Characteristics of Buck Semen with Regard to Ejaculate Numbers, Collection Intervals, Diluents and Preservation Periods

To determine the number of ejaculates which can be collected within a 20‐min period after the smallest number of days of sexual rest, and a good diluent to preserve semen for routine AI, five mature Black Bengal bucks were used in three experiments. In experiment 1, semen from the bucks were collected by using artificial vagina at homosexual mounts as many times as possible during 20 min. The ejaculate numbers 1, 3 and 4 (or 5 when the buck could produce it) were examined for important semen characteristics. The mean ejaculate volume, density, mass activity, sperm motility, sperm concentrations, total spermatozoa/ejaculate, proportion of spermatozoa with normal acrosome, midpiece and tail, and the proportion with normal head morphology varied between 267 and 342 µl, 4.1–4.5 (1–5 scale), 4.1–4.2 (1–5 scale), 77–79%, 4187 × 10⁶–5064 × 10⁶/ml, 1140 × 10⁶–1746 × 10⁶, 91–94% and 99%, respectively, depending on the collection number of the ejaculate. The difference between the ejaculates was significant only with respect to volume (p < 0.05). In experiment 2, semen was collected from the bucks successively during 20 min after 1, 2, 3 and 4 day intervals, and the first ejaculates were evaluated for the above‐mentioned semen characteristics. Semen collected after 2 or more day intervals had significantly higher volume, sperm concentration and total spermatozoa/ejaculate (p < 0.05). In experiment 3, pools of two to three ejaculates were diluted (1 : 5; semen : diluent) in splits with glucose‐citrate‐egg yolk (GCEY), Tris‐fructose‐egg yolk (TFEY) or skim milk (SM) and preserved at +4 to +7°C. Before chilling or after 0 (15 min chilling), 1, 2, 3 and 4 days of preservation, semen was evaluated for motility and proportion of normal spermatozoa with respect to acrosome, midpiece and tail. In data pooled across the bucks, the sperm motility was better in GCEY and TFEY than in SM, and the proportion of normal spermatozoa was higher in SM than in the others (p < 0.05). However, the differences in proportion of normal spermatozoa between diluents were not significant when the data were analysed separately within preservation periods. The sperm motility consistently dropped after 1 day of preservation (p < 0.01); the motility remained 50% or more up to 4 days in TFEY, 3 days in GCEY and only 2 days in SM. The proportion of spermatozoa with normal acrosome, midpiece and tail, which was generally quite high ( 90%), decreased after 3 days of preservation (p < 0.01). We conclude that Black Bengal bucks can be collected three times during 20 min, every 3 days, and that buck semen holds good motility and proportion of normal spermatozoa up to 3 days in GCEY or TFEY at 4 to 7°C.     

Assessment of chosen semen characteristics of two colour morphs of the Arctic fox Alopex lagopus L.

The aim of the study was to evaluate semen quality in the two most popular colour morphs of the Arctic fox Alopex lagopus L., blue and white, based on ejaculate parameters, acrosin activity and analysis of sperm morphology. The research material consisted of ejaculates collected once by manual stimulation from 20 one‐year‐old male Arctic foxes (10 individuals of the blue morph and 10 of the white morph). Ejaculates were evaluated in terms of volume, sperm concentration, total number of spermatozoa and the percentage of spermatozoa with major and minor defects. The study revealed that male blue Arctic foxes produce ejaculates with much higher concentration (148.75 × 10⁶/ml) and total number of spermatozoa (98.16 × 10⁶) compared to white Arctic foxes (42.88 × 10⁶/ml and 35.2 × 10⁶ respectively). The level of acrosin activity from white foxes seemed to be higher compared to blue foxes but the difference was not statistically confirmed. Semen from Arctic foxes is characterized by high inter‐individual variability in sperm morphology. The frequency of morphological changes in sperm from Arctic foxes does not significantly depend on ejaculate volume, sperm concentration or the total number of spermatozoa in the ejaculate, but is associated with acrosin activity.     

