Poor Prediction Value of Sperm Head Morphometry for Fertility and Litter Size in Rabbit

This study was conducted to investigate the predictive capacity of fertility and litter size of sperm head morphometric measurements when the ejaculates fulfilled the minimum requirements commonly used in artificial insemination (AI). Semen samples from 11 rabbits (77 ejaculates) were evaluated for sperm motility, abnormal spermatozoa and sperm head morphometry using computer automated sperm analysis system. Morphometric dimensions for length, width, area and perimeter were analysed. Only ejaculates with more than 70% of motility rate and <15% of abnormal sperm were used for AI. A total of 1031 individual AI were performed in commercial rabbitries. Our results showed significant differences among animals for all sperm head measurements. The mean values for fertility and litter size obtained were 68.4 ± 0.01% and 9.3 ± 0.1% respectively. To assess the predictive value of morphometric dimensions in fertility, a logistic regression analysis was applied. Moreover, multiple linear regression analyses were used to examine the relationship between litter size and sperm head morphometric parameters. Logistic regression analysis rendered a significant model between fertility and area and perimeter, explaining the 0.65% variation. Multiple linear regression analysis rendered a significant model between litter size and width, area and perimeter that explained the 1.3% variation. By conclusion, the sperm head morphometric parameters assay showed low potential to predict fertility and litter size when the ejaculates fulfilled the minimum requirements commonly used in AI (motility and abnormal spermatozoa) in rabbit.     

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%).     

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).     

Influence of Random Factors and Two Different Staining Procedures on Computer-assisted Sperm Head Morphometry in Bulls

Contents Computer-assisted sperm analysis has the potential to improve reproducibility and objectivity in the assessment of sperm morphology. The aim of this study was to evaluate the use of a computer-assisted sperm morphometry analysis system for the determination of sperm head dimensions in bulls. Two experiments were performed to determine the variability caused by random factors and the influence of two different staining procedures. In the first experiment, three ejaculates were collected from each of five clinically healthy bulls. Air-dried semen smears were stained using a modified Farelly staining. The slides were observed via bright field microscopy with green filter using a 100× oil immersion objective. A video camera attached to the microscope transmitted images to a personal computer. Each sperm head was identified and analysed by the computer software (Morphology Analyzer V. 1.5; Mika Medical GmbH, Ismaning, Germany). Area, length and width of each sperm head were calculated and stored in a database for further statistical analysis. A minimum of 100 sperm heads were evaluated per slide. In experiment 2, the influence of two different staining procedures (Farelly and Papanicolaou) on sperm head dimensions was determined. The mean spermatozoal head measurements across all slides for area, length and width were 40.49 μm², 9.70 μm and 5.30 μm, respectively. On the basis of these results, the variability between slides, ejaculates and bulls using variance component estimation was calculated. All random factors (bull, ejaculate and slide) had a significant effect (p < 0.001) on sperm head dimensions. However, the variability attributable to bull (18.89–51.72%) was considerably higher compared with that of slide and ejaculate (0.17–5.27%). Additionally, differences existed between bulls concerning the shape and normality of histograms of their sperm head dimensions. The minimum number of spermatozoa required for analysis of sperm head dimensions was found to be about 60 spermatozoa per sample. The use of Papanicolaou stain resulted in significantly smaller sperm head dimensions, e.g. sperm head area 31.48 versus 38.35 μm² (p < 0.001). In conclusion, computer-assisted sperm head morphometry provides an objective, precise and reproducible tool. Comparisons of results from different studies should consider the influence of random and experimental factors to avoid misinterpretation.     

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.     

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 collection frequency on quantitative and qualitative characteristics of pigeon (Columba livia) semen

The aim was to estimate the optimal frequency of semen collection from pigeons in relation to ejaculate volume, sperm concentration, total spermatozoa in ejaculate and percentage of live morphologically normal cells. The study was carried out on 455 ejaculates collected from two groups of pigeons, each of 10 males (group I: meat-type breed; group II: fancy pigeon). The birds were selected and kept individually in cages under a natural photoperiod. A two-person technique was used for semen collection (lumbo-sacral and cloacal region massage). Semen was collected once, twice or three times per week. Colour, consistency and volume of ejaculates were evaluated macroscopically immediately after collection. Sperm concentration and total number of cells in the ejaculate were estimated after dilution with Ringer's solution. A live-dead stain technique (nigrosin-eosin) was used to determine the percentage of live and normal spermatozoa. Semen collected 3x/week was of high quality. The average volume of a single ejaculate was small (21 microl in group I and 19 microl in group II), but sperm concentration was high--1.58 x 10(9)/ml and 1.96 x 10(9)/ml, respectively. The mean number of spermatozoa per ejaculate was 30.48 x 10(6) in group I and 39.49 x 10(6) in group II. An increased percentage of live and normal spermatozoa in semen collected more frequently was also observed. Collecting pigeon semen 3x/week provides spermatozoa in larger amounts and of better quality than less frequent collections (1x/week or 2x/week) and is recommended for obtaining more insemination doses.     

