The effect of cryopreservation on sperm head morphometry in Florida male goat related to sperm freezability

The Sperm Class Analyzer was used to investigate the effect of freeze-thawing procedure on Florida buck sperm head morphometry, and to relate possible changes in sperm head dimensions to cryopreservation success. Semen samples (n=76) were frozen with tris and milk-based extenders and thawed. Sperm quality samples (motility, morphology, acrosome), and sperm head morphometric values (length, width, area, perimeter, ellipticity) were compared between fresh and frozen-thawed samples. Sperm freezability was judged according to the sperm quality parameters assessed. Fertility data was obtained after artificial insemination with cryopreserved semen. Cryopreservation success was different between freezing methods. Sperm head dimensions were significantly (p<0.05) smaller in cryopreserved tris and milk spermatozoa respectively than in those of the fresh samples. The sperm head morphometric parameters that had changed after cryopreservation were lower in suitable semen samples after thawing and with successful pregnancies after artificial insemination. These data suggest that changes in sperm head morphometry might reflect spermatozoa injury occurred during cryopreservation.     

Computer automated sperm head morphometry analysis (ASMA) of goat spermatozoa

The development of computer automated sperm morphometry analysis (ASMA) allows for the objective analysis of sperm head dimensions. A number of studies have been performed to optimize the efficiency of these systems when analyzing spermatozoa from a variety of species. In this study, frozen semen from 10 fertile goat bucks was thawed and prepared on slides for morphometric analysis to evaluate technical variation and to standardize ASMA procedures for goat spermatozoa. Methods of staining, the number of spermatozoa necessary to sample and optimal microscopic magnification were assessed. Staining for 20 min in hematoxylin (HEM) was found to be optimal. The most efficient method of analyzing goat sperm morphometry was to evaluate 100 sperm cells at x20 objective magnification. Using these techniques, a sample could be analyzed in approximately 2 min. The system properly recognized and digitized spermatozoa 96% of the time with a target recognition error rate of less than 1%. The morphometric measurements of sperm heads for all 10 bucks were the following: length = 7.69microm, width = 3.80microm, width/length ratio = 0.5, area = 22.82microm and perimeter = 20.15microm. The mean coefficients of variation (CV) for all bucks ranged from 3.4% for length to 5.8% for area. Standardized sample preparation techniques and analysis were found to improve the efficiency of ASMA.     

Cryopreservation effects on canine sperm morphometric variables and ultrastructure: Comparison between vitrification and conventional freezing

Semen cryopreservation is an increasingly demanded technique in canids, particularly in order to preserve and spread high genetic value material. Sperm vitrification may represent an interesting alternative to costly and time consuming conventional freezing. The objective of this study was to evaluate the effect of sperm vitrification on sperm morphometry and ultrastructure compared to conventional freezing. Pools of nine beagle dogs were both frozen and vitrified. Computerized morphological parameters (length, wide, area and perimeter) and sperm ultrastructure, using scanning and transmission microscopy, were analysed in both fresh and in thawed/warmed samples. There were no differences (p > 0.05) between post-thaw and fresh morphometric variables of the sperm heads. However, cluster analysis revealed that sperm-heads turned out to be smaller after thawing (p < 0.05) in two of the four subpopulations. Vitrification-warming process led to an overall increase in sperm-head size. Furthermore, the sperm head size increased after warming in two subpopulations (p < 0.05). In conclusion, the variations in the sperm head area depended on the cryopreservation procedure (conventional freezing or vitrification). Conventional freezing tended to decrease the head dimensions, at least in some subpopulations, and vitrification led to an overall increase in the sperm head size. Decondensation of chromatin and plasma membrane blebbing in the head region was observed by transmission electron microscopy in several vitrified sperm, which might explain the increase of head dimensions detected by CASA-Morph system.     

