Sperm Head Area Research Articles

P-038 Sperm head morphometric reference values of 1856 sperms in fertile Asian males

Abstract Study question To explore comprehensive normal sperm morphometric reference values by measuring 1856 sperms in fertile males using artificial intelligence (AI) technique. Summary answer Four novel describing parameters of sperm head (size, shape, area, and uniformity) and 14 other features for normal sperm head were extracted and characterized. What is known already Sperm head morphology is crucial for male factor infertility diagnosis and assessment of male reproductive potential. The human spermatozoon is one of the most diverse cell types known. However, most assessment criteria have only 3 morphometric parameters (Length, width, and length/width ratio) for sperm head morphometric measurement. We detailed the features of sperm head by defining and characterizing 14 parameters based on AI. Study design, size, duration This is a prospective observational study. The study included 1856 normal sperm images from 23152 stained sperm acquired by an Automated Sperm Morphology Analysis (ASMA) system. The measured sperm morphometry data were acquired from May 2020 to December 2021. Participants/materials, setting, methods All participants (54 fertile males) had fathered a child naturally during the past year. The measured sperm morphometry data were acquired from candidate donors of the Sichuan Province Human Sperm Bank (SPHSB, Chengdu, China). Images of 23,152 stained sperm captured by ASMA. All sperm images were assessed by three experienced technicians. Finally, we concluded 14 morphometric data from normal sperm by measuring sizes in pixels. Main results and the role of chance (1) Characteristics of sperm head size and shape: We present eight sperm head size and shape parameters for the 1856 visually normal spermatozoa. The median head size parameters included head length of 4.02 (95% CI: 4.01–4.05) μm, head width of 2.52 (95% CI: 2.52–2.54) μm, length to width ratio of 1.59 (95% CI: 1.59–1.62), and head girth of 11.18 (95% CI: 11.17–11.25) μm. The median head symmetry parameters included short axis symmetry (SAS) of 92.1% (95% CI: 88.3%–89.2%) and the long axis symmetry (LAS) of 94.3% (95% CI: 91.6%–92.3%). The median head shape parameters included median of ellipse intersection over union (EIoU) of 88.9% (95% CI: 85.4%–86.3%) and girth intersection over union (GIoU) of 102.4% (95% CI: 102.4%–102.5%). (2) Characteristics of sperm head area and acrosome uniformity: Descriptive analysis of the sperm head area characteristics included a median sperm head area of 7.72 (95% CI: 7.74–7.85) μm2, acrosome area of 3.67 (95% CI: 3.77–3.88) μm2, acrosome area ratio of 48.2% (95% CI: 47.8%–48.6%), post-acrosomal area of 3.95 (95% CI: 3.95–4.00) μm2, acrosome uniformity of 88.6% (95% CI: 88.6%–89.1%), and nuclear uniformity of 100.0% (95% CI: 99.8%–99.9%). Limitations, reasons for caution This study had several limitations that should be noted. The use of samples from a single center, covering relatively young participants from only seven provinces in China, could limit the usage of our findings. The use of only three technicians to assess the samples may have introduced a subjective bias. Wider implications of the findings The measurement technique was accurate, objective, and highly informative by AI. The data provide extremely detailed information for reference studies and comparison of sperm head parameters among different male groups. These new parameters can be applied in routine semen analysis when assessing sperm morphology and male fertility potential. Trial registration number not applicable

Normal sperm head morphometric reference values in fertile