Single layer centrifugation of stallion spermatozoa consistently selects the most robust spermatozoa from the rest of the ejaculate in a large sample size

Reasons for performing study: An improvement in sperm quality after single layer centrifugation (SLC) has been seen in previous studies using small sample sizes (for example, n = 10 stallions). There is a need to investigate whether this improvement is repeatable over several breeding seasons with a larger number of stallions (n ≥ 30 stallions). Objective: To make a retrospective analysis of the results of SLC performed on more than 250 sperm samples (176 ejaculates) from 31 stallions in 3 consecutive breeding seasons. Methods: Sperm quality (motility, proportion of morphologically normal spermatozoa and the proportion of spermatozoa with undamaged chromatin) was assessed before and after SLC. Results: All parameters of sperm quality examined were significantly better in sperm samples after SLC than in their unselected counterparts (P<0.001 for each parameter). The yield of spermatozoa obtained after SLC was influenced by the type of extender used and also by the concentration of spermatozoa in the original ejaculate, with fewer spermatozoa being recovered when the loading dose contained a high concentration of spermatozoa. The optimal concentration was approximately 100 × 10⁶/ml. Sperm concentration in the samples loaded on to the colloid influenced the sperm yield while the type of semen extender affected sperm quality and survival. Furthermore, the scaled‐up SLC method was found to be suitable for use with a range of ejaculates, with similar sperm kinematics being observed for standard and scaled‐up preparations. Conclusions: SLC consistently improved the quality of stallion sperm samples from a large number of ejaculates. The method could be scaled‐up, allowing larger volumes of ejaculate to be processed easily from a wide range of stallions.     

Effect of Collection Rhythms and Season on Rabbit Semen Production

The effects of collection regimen and time of year on rabbit semen production were determined in this study. A total of 14 crossbreed Hyla bucks were used in winter and summer. In each season, rabbits were assigned to two groups. In group 1, (n = 7) rabbits were subjected to an extensive collection regimen (two ejaculates per male, once daily/week) and in group 2, (n = 7) a semi‐intensive semen collection regimen was performed (two ejaculates per male, twice weekly). The traits recorded for each sample were libido, volume, pH, motility, sperm concentration, percentage of alive spermatozoa and sperm abnormalities. The results obtained in this study indicate that when increasing collection frequency, the rate of useful collections decreased (from 0.81 ± 0.017 to 0.69 ± 0.016; p < 0.01). The rate of useful collection also decreased in the transition from winter to summer (from 0.79 ± 0.018 to 0.70 ± 0.017; p < 0.01). Among the ejaculate characteristics studied, only volume/ejaculate (from 0.64 ± 0.015 to 0.53 ± 0.017; p < 0.01) and spermatozoa/ml (from 406 ± 15 to 359 ± 13 million; p < 0.01) appeared negatively affected by collection. In winter fewer volume/ejaculates were produced (0.55 ± 0.015 vs 0.60 ± 0.016 ml; p < 0.01) and fewer spermatozoa/ml (360 ± 14 vs 394 ± 16 million; p < 0.01) than in summer. The doses produced per ejaculate decreased as collection frequency increased, but the number of doses produced per week was higher in the semi‐intensive than the extensive rhythm (26.5 ± 2.1 vs 20.9 ± 1.5; p < 0.01). The results suggest that a semi‐intensive rhythm may be viewed favourably.     

The Effect of Glycerol Concentrations on the Post‐thaw In Vitro Characteristics of Cryopreserved Sex‐sorted Boar Spermatozoa

The objective of this study was to optimize protocols for the cryopreservation of sex‐sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex‐sorted sperm frozen at low sperm concentrations (20 × 10⁶ sperm/ml; S20 group). Non‐sorted spermatozoa frozen at 1000 × 10⁶ (C1000 group) and 20 × 10⁶ (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post‐thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non‐sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post‐thaw motility of sex‐sorted spermatozoa frozen at low concentrations.     