Successful pregnancies with directional freezing of large volume buck semen

Artificial insemination with frozen-thawed buck semen shows variable results which depend on many factors related to semen quality and the cryopreservation processing. We conducted experiments based on a new freezing method, directional freezing, of large volumes (8 ml). In the first experiment semen from three Saanen bucks, ages 1-2-years-old and genetically selected for milk improvement, was frozen individually. Two to three-years-old Saanen females (n = 164) were synchronized with controlled internal drug release (CIDR), pregnant mare serum gonadotrophin (PMSG) and prostaglandin. Double cervical inseminations were performed with frozen-thawed semen and fresh semen as control. In the second experiment we used pooled, washed frozen semen to examine the effect of washed seminal plasma. The motility after washing was 80-90% and after thawing was 55-65% for all bucks. The sperm concentration increased with the collections and the advance into the breeding season from 1.9 x 10(9) to 4.4 x 10(9) cell/ml average. Two inseminations were carried out at 8h intervals. The first insemination was performed at 32 h after CIDR withdrawal with fresh and frozen-thawed semen. Pregnancy rates were assessed by ultrasonography conducted 40 and 90 days post-insemination (from three bucks). Results were 58, 67, 50% with fresh semen, and for frozen semen were 33, 37 and 53%; these results were significantly different in one of the three bucks (P < 0.005). In the second experiment with pooled, washed semen the pregnancy rate was 41.6%, which compared with the average results of the frozen semen in the first experiment 38.9% no significant difference was found. We conclude that freezing buck semen in large volumes (8 ml) is possible. Cryobanking of buck semen will facilitate a genetic breeding program in goats and preservation of biodiversity. Washed semen did not improve the fertility of the semen when Andromed bull extender is used.     

Fertility of fresh and frozen rabbit semen inseminated at different times is indicative of male differences in capacitation time

Some reports indicate that sperm from different males differ in capacitation time, and other reports suggest that freezing sperm may affect their capacitation time. These two variables were specifically studied in rabbits in a fertility trial with 96 does inseminated with approximately 1.6 million motile fresh or frozen sperm from three different bucks at 15, 10, 5, and 0 h before expected ovulation. Fresh semen averaged 84% live (unstained) sperm and 88% had normal acrosomes; corresponding values for frozen sperm were 44% and 54%. On the basis of does that became pregnant, average litter size with fresh semen was 5.5 and with frozen semen was 4.8 (p greater than 0.05), but overall, does bred with frozen semen produced fewer young (p less than 0.05). On the basis of total does and total semen, average litter size from insemination at 15, 10, 5, and 0 h was 2.8, 4.2, 3.8, and 1.7, and average litter size for the three bucks was 4.0, 1.8, and 3.6. There was no interaction of type of semen (fresh or frozen) with the other variables in the model (p greater than 0.05). Bucks and time of insemination affected both the proportion of does that were pregnant and litter size (p less than 0.01). A major interaction between buck and time of insemination (p less than 0.01) was due apparently to both differential sperm survival and probable capacitation time among bucks. This major interaction should be considered in designing in vitro and in vivo fertility studies, and for selecting males for use in artificial insemination.     

Sperm characteristics in plains (Bison bison bison) versus wood (Bison bison athabascae) bison

The objective was to compare sperm characteristics between the two subspecies of North American bison, plains bison (Bison bison bison) and wood bison (Bison bison athabascae). Frozen-thawed ejaculated sperm from age-matched plains (n = 3) and wood (n = 2) bison were evaluated for morphometry, motility, viability, protein profile, and in vitro fertilization characteristics. Sperm morphometry and motility were assessed with computer-based systems, viability was assessed with SYBR-14 and propidium iodide, and fertilizing ability was determined using a heterologous in vitro fertilization system (using bovine oocytes). For plains versus wood bison, there were significant differences for head width (4.76 ± 0.22 vs 4.71 ± 0.19 μm; mean ± SD), head area (35.64 ± 1.91 vs 34.72 ± 2.64 μm²), head perimeter (23.61 ± 0.68 vs 23.31 ± 0.98 μm), midpiece length (14.58 ± 0.4 vs 14.36 ± 0.51 μm), midpiece width (0.81 ± 0.06 vs 0.79 ± 0.07 μm), and tail length (46.61 ± 2.15 vs 45.98 ± 2.08 μm). However, there was no significant difference in head length (overall, 9.04 ± 0.37 μm), progressive motility (41.16 ± 8.39%), or viability (41.58 ± 5.58%). Based on two-dimensional gel electrophoresis, 93 out of 113 protein spots were similar in their expression patterns. Furthermore, we inferred that differences in sperm biometry between these subspecies did not affect in vitro fertilization percentage (overall, 82.62 ± 12.13%). Based on these findings, we concluded that plains bison were an appropriate research model for developing reproductive technologies for wood bison.     

