine artificial insemination programmes and to the potential benefits of PureSperm density gradient centrifugation for the application of cryopreserved bull spermatozoa to other biotechnological procedures such as flow cytometric sex sorting followed by re-freezing and thawing.     

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.     

Influence of season on the freezability of free-range poultry semen

The purpose of the present study was to examine the seasonal variation in freezing damage in free-range rooster sperm. Over a period of 1 year, heterospermic semen samples were collected weekly by massage from the roosters of 14 Spanish chicken breeds, all housed under natural photoperiod and climatic conditions. All samples were frozen in straws using DMA as a cryoprotectant, placing them first in nitrogen vapour and then plunging them into liquid nitrogen. No seasonal effects on fresh sperm quality were found. Neither did season affect the percentage of viable frozen-thawed spermatozoa nor the percentage with an intact acrosome. However, the collection season influenced (p < 0.05) most frozen-thawed sperm motility values. The percentage of immotile frozen-thawed spermatozoa was lower (p < 0.05) in spring-collected sperm than in summer- or autumn-collected samples. The percentage of spermatozoa showing progressive motility was higher in spring-collected sperm compared with winter-, summer- or autumn-collected samples (p < 0.05). The curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) values of spring-collected sperm were also higher (p < 0.05). In conclusion, spring would appear to be the best season for collecting and freezing the semen of free-range Mediterranean chicken breeds.     

Genetic Parameters for Semen Traits in AI Boars Estimated from Data on Individual Ejaculates

Semen volume, sperm concentration, percentage progressive motility, percentage of abnormal spermatozoa and total number of sperms from 215 830 ejaculates were analysed. The ejaculates were collected between 2000 and 2005 and originated from 3675 boars of different breeds and crossbred combinations from 23 AI centres of the Czech Republic. Genetic parameters were estimated using multiple- and single-trait animal models. Factors included in the models were: breed or crossbred combination, year–month, age class of the boar, interval between subsequent semen collections, joint effect of AI centre and year, permanent environment and additive genetic effect of the boar. The estimated heritabilities for semen volume and sperm concentration were approximately 0.20, whereas the estimates were somewhat lower for motility, percentage of abnormal sperm and total number of sperms. High negative genetic correlations were estimated between semen volume and sperm concentration (−0.68 for dam breeds, −0.69 for sire breeds) and between motility and percentage of abnormal sperm (−0.93 for dam breeds and −0.59 for sire breeds). The correlations between both semen volume or sperm concentration and motility or percentage of abnormal sperm were mostly small and negligible. Repeatabilities of 0.43–0.46, 0.37–0.38, 0.29–0.35, 0.42–0.50 and 0.29–0.30 were estimated for semen volume, sperm concentration, motility, percentage of abnormal sperm and the total number of sperms, respectively. On the basis of the estimated genetic parameters presented here, effective selection on sperm characteristics, especially for volume and concentration, should be possible using an animal model.     

Influence of the Freezing Technique (Nitrogen Liquid vs Ultrafreezer of −152°C) and Male-to-Male Variation Over the Semen Quality in Canarian Mastiff Breed Dogs

This study was carried out to assess the in vitro quality of canine semen frozen in an ultrafreezer at -152 degrees C and to evaluate the male-to-male variation of frozen semen in five male dogs of the Canarian Mastiff breed. Four ejaculates of each dog were processed individually (5% glycerol and 0.5% Equex) to reach a final concentration of 100 x 10(6) spermatozoa/ml. Then, two freezing techniques were tested to assess the seminal quality (sperm motility, live spermatozoa and abnormal sperm cell percentages) at 1, 30, 60, 120 and 360 days after freezing: (i) semen was frozen and stored in liquid nitrogen; (ii) semen was frozen and stored in the ultrafreezer at -152 degrees C. After freezing-thawing, both freezing protocols showed no significant differences in sperm motility and the percentages of live and abnormal spermatozoa. On the other hand, the microscopic characteristics of spermatozoa in fresh semen were practically similar among males; however, after the semen processing and freezing, significant differences were observed (p < 0.05) among males, especially as regards sperm motility. This inter-individual variability was detected in both freezing protocols, showing that the male-to-male variation in the seminal quality post-freezing was independent of the freezing technique used. The in vitro results obtained in the Canarian Mastiff breed confirmed that the use of ultra-freezers at -152 degrees C is a potential alternative to liquid nitrogen for storing canine semen for long periods of time.