Bestimmung der osmotischen Resistenz von Eberspermien und deren Beziehungen zur Konservierungsfähigkeit von Samenproben

Contents Determination of the osmotic resistance of boar spermatozoa and the relations to sperm freezing and long storage The osmotic behaviour of fresh boar spermatozoa (482 ejaculates from 34 A.I. boars) was examined in a standard medium but with different osmotic pressures ranging from 150 mosm/kg till 500 mosm/kg. The percentage of spermatozoa with normal acrosomal ridge (NAR) was determined after incubation of 0.2 ml fresh semen in 3 ml of the osmotic test solutions at 39° C for 15 and 120 min. In the non-isotonic media % NAR decreased and could be quantified depending to incubation time and to the different osmolalities; NAR decreased strongest at 150 or 500 mosm/kg and after 120 min incubation time. Differences in % NAR were large between ejaculates and between boars but low within boars and statistically significant correlations had been found to % NAR and motility in froren/thawed semen samples or stored samples. Still higher correlations were found by calculation of an osmotic resistance test-value (ORT-value) which is defined as follows: ORT = 1/2 % NAR in isotonic medium/(after 15 min) +% NAR in 150 or 500 mosm/kg/(after 15 or 120 min) ORT-values can be calculated for hypotonic (150 mosm/kg) as well as for hypertonic (500 mosm/kg) test solutions and for 15 and 120 minutes incubation time. The hypotonic ORT-value for 120 min. is the most accurate; the values varied from 34 till 83 between ejaculates and from 44 till 76 between boars with a low variability within boars. Grouping of boars by their ORT-values in “good”, “moderate” and “poor” corresponded to a similar grouping of these boars by their semen quality (% NAR and motility) in preserved semen samples. Also a grouping of single ejaculates by their ORT-values corresponded to a similar qualitative classification of these ejaculates. ORT seems to become a good and practicable assessment test for the selection of boars suitable dor deep freezing or long storage of their spermatozoa. On the other hand preservation techniques too can be examined by ORT. But more investigations with this test must be done especially its relation to the fertilization capacity of semen samples with different ORT-values.     

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.     

The ostrich (Struthio camelus) ejaculate--effects of the method of collection, male age, month of the season, and daily frequency

1. Over three breeding seasons on a farm in Poland, semen was collected from 11 ostriches using the dummy and the teaser method to study the effects of the method of collection, male age, month in the breeding season, and daily collection frequency on ejaculate characteristics.

2. A total of 259 ejaculates were collected, with an average volume of 1·28?±?0·6 (±SEM) ml. Sperm concentration was 3·34?±?0·08?×?10⁹/ml, the total number of spermatozoa 4·32?±?0·22?×?10⁹, and motility 4·56?±?0·04.

3. There was no difference in ejaculates collected by the dummy and teaser methods, but the between-individual variation was considerable. Ejaculate characteristics increased with male age and varied between months, with little evidence for seasonal decline. Daily collections for 10 days did not affect sperm output.

4. The results open up avenues for further research on development of a viable protocol for artificial insemination in ostriches and efficient semen storage.

5. The between-male variation suggests that the ejaculate output could be maximized through selection.     

The Effect of Sperm Concentration and Storage Vessel on Quercetin‐Supplemented Rabbit Semen During Chilled Storage

Extending the shelf life of chilled rabbit spermatozoa is vital for the expansion of the farmed rabbit industry. This study evaluated the relationship between sperm concentration and packaging on in vitro quality of chilled rabbit semen over 96 h. Semen was collected from adult bucks (n = 4) and pooled at 37°C following evaluation. Pooled ejaculates were diluted with a Tris‐based extender supplemented with 100 μm quercetin to a concentration of 15, 30 or 60 × 10⁶ spermatozoa/ml, packaged into plastic tubes or 0.5‐ml straws and stored at 15°C. Sperm quality was assessed by computer‐assisted sperm Analysis [total motility (tMOT)] and flow cytometry [viability, acrosome integrity, H₂O₂ production, plasma membrane disorder, apoptosis and DNA fragmentation index (DFI)] at 0, 48, 72 and 96 h. From 48 h, concentrations of 30 and 60 × 10⁶ spermatozoa/ml reported the highest tMOT, irrespective of storage vessel (p < 0.05). Storage in straws reduced oxidative stress and improved plasma membrane stability. The %DFI, mean DFI and SD‐DFI were increased in spermatozoa stored in tubes compared with straws (p < 0.05). Although the use of low sperm concentrations in artificial insemination doses would facilitate greater dispersion of genetically superior rabbit bucks, dilution to 15 × 10⁶ spermatozoa/ml had a detrimental impact on motility. As such, chilled storage at 30 × 10⁶ spermatozoa/ml may provide a suitable balance between motility and H₂O₂ production to best maintain overall sperm function and should be evaluated in a large‐scale AI trial.     