The effect of optimal and suboptimal concentrations of sperm on the fertility of fresh and frozen bovine semen and a theoretical model to explain the fertility differences

In vitro trials on the survival of sperm stored fresh or rediluted after freezing showed quite significant differences in the total survival time of sperm incubated at 37°C. Even after adjusting for different sperm concentrations, survival was still superior in the fresh compared with rediluted frozen sperm (124 ± 5.5 h vs. 75.8 ± 3.1 h). There was no significant difference in fertility between rediluted frozen sperm (RDF) and fresh sperm, both stored in Caprogen, when the insemination dose was 20 × 10⁶ and 2.5 × 10⁶ sperm, respectively. Reduction of the sperm concentration in the insemination dose from 20 × 10⁶ to 5 × 10⁶ sperm in RDF and from 2.5 × 10⁶ to 0.5 × 10⁶ sperm in fresh semen reduced non return rates by 7.9% and 7% respectively (P < 0.001). The bull × dose rate interaction for non return rate was not significant for fresh semen, but significant for RDF (P < 0.001). Two theoretical models were used to examine the effects of freezing on the survival of sperm in the female reproductive tract and the probability of fertilisation. There is a suggestion that freezing had no effect on survival time of sperm in the female reproductive tract, but either reduced the probability of fertilisation by a single spermatozoon or altered the pattern of sperm survival in the female reproductive tract.     

Morphometric differences in sperm head dimensions of fertile and subfertile stallions

Gross morphological evaluation of stallion spermatozoa is of clinical value in assessing male fertility in the horse. While of value, methods of subjective sperm classification yield highly variable results. Recent development of computer-assisted sperm morphometry analysis (ASMA) technology has allowed for the objective analysis of sperm head morphometry. In the current study, ASMA was employed to determine morphometric differences in sperm head dimensions between fertile and subfertile stallions. At least 200 spermatozoa from each of 10 fertile and 10 subfertile stallions were analyzed by a commercial ASMA instrument. The mean measurements for length, width, area, perimeter, and width/length for each stallion were recorded and group means compared by a two-sample t-test. The mean measurements for length, area and perimeter were significantly larger in the subfertile than the fertile group (5.77 microm vs 5.33 microm, 12.66 microm vs 11.37 microm and 14.59 microm vs 13.64 microm, respectively). The width of sperm heads from stallions in the subfertile group also tended to be larger than those of fertile stallions. The data suggest that differences in the dimensions of sperm heads may exist between fertile and subfertile stallions.     

Morphometric characterization of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa using computer-assisted spermatozoa analysis (ASMA)