Some aspects of reproductive function in the male Beagle at puberty

Three male Beagle litter-mates, which were kept in the same environment and fed on the same diet, were studied from 20 to 45 weeks of age. The first ejaculates were obtained at 33 weeks in one and 34 weeks in two of the puppies. Ejaculatory volume, sperm concentration, libido increased quickly and although the early ejaculates contained large numbers of abnormal spermatozoa they soon decreased. By 45 weeks of age the seminal characteristics were comparable with those reported for adults. Plasma testosterone concentrations increased at the time that the first ejaculate was obtained.     

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.     

Evaluation of sperm morphometry of rabbits (Oryctolagus cuniculus f. domesticus)

Sperm morphology and morphometry are considered parameters in fertility diagnosis. They are especially important in the case of species for which there is no standard with respect to morphometric sperm parameters. It is then crucial to apply the staining technique that has the least influence on the sperm structure and provides the most detailed image, so as to enable measurements. The aim of the research was to assess the morphometric parameters of rabbit sperm using silver nitrate staining. The staining process revealed a detailed image of the spermatozoon head and tail, thus enabling precise measurements. From these basic morphometric parameters, four additional shape indices characterizing the sperm head were calculated: ellipticity, elongation, roughness and regularity. These parameters more precisely characterize the shape of the sperm head. Silver nitrate staining can be used as an independent technique in assessment of sperm structure or to supplement routine diagnostics.     

Influence of Sample Preparation, Staining Procedure and Analysis Conditions on Bull Sperm Head Morphometry using the Morphology Analyser Integrated Visual Optical System

The importance of standardizing the procedures of sample and slide preparation for computer‐assisted morphologic analysis has been emphasized in human and veterinary andrology. The purpose of this study was to optimize slide preparation (dilution grade and sperm washing), staining procedures and analysis conditions (colour of light source and objective magnification) for the morphometric analysis of bull spermatozoa using the Hamilton Thorne morphology analyzer integrated visual optical system (IVOS). For experiment 1, one ejaculate was collected from one bull and diluted to 200 000–300 000 spermatozoa/μl. Slides were prepared and stained using seven different procedures: rapid Papanicolaou (PAP), rapid Papanicolaou with prolonged staining times (PAP+), Diff‐Quik (DIF), haematoxylin (HEM), Farelly (FAR), Spermac (SPER) and the modified GZIN (MGZIN) staining. All slides were analysed using a Hamilton Thorne Morphology Analyser IVOS equipped alternatively with a red, green or blue light source, and a 40× or 100× oil immersion objective. Recognition and digitization errors as well as morphometric parameters were determined. The IVOS was unable to detect DIF‐stained spermatozoa. The GZIN and the SPER staining as well as the blue light source led to unsatisfactory results. Among the staining methods examined, the FAR, HEM, PAP+, and PAP staining, preferably in combination with the green light source, and the 40× objective yielded optimal results concerning sperm recognition and digitization. The 100× objective did not allow reliable analysis of the sperm heads because of a frequently appearing digitization error. For experiment 2, three ejaculates were collected from each of three bulls and diluted to five dilution grades (100 000–500 000 spermatozoa/μl). An aliquot of each dilution grade was washed additionally. The percentage of correctly digitized sperm heads decreased with increasing spermatozoal concentration. However, the evaluation speed increased. The range of 200 000–300 000 spermatozoa/μl appeared to be a reasonable compromise for both criteria. Sperm washing failed to further improve the analysis results. Sperm head dimensions were influenced significantly by all variations of the methods in both experiments. In conclusion, using the proposed methods, the IVOS allows precise and reliable morphometric analyses of bull spermatozoa. The consistent application of these procedures may lead to an inter‐laboratory standardization and to further establishment of generally accepted morphometric criteria used in human andrology (e.g. World Health Organisation or strict criteria).     

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.     