Sperm Production and Sperm Morphology of Swedish Warmblood Stallions

The present study investigated daily sperm output and sperm morphology of fresh semen in eight Swedish Warmblood stallions aged 5–8 years. They were used for artificial insemination, and their fertility during the breeding season of semen collection exceeded 60% per cycle. One ejaculate of semen was collected daily for 10 consecutive days from each stallion. The gel-free volume was measured, and the sperm concentration was assessed with a Bürker chamber. The volume of gel-free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). Sperm morphology was examined in ejaculates collected on days 2, 5 and 10. An aliquot from each ejaculate was fixed in 1 ml formol–saline immediately after collection and examined under a phase-contrast microscope (magnification 1000×) to assess morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin–eosin) for a more detailed examination of the sperm heads under a light microscope (magnification 1000×). Analysis of variance was applied to data. Total spermatozoa number decreased progressively during the first 8 days of collection, and daily sperm output (DSO) was calculated as mean TSN of collections on days 8–10, being 6.4 × 10⁹ spermatozoa. The overall percentages of morphologically normal spermatozoa in ejaculates collected on days 2, 5 and 10 were above 70%, being significantly lower in ejaculate 2 (68.6%) compared with ejaculates 5 and 10 (72.9% respectively 75.3%).     

Effect of Ram Age on Structural and Functional Competence of Frozen–Thawed Spermatozoa in Dairy Sheep

The objectives of this study were to investigate the influence of ram age on structural and functional competence of frozen–thawed spermatozoa and to test the hypothesis that increasing number of sperm bound to the zona pellucida in vitro was associated with decreasing in vivo fertility of frozen semen. Rams were allocated into two groups. Each group consisted of five rams aged either 1–2 years (young) or 4–5 years (mature). Three successive ejaculates were collected from each ram using an artificial vagina. Only ejaculates of ≥ 2.5 × 10⁹ sperm/ml and 80% sperm progressive motility were pooled per ram, diluted with Bioxcell^® medium and frozen in 0.25 ml straws. The end points of post‐thawing semen evaluation were computer‐assisted cell motility analysis, sperm capacitation (chlortetracycline assay), simultaneous assessment of plasma membrane integrity, mitochondrial membrane potential and condensation status of nucleus, per‐cell analysis of lipid peroxidation using C11‐BODIPY^581/591, sperm‐hemizona binding (HZB) ability and sperm fertility after laparoscopic insemination of ewes (n = 114) in the progestagen‐synchronized oestrus. The results showed that mature rams had significantly lower values of sperm hyperactivated motility and peroxidized sperm, higher percentages of live non‐capacitated sperm and sperm cells with intact plasma membrane, functional mitochondria and condensed chromatin, as well as, greater lambing rate and ewe prolificacy. Sperm HZB binding ability was higher (p < 0.05) for young than for mature rams. Significant correlations were found between number of spermatozoa bound to the zona pellucida and semen fertility (r = ?0.63 to ?0.71). In conclusion, mature rams have better semen quality and in vivo fertility than young rams. Cryocapacitation can be involved in decreasing ram semen fertility as evidenced by the high number of spermatozoa bound to the zona pellucida in vitro.     