As part of a larger study on sperm quality and cryopreservation methods, the present study characterized the head morphometry of sharpsnout sea bream (Diplodus puntazzo) and gilthead sea bream (Sparus aurata) spermatozoa, using both scanning electron microscopy (SEM) and computer‐assisted morphology analysis (ASMA). The latter method has been used rarely in fish and this is its first application on sharpsnout sea bream and gilthead sea bream spermatozoa. Results obtained using SEM are expensive and time‐consuming, while ASMA provides a faster and automated evaluation of morphometric parameters of spermatozoa head. For sharpsnout sea bream spermatozoa, similar head measurement values were obtained using both ASMA and SEM, having a mean ± standard error length of 2.57 ± 0.01 μm vs 2.54 ± 0.02 μm, width of 2.22 ± 0.02 μm vs 2.26 ± 0.04 μm, surface area of 4.44 ± 0.02 μm² vs 4.50 ± 0.04 μm² and perimeter of 7.70 ± 0.02 μm vs 7.73 ± 0.04 μm using ASMA and SEM, respectively. Although gilthead sea bream spermatozoa were found to be smaller than those of sharpsnout sea bream, spermatozoal head morphometry parameters were also found to be similar regardless of evaluation method, having a mean head length of 1.97 ± 0.01 μm vs 1.94 ± 0.02 μm, head width of 1.80 ± 0.01 μm vs 1.78 ± 0.02 μm, surface area of 3.16 ± 0.03 μm² vs 3.18 ± 0.06 μm² and perimeter of 6.52 ± 0.04 μm vs 6.56 ± 0.08 μm using ASMA and SEM, respectively. The results demonstrate that ASMA can be considered as a reliable technique for spermatozoal morphology analysis, and can be a useful tool for studies on fish spermatozoa, providing quick and objective results.     

Effect of sequence of insemination after simultaneous thawing of multiple semen straws on conception rate to timed AI in suckled multiparous Nelore cows

The objective was to determine the effect of sequence of insemination after simultaneous thawing of multiple 0.5 mL semen straws on conception rate in suckled multiparous Nelore cows. The effect of this thawing procedure on in vitro sperm characteristics was also evaluated. All cows (N = 944) received the same timed AI protocol. Ten straws (0.5 mL) of frozen semen from the same batch were simultaneously thawed at 36 °C, for a minimum of 30 sec. One straw per cow was used for timed AI. Frozen semen from three Angus bulls was used. Timed AI records included sequence of insemination (first to tenth) and time of semen removal from thawing bath. For laboratory analyses, the same semen batches used in the field experiment were evaluated. Ten frozen straws from the same batch were thawed simultaneously in a thawing unit identical to that used in the field experiment. The following sperm characteristics were analyzed: sperm motility parameters, sperm thermal resistance, plasma and acrosomal membrane integrity, lipid peroxidation, chromatin structure, and sperm morphometry. Based on logistic regression, there were no significant effects of breeding group, body condition score, AI technician, and sire on conception rate, but there was an interaction between sire and straw group (P = 0.002). Semen from only one bull had decreased (P < 0.05) field fertility for the group of straws associated with the longest interval from thawing to AI. However, the results of the laboratory experiment were unable to explain the findings of the field experiment. Sperm width:length ratio of morphometric analysis was the single sperm characteristic with a significant interaction between sire and straw group (P = 0.02). It was concluded that sequence of insemination after simultaneous thawing of 10 semen straws can differently affect conception rates at timed AI, depending on the sire used. Nevertheless, the effects of this thawing environment on in vitro sperm characteristics, remain to be further investigated.     

Objective assessment of the cryoprotective effects of dimethylformamide for freezing goat semen

The aim of this work was to assess the cryoprotective effects of dimethylformamide (DMF) for freezing goat semen, using an objective analysis by computer-assisted sperm analysis (CASA). Twenty-one ejaculates (seven per animal) were collected from three stud bucks with the aid of an artificial vagina and immediately evaluated for gross and microscopic characteristics. The semen was diluted in two steps with a Tris–egg yolk extender containing 6% glycerol or 6% DMF, frozen in 0.50-mL straws, and stored in liquid nitrogen. Samples were accessed for sperm morphology, sperm membrane structural and functional integrity, and by CASA, immediately after thawing. There were differences (P < 0.05) between glycerol and DMF with regard to subjective progressive motility (23.9 ± 2.2% vs. 16.6 ± 2.0%), objective progressive motility (3.5 ± 0.4% vs. 1.8 ± 0.3%), linearity (53.9 ± 1.6% vs. 48.1 ± 1.4%) and amplitude of lateral head (2.3 ± 0.1 vs. 2.9 ± 0.1 mm), which confirmed the efficiency of glycerol. In conclusion, dimethylformamide could be used as an alternative cryoprotectant for goat semen freezing. However it was showed that no benefits were derived by using dimethylformamide to replace glycerol at an equal 6% concentration.     