Assessment of Suitable Porcine Semen for Freezing,According to the Ejaculate Characteristics in the Iberico × Landrace Breed

A total of 35 ejaculates were studied in order to assess the suitability of porcine semen for freezing according to the ejaculate characteristics. The effects of the freezing procedure were identified; a decrease in motility and acrosome quality was found after thawing. The best results on motility were linked to the ejaculates with a volume of less than 100 ml of the sperm‐rich fraction, a concentration lower than 450 × 10⁶ spermatozoa/ml and an agglutination score below 2. However, the best normal apical ridge (NAR) was found when the volume of the sperm‐rich fraction was greater than 150 ml. For this reason, an intermediate volume of the sperm‐rich fraction of the ejaculate for the best motility and the best NAR, a concentration lower than 450 × 10⁶ spermatozoa/ml and a rate of agglutination below 2 should provide the best quality after freezing. This study also attempted to determine whether a positive effect of ejaculate selection on the overall freezing performance might be expected.     

Effects of Cryopreservation on Bull Spermatozoa Distribution in Morphometrically Distinct Subpopulations

Assisted sperm morphometry analysis (ASMA) was used in this study to determine the effects of cryopreservation on bull spermatozoa distribution in morphometrically distinct subpopulations. Ejaculates were collected from five bulls and were divided. One portion was diluted at 30 degrees C in a skim milk-egg yolk medium, containing glycerol. A microscope slide was prepared from single extended sperm samples prior to freezing. The remainder of each sample was frozen in nitrogen vapours. After thawing, sperm smears were prepared as described above. All slides were air dried and stained with Hemacolor. The sperm-head dimensions for a minimum of 200 sperm heads were analysed from each sample by means of the Sperm-Class Analyser (SCA), and the mean measurements recorded. Our results showed that applying the ASMA technology and multivariate cluster analyses, it was possible to determine that three separate subpopulations of spermatozoa with different morphometric characteristics coexist in bull ejaculates (large, average and small spermatozoa). The mean values of each sperm head dimension among the three subpopulations of spermatozoa were significantly different (p < 0.001). Besides, there were significant (p < 0.001) differences in the distribution of these three sperm subpopulations between fresh and thawed samples. Thus, the percentage of representation of the subpopulation that includes those spermatozoa whose dimensions are the biggest, decreased from 52.06% in extended fresh samples to 15.51% in the thawed ones. Contrarily, the percent of representation of the subpopulation containing the smallest spermatozoa, increased from 8.70% in extended fresh samples to 34.04% in the thawed ones. In conclusion, the present study confirms the heterogeneity of sperm head dimensions in bull semen, heterogeneity that vary through the cryopreservation procedure.     

Morphological features of spermatozoa of swamp buffalo AI bulls in Thailand

The appearance and incidence of sperm abnormalities was studied in 115 ejaculates, collected periodically over 1 year covering all seasons from five mature, healthy swamp buffalo (Bubalus bubalis) bulls reared under tropical conditions and serving as the current source of semen for artificial insemination (AI) in Thailand. Light microscopy of stained smears was used to investigate sperm head shape morphology, while unstained wet smears were used to examine other sperm abnormalities. The most commonly found morphological aberrations were pear-shaped spermatozoa, knobbed acrosomes, proximal cytoplasmic droplets, simple bent tails and coiled tails under the head, whose ultrastructure (scanning electron microscopy) corresponded to what has been found in other species of bovidae, including varieties of buffalo. The mean prevalence (as least squares mean +/- SEM) of sperm abnormalities was low (below 15%), corresponding to healthy spermiograms. The younger bulls (<10 years old, n = 3) had less abnormalities than the older ones (10.1 +/- 0.6% versus 14.1 +/- 0.8%, P < 0.001, n = 2), including abnormalities of sperm head shape (1.1 +/- 0.3% versus 3.6 +/- 0.3, P < 0.001), acrosome defects with knobbed acrosomes (1.1 +/- 0.2% versus 1.2 +/- 0.3%, P < 0.001), spermatozoa with proximal cytoplasmic droplets (2.7 +/- 0.1% versus 1.4 +/- 0.2%, P < 0.001), defective mid-pieces (0.2 +/- 0.1% versus 0.3 +/- 0.1%) and abnormal sperm tails (3.1 +/- 0.3% versus 5.7 +/- 0.4%, P < 0.001). The within-bull effect of the year solely affected the incidence of pear-shaped spermatozoa while the incidences of abnormal contour, variable size of sperm head shapes, abnormal mid-piece and simple bent tail among bulls were affected by ejaculate (week of collection). Interaction between age and ejaculate affected only the prevalence of spermatozoa with proximal cytoplasmic droplets. In conclusion, the types of defects encountered were similar to those found in other bovidae, with a very low prevalence over the year the AI sires were followed through.     