Use of the Sperm Quality Analyzer (SQA II‐C) for the Assessment of Dog Sperm Quality

In the present study, an automated system for sperm analysis, the Sperm Quality Analyzer (SQA II‐C), was tested as a potential tool for the assessment of dog sperm quality. In the first experiment the device displayed a good repeatability of measurements for semen of medium and high quality, as evidenced by a low coefficient of variance (CV; 0.08), whereas a high CV (0.46) was obtained for one dog with semen of inferior quality. In the second experiment, seven different sperm concentrations (25–300 ×10⁶/ml), obtained by dilutions in Hepes‐TALP medium were stored for 48 h at room temperature. A concentration dependent increase in sperm motility index (SMI) was shown, reaching a plateau at 150×10⁶ spermatozoa/ml. For all sperm concentrations, the SMI value decreased significantly after 24 h, indicating the importance of sperm motility for SMI values. For sperm concentrations lower than 150×10⁶/ml, highly significant correlations [r=0.80;p<0.05] were established between SMI values on one hand and sperm concentration, and semen parameters expressing the overall semen sample quality on the other hand (experiment 3) while non‐significant or low correlations were found between SMI values and other individual sperm parameters. In experiment 4, significantly high correlations (r=0.97) were found between mean SMI values and post‐thaw motility and progressive motility assessed subjectively. In conclusion, our study indicates that both motility and concentration largely influence SMI values and that the SQA II‐C saturates at 150×10⁶ fresh spermatozoa/ml. In our opinion, the SQA II‐C may be a useful and objective device to assess the post‐thaw motility of dog sperm.     

Fractions of the semen of red deer (Cervus elaphus)--their occurrence and characteristics in different periods of season

During a complete season 145 ejaculates were collected from 4 red deer stags (Cervus elaphus) using a modified artificial vagina. Differences found in characteristics of ejaculates allowed to identify four different types of ejaculates: pre-mating, mating, transitory, and post-mating ejaculate. These types of ejaculates occurred exclusively during corresponding periods of the season in the sequence mentioned above. Three fractions of ejaculates were identified: grey, white, and yellow. Pre- and post-mating ejaculates were homogenous and grey. The mating- and transitory ejaculate consisted of two fractions; the mating ejaculate consisted exclusively of white and yellow fractions and the transitory one contained exclusively the white and grey fraction. Respective fractions of ejaculates differed also in consistency. Pre- and post-mating ejaculates as well as the grey fraction of the transitory ejaculate were watery, the white fraction of the mating and transitory ejaculate were milky and the yellow fraction was honey-like. All respective fractions of ejaculates contained spermatozoa, except the yellow fraction. Volume, sperm concentration and pH of the respective fractions of ejaculates were examined. The yellow fraction had the largest volume and contributed most to differences between the ejaculates as concerns the volume. Sperm concentration was highest in the white fraction of the mating and the transitory ejaculate. The objective of this report was to confirm the previous findings about occurrence of distinguishable fractions and to describe their characteristics in respect to different periods of the season.     

Factors influencing boar sperm cryosurvival

Optimal sperm cryopreservation is a prerequisite for the sustainable commercial application of frozen-thawed boar semen for AI. Three experiments were performed to identify factors influencing variability of postthaw sperm survival among 464 boar ejaculates. Sperm-rich ejaculate fractions were cryopre-served using a standard freezing-thawing procedure for 0.5-mL plastic straws and computer-controlled freezing equipment. Postthaw sperm motility (assessed with a computer-assisted semen analysis system) and viability (simultaneously probed by flow cytometry analysis after triple-fluorescent stain), evaluated 30 and 150 min postthaw, were used to estimate the success of cryopreservation. In the first experiment, 168 unselected ejaculates (1 ejaculate/boar), from boars of 6 breeds with a wide age range (8 to 48 mo), were cryopreserved over a 12-mo period to evaluate the predictive value of boar (breed and age), semen collection, transport variables (season of ejaculate collection, interval between collections, and ejaculate temperature exposure), initial semen traits, and sperm quality before freezing on sperm survival after freezing-thawing. In Exp. 2, 4 ejaculates from each of 29 boars, preselected according to their initial semen traits and sperm quality before freezing, were collected and frozen over a 6-mo period to evaluate the influence of interboar and intraboar ejaculate variability in the survival of sperm after cryopreservation. In Exp. 3, 12 ejaculates preselected as for Exp. 2, from each of 15 boars with known good sperm cryosurvival, were collected and frozen over a 12-mo period to estimate the sustainability of sperm cryosurvival between ejaculates over time. Boar and semen collection and transport variables were not predictive of sperm cryosurvival among ejaculates. Initial semen traits and sperm quality variables observed before freezing explained 23.2 and 10.9%, respectively, of the variation in postthaw sperm motility and viability. However, more that 70% of total variance observed in postthaw sperm quality variables among ejaculates was explained by boar. This indicates that boar is the most important (P < 0.001) factor explaining the variability among ejaculates in sperm cryosurvival, with most (14 of the 15 boars in Exp. 3) showing consistent (P > 0.05) sperm cryosurvival over time.     