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.     

Cryopreservation of semen from endangered pheasants: the first step towards a cryobank for endangered avian species

In order to improve the genetic management of bird species within the European Endangered Programs (EEP), a research project on artificial insemination and cryopreservation of Galliformes semen has been developed. The aim of the program is to create a sperm cryobank for threatened bird species. During this study, semen was collected from 17 pheasant species and specific characteristics of ejaculates were analyzed (volume, sperm concentration, motility, pH). Artificial insemination with fresh semen was performed in nine species and with frozen semen in eight species. Inseminations with frozen and thawed semen were made in 17 species. Viability of fresh and frozen semen was assessed in vitro using double stains, eosin and nigrosin. The effect of pH (7-8.5) on viability of fresh and frozen/thawed spermatozoa was also studied. Chicks hatched in eight and three species after insemination with fresh and frozen/thawed semen, respectively. Species varied widely in semen viability: 1-30% of spermatozoa survived freezing and thawing. There was a negative correlation between the viability of frozen spermatozoa and semen pH. In our experimental conditions, the pH of diluents had no effect on semen viability. However, semen with the highest pH had the lowest quality after freezing and thawing. These experiments demonstrated the feasibility of using a very simple and inexpensive method to achieve artificial insemination and cryopreservation of semen in endangered pheasant species.     

Assessment of goat semen freezability according to the spermatozoa characteristics from fresh and frozen samples

A total of 110 ejaculates were assessed in order to determine the influence of the physical parameters of goat semen on post-thaw motility and acrosome integrity. Sperm ejaculate characteristics, sperm motility, morphology and acrosome integrity were assessed in fresh and frozen samples by the sperm class analyzer (SCA) and Spermac staining technique. A decrease in acrosome integrity and sperm motility was found after thawing (P<0.01). Six semen parameters assessed before freezing were identified as predictors of sperm freezability (P<0.01). The percentage of morphological abnormalities (R=0.856) and motile sperm cells (R=0.655) in fresh semen are the best predictors to know the total post-thaw variability parameters.     

Dietary flax seed oil and/or Vitamin E improve sperm parameters of cloned goats following freezing-thawing

Semen cryopreservation is affected by individual differences and use of clones animal from the same source is the main tool to eliminate genetic variation. Among many nutrients that are necessary for fertility, essential fatty acids and antioxidants are vital for production of healthy sperm by improving sperm membrane integrity and protecting sperm from oxidative stress. The goal of the current study was to investigate whether a flax seed oil or/and Vitamin E dietary supplementation could improve semen quality of cloned bucks following semen cryopreservation. Accordingly, eight adult cloned Bakhtiari bucks were divided randomly into four groups. Bucks were offered a base diet of hay and concentrate. The concentrate was enriched with flax seed oil, 30 gr/kg body weight/day (OIL), Vitamin E (VIT), 3 gr/kg body weight/day, or combined flax seed oil and the vitamin E (OIL-VIT). The concentrate with no supplements was considered as control group (CONT). Both flax seed oil and Vitamin E supplements were added to the total diet. The bucks were fed with their corresponding diets for a total of 9 weeks while sperm collection was carried out within 10–14 weeks. Ejaculates were diluted with Andromed^® and were frozen in liquid nitrogen. Sperm parameters and reactive oxygen species (ROS) contents were evaluated following freezing/thawing. According to the results of our study, dietary supplementation with flax seed oil, or/and Vitamin E can improve sperm motility, vitality and number of sperm with intact plasma membrane following freezing-thawing. But the degree of improvement in these parameters was significantly higher when Flax seed oil and vitamin E were co-supplemented.     