Exploratory analysis of differences in sperm morphology in Nelore and Gir (Bos indicus) bulls

The aim of this study was to evaluate the effects of breed and season on semen quality parameters of zebu bulls. Data (1,632 registers) of semen production from Gir (n?=?4) and Nelore (n?=?15) bulls were collected between October 2005 and November 2009. The ejaculates were collected twice a week during various seasons (summer, fall, winter, and spring) and evaluated for the following semen parameters: ejaculate volume, sperm concentration, sperm motility, forward progressive motility (FPM), and sperm morphology. Factor analysis was used to determine the relationship among variables. The effect of breed (Gir and Nelore) and season and their cross effect on each parameter and extracted factor were tested using ANOVA. A negative correlation (P?<?0.05) was observed between FPM and proximal droplet, as well as with abnormal loose head, abnormal small head, pouch formation, abnormal mid-peace, and strongly folded tail. Gir bull sperm showed more major defects, detached acrosome, and minor FPM (P?<?0.01), whereas Nelore bulls showed a higher number of sperm with normally loose head.     

Morphological and Ultrastructural Studies of Moose Spermatozoa

Epididymal spermatozoa from moose were studied in phase contrast, light interference and electron microscope. Some samples taken from cauda were diluted and frozen in liquid N₂. The motility of the sperms after thawing was good.The concentration of spermatozoa in cauda was calculated to 10 × 10⁶ cells per µl.Morphologically the spermatozoa of moose were found to be quite similar to those collected from bulls. The length of the sperm head was found to be approx. 8.8 µ and the average maximal width 5.2 µ. The average length of the tail was 54.7 µ and the entire length of the spermatozoon varied from 60 to 64 µ. Compared with sperm cells from bulls the moose spermatozoa appeared to have a somewhat shorter and broader head and a slightly shorter tail.The migration of the cytoplasmatic droplets, which was found to be completed in caput, seemed to follow the same pattern as in bulls and boars. As found in these species there was also in the moose a higher frequency of secondary abnormalities in the spermatozoa from cauda than in those from the other parts of epididymis.Studies of the fine structure of the moose spermatozoa seemed to indicate that these are of the same type as the spermatozoa of bulls, rams and boars. In sagittal sections the sperm head was thin, but in contrast to the sperm cells of the species mentioned above no typical waist-like narrowing in the equatorial region was found. The equatorial segment also seemed to be less arched than in the spermatozoa from bulls, rams and boars. Otherwise, no principal difference was found between ultrastructure of the moose spermatozoa and that of the spermatozoa collected from domestic species.     

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.     

Does the Microbial Flora in the Ejaculate Affect the Freezeability of Stallion Sperm?

In an attempt to evaluate the possible relationship between the microbial flora in the stallion ejaculate and its ability to freeze, three ejaculates from five stallions were frozen using a standard protocol. Before freezing, an aliquot was removed for bacteriological analysis. Bacterial growth was observed in all the ejaculates studied. The isolated microorganisms were: Staphylococcus spp. and Micrococcus spp. (in all the stallions), β-haemolytic Streptococcus (in stallions 3 and 4), Corynebacterium spp. (in stallions 1, 3–5), Rhodococcus spp. (in stallion number 2), Pseudomonas spp. (in stallion number 1) and Klebsiella spp. (in stallions 1, 3 and 5). The presence and richness of Klebsiella and β-haemolytic Streptococcus in the ejaculate were related to two sperm variables post-thaw, namely the proportion of dead spermatozoa (ethidium+ cells; r = 0.55, p < 0.05) and the amplitude of lateral displacement of the sperm head (ALH, μm; r = −0.56, p < 0.05), respectively. The degree of growth of Corynebacterium spp. in the ejaculate was positively correlated with the percentage of spermatozoa showing high caspase activity post-thaw (r = 0.62, p < 0.05). The presence and number of colonies of β-haemolytic Streptococcus were negatively correlated (r = −0.55, p < 0.05) with low sperm caspase activity. It is concluded that the microbial flora of the equine ejaculate may be responsible for some of the sublethal damage experimented by the spermatozoa during cryopreservation.