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.     

In Vitro Evaluation of Liquid‐stored Buffalo Semen at 5°C Diluted in Soya Lecithin Based Extender (Bioxcell®), Tris‐Citric Egg Yolk,Skim Milk and Egg Yolk‐Citrate Extenders

This study was designed to compare the quality of liquid‐stored buffalo bull spermatozoa in soya lecithin based extender Bioxcell^® (BIOX), milk (MILK), tris‐citric egg yolk (TEY) and egg yolk‐citrate (EYC) extender at 5°C. Semen was collected from five Nili‐Ravi buffalo (Bubalus bubalis) bulls of 6–7 years of age with artificial vagina over a period of 3 weeks (two consecutive ejaculates once in a week). Semen ejaculates having more than 60% motility were pooled, split into four aliquots, diluted (37°C; 10 × 10⁶ motile spermatozoa/ml), cooled from 37 to 5°C in 2 h (0.275°C/min) and stored for 5 days. Sperm motility, viability, plasma membrane integrity (PMI) and normal acrosomal ridge were studied at first, third and fifth day of storage. Higher values of progressive sperm motility (%), sperm viability (%), sperm PMI (%) and normal apical ridge (%) were observed in BIOX, MILK and TEY extenders at first, third and fifth day of storage than EYC extender. Progressive sperm motility, sperm viability and sperm PMI in BIOX^® extender were not different from MILK and TEY extenders at 1st and third day storage period. However, at fifth day of storage, the values for these parameters remained significantly higher (p < 0.05) in BIOX^® compared with MILK, TEY and EYC extenders. At fifth day of storage, the semen quality parameters for Bioxcell^® were comparable to those with MILK and TEY extenders at third day of storage. In conclusion, motility, viability and PMI of buffalo bull spermatozoa remained similar in Bioxcell^®, milk and TEY extender at first and third days of storage at 5°C. Yet, the values for the aforementioned parameters in Bioxcell^® were higher compared with milk, TEY and EYC extender at fifth day of storage at 5°C.     

Seminal characteristics of two- and three-year-old quarter horse stallions

Seminal and testicular characteristics were compared in 19 three-year-old and 23 two-year-old Quarter Horse stallions. Semen was collected, and testicular evaluations made on all stallions once in April or May and again 60 days later. Semen was collected from stallions twice, one hour apart, at each evaluation. Average testicular tone and scrotal width were greater in three-year-olds than in two-year-olds. Ejaculates of three-year-olds had higher motility scores (66% vs 47%) sperm movement (3.2 vs 2.4), normality (77 vs 71), and total number of normal, motile spermatozoa per ejaculate (4.3 × 10⁹ vs 2.2 × 10⁹). Two-year-olds had twice as many cytoplasmic droplets (11% vs 5%) as three-year-olds. Average sperm concentrations per ml of gel-free semen and gel-free volume of ejaculates were not different. Volume of ejaculate and motility of spermatozoa were positively correlated with pregnancy rate, whereas percent cytoplasmic droplets was negatively correlated with pregnancy rate in both groups of stallions. Pre- and post-ejaculatory urethral, seminal, and preputial cultures were examined for pathologic organisms on all stallions. Eighteen of 42 had positive cultures for Klebsiella spp., Pseudomonas spp., β-Strep spp., or E. coli. Nine two-year-olds and 3 three-year-olds had positive cultures for Klebsiella spp. All but one of these stallions were housed on wood shavings and allowed limited exercise. Three-year-old stallions had superior seminal quality as compared with two-year-olds.     