Morphological characterization of ejaculated cynomolgus monkey (Macaca fascicularis) sperm

The aim of this study was to give reference values for the frequency of morphological sperm abnormalities present in the semen from nonexperimental cynomolgus monkeys as well as for the dimensions of sperm heads. Spermatozoa from the liquid portion of electroejaculates from 14 cynomolgus monkeys were air‐dried as smears, fixed, and stained with Harris's Haematoxylin and subjected to visual analysis of morphology and computer‐aided analysis of ten morphometric variables. The majority (83%) of sperm were morphologically normal. Tail defects were the most common (11%), and showed the highest variation between individuals, the values ranging between 4 and 23%. Head abnormalities consisted of large, tapering, and amorphous forms but were not frequent (0.4%), the values ranging between 0 and 1.3%. Midpiece imperfections were found in all the individuals; the mean percentage was 5%, and the range varied between 3 and 9%. Tail plus midpiece was the only multiple abnormality observed, with a mean value of 1.5% and a range between 0 and 8%. The majority of these double defects consisted of a coiled tail together with a coiled midpiece. Mean values for the morphometric parameters characterizing sperm heads were as follows: area 17.2 μm², perimeter 15.2 μm, length 5.8 μm, width 4.0 μm, L/W ratio 1.5, gray‐level 98, ellipticity 0.2, first shape factor 0.9, second shape factor 1.4, and third shape factor 1.1. Overall coefficients of variation for the majority of parameters were below 7%, showing the great homogeneity in the dimensions of cynomolgus sperm heads. Most useful parameters for sperm characterization, according to their low variability, were perimeter, length, width, L/W ratio, and shape factors. Differences in these parameters were, however, observed between monkeys. Am. J. Primatol. 47:105–115, 1999. © 1999 Wiley‐Liss, Inc.     

Assessment of sperm damages during different stages of cryopreservation in water buffalo by fluorescent probes

The present study was designed to investigate the sperm damages occurring in acrosome, plasma membrane, mitochondrial activity, and DNA of fresh, equilibrated and frozen–thawed buffalo semen by fluorescent probes. The stability of sperm acrosome and plasma membrane stability, mitochondrial activity and DNA status were assessed by fluorescein conjugated lectin Pisum sativum agglutinin, Annexin–V/propidium iodide, JC-1 and TUNEL assay, respectively, under the fluorescent microscope. The damages percentage of acrosome integrity was significantly increased during equilibration and freezing–thawing process. The stability of sperm plasma membrane is dependent on stability of phosphatidylserine (PS) on the inner leaflet of plasma membrane. The frozen–thawed sperm showed externalization of PS leading to significant increase in apoptotic, early necrotic and necrotic changes and lowered high mitochondrial membrane potential as compared with the fresh sperm but all these parameters were not affected during equilibration. However, the DNA integrity was not affected during equilibration and freezing–thawing procedure. In conclusion, the present study revealed that plasma membrane and mitochondria of buffalo sperm are more susceptible to damage during cryopreservation. Furthermore, the use of fluorescent probes to evaluate integrity of plasma and acrosome membranes, as well as mitochondrial membrane potential and DNA status increased the accuracy of semen analyses.     

Effect of cooling and freezing,the two first steps of a freezing protocol,on the fertilizing ability of the rabbit sperm

The effect of different phases of a freezing protocol on the fertilising ability of rabbit spermatozoa was evaluated. An extender containing 1.75 M DMSO and 0.05 M sucrose (final concentration) was used to freeze rabbit sperm. In the first experiment, the results obtained with fresh and cooled (5 degrees C for 45 min) sperm were compared; no differences were observed between fresh and cooled semen for any of the parameters studied: fertility rate (78% vs. 91% for fresh and cooled sperm, respectively), and normal embryos two days after insemination (6.8 vs. 8.5 normal embryos for fresh and cooled sperm). In the second experiment, the results obtained with fresh semen and sperm which had passed the first two steps of a freezing protocol (5 degrees C for 45 min and -30 degrees C for 30 min, and thawed at 50 degrees C for 15 s) were compared; the differences between them were obtained for fertility rate (94% vs. 61% for fresh and frozen sperm, respectively) and normal embryos two days after insemination (7.8 vs. 3.8 fresh and frozen sperm). These observations indicated that the differences in the results obtained with fresh and cryopreserved sperm were produced during the second step of the freezing protocol, and that apparently no toxic effect of DMSO was produced.     