Semen quality after vasectomy in the ramQualité du sperme du bélier vasectomiséde]Samenqualität nach vasektomie beim schafbock

Twenty four yearling rams which were trained for use with the artificial vagina were used to study changes in the volume, motility and density of ejaculates collected between 3 h and 6 days after vasectomy. Control rams underwent sham operations. On the day of vasectomy only, there was a decrease in the speed of ejaculation and an increase in sniffing and pawing activity.Ejaculate volume was unaffected by sham operation, but declined by 40–50% to 0.23–0.62 ml after vasectomy (P < 0.05).Irrespective of the timing of semen collections, motility declined to zero by the third ejaculate after vasectomy and density at that point had fallen to less than 0.1 × 10⁹ sperm/ml (less than 7% of pre-vasectomy levels).It is concluded that rams should be ejaculated at least 3 times after vasectomy if semen quality is to be reduced to the low levels normally associated with sterility, and that if rams are required for immediate use this may be achieved on the day of vasectomy.     

Effects of breed and ejaculate volume on sperm morphology and semen parameters of boars

The aim of the study was to determine the relation between the semen quality, frequency of sperm defects, sperm dimensions and shape, and the ejaculate volume of Large White and Landrace boars. A total of 648 ejaculates collected from 31 Large White and 30 Landrace boars were divided into three groups according to the criterion of the ejaculate volume. In this study Landrace boars produced ejaculates with higher volume, sperm concentration, and total numbers of spermatozoa than Large White boars. Landrace boars also showed a lower frequency of sperm with morphological abnormalities (P < 0.05). Landrace boars sperm had larger heads, which were by 0.15 μm longer, and by a larger perimeter and area (P < 0.05). Landrace boar spermatozoa also had a longer flagellum and were generally larger and by 2.07 μm longer than Large White boar sperm (P < 0.05). Significant differences were also found in the shape of sperm of the two breeds (P < 0.05). Landrace boars sperm had more elongated heads, and the ratio of head size to flagellum length was lower than in Large White boars sperm (P < 0.05). Sperm from ejaculates with low volume had a shorter flagellum and a greater head length/flagellum length ratio than sperm from medium- and high-volume ejaculates (P < 0.05).     

The benefits of cooling boar semen in long‐term extenders prior to cryopreservation on sperm quality characteristics

This study investigated the effects of long‐term extenders on post‐thaw sperm quality characteristics following different holding times (HT) of boar semen at 17 and 10°C. Sperm‐rich fractions, collected from five boars, were diluted in Androhep^® Plus (AHP), Androstar^® Plus (ASP), Safecell^® Plus and TRIXcell^® Plus (TCP) extenders. The extended semen samples were held for 2 hr at 17°C (HT 1) and additionally for 24 hr at 10°C (HT 2), after they were evaluated and frozen. CASA sperm motility and motion patterns, mitochondrial membrane potential (MMP), plasma membrane integrity (PMI) and normal apical ridge (NAR) acrosome integrity were assessed in the pre‐freeze and frozen‐thawed semen. The Vybrant Apoptosis Assay Kit was used to analyse the proportions of viable and plasma membrane apoptotic‐like changes in spermatozoa. Results indicated that boar variability, extender and HT significantly affected the sperm quality characteristics, particularly after freezing‐thawing. Differences in the pre‐freeze semen were more marked in the sperm motion patterns between the HTs. Pre‐freeze semen in HT 2 showed significantly higher VCL and VAP, whereas no marked effects were observed in the sperm membrane integrity and viability (YO‐PRO‐1^?/PI^?) among the extenders. Post‐thaw sperm TMOT and PMOT were significantly higher in the AHP and ASP extenders of HT 2 group, whereas VSL, VCL and VAP were markedly lower in the TCP extender. Furthermore, spermatozoa from the AHP‐ and ASP‐extended semen of HT 2 group were characterized by higher MMP, PMI and NAR acrosome integrity following freezing‐thawing. In most of the extenders, the incidence of frozen‐thawed spermatozoa with apoptotic‐like changes was greater in HT 1. The findings of this study indicate that holding of boar semen at 10°C for 24 hr in long‐term preservation extenders modulates post‐thaw sperm quality characteristics in an extender‐dependent manner. These results will further contribute to the improvement in the cryopreservation technology of boar semen.