Automatic evaluation of ram sperm morphometry

This study was designed to develop a new method based on fluorescence microscopy and image analysis for the automatic assessment of sperm morphometry and to study separately the effect of drying and fixation on the parameters of head sperm morphometry in the ram. The study was divided into two experiments. In the first experiment, ejaculates from 25 adult males were collected using an artificial vagina, diluted and divided into four sample aliquots. The first was labeled directly with Hoechst 33342 (FRESH), and the others were processed as smears. Between smears, one group was directly labeled with Hoechst after air drying (DRIED), and the other were fixed either with glutaraldehyde (GLUT), or with methanol (MET), and labeled with Hoechst afterward. Digital images of the fluorescence-labeled sperm were recorded with a digital camera, and sperm heads were automatically captured and analyzed using the ImageJ program. The method used allowed a fast and automatic selection of most sperm heads for a given image with high precision. There was a general trend toward significant decrease in head length, width, area and perimeter of air-dried sperm compared with fresh sperm. On average, this decrease was of 4.1% in length, 4.3% in width, 9.1% in area, and 2.8% in perimeter. Between semen smears, fixation with glutaraldehyde significantly increased head sperm dimensions. The smears fixed with glutaraldehyde method is recommended for a more practical use than with fresh samples, providing better quality images than the other methods, and because the morphometric results obtained were more similar to the FRESH group than those of the DRIED and MET. In the second experiment, ejaculates from adult males were used to compare the sperm head morphometric results obtained with the new method developed (using the GLUT treatment as reference) with a more conventional CASMA method (semen smears stained with Hemacolor and processed with the ISAS commercial software, HEM). The GLUT method allowed the analysis of 100% of sperm, whereas only 93% of sperm could be analyzed using HEM. Spermatozoa displayed a bigger size when processed with HEM than with GLUT method in all primary sperm head morphometric parameters. A significant correlation was observed between the two methods used in this experiment for all morphometric size parameters. The new method developed allows automatic determination of sperm head morphometry in a reduced time, which facilitates its use in routine semen analysis. It was concluded that the automation of sperm morphometry is feasible using fluorescence microscopy and image analysis and that the effect of drying and fixation was less important than previously stated.     

Integration of future biotechnologies into the equine industry

There has and will continue to be reproductive techniques available that have a positive impact upon the equine breeding industry. This review focuses on semen technologies that have been developed or are in the process of being developed. The use of fluorescent dyes and flow cytometry has provided the researcher and clinician with powerful tools to evaluate several sperm attributes. These procedures have been utilized to evaluate sperm viability, acrosome status, mitochondrial status, DNA integrity and stages of capacitation. Flow cytometry allows several sperm attributes to be evaluated on thousands of spermatozoa in a matter of seconds. Development of procedures for insemination of mares with relatively small numbers of spermatozoa has the potential to change how stallions and their semen are managed. This review discusses the use of insemination of fresh, frozen and sex-sorted spermatozoa in relatively small numbers compared with conventional insemination technologies. The recent acceptance of frozen-thawed semen by many of the major breed registries has stimulated an increase in research on frozen semen. Many of the studies have focused on identifying damage during the freezing and thawing process. Numerous studies also have been conducted to modify freezing extenders so that the sperm are protected during the freezing and thawing process. The production of in vitro-produced embryos is extremely limited in the horse due to the failure of in vitro fertilization. However, intracytoplasmic sperm injection (ICSI) has been used for the production of foals from stallions that have less than typical sperm numbers or from stallions that have died and a limited quantity of frozen semen is available. This technique has been used by several laboratories to produce embryos in vitro. The breeder and veterinarian now have access to techniques that allow assessment of semen quality, improvement of procedures for freezing and thawing and insemination of mares with fewer numbers of spermatozoa. It is likely that the next decade will also produce tremendous advances in semen technologies that can be utilized in the horse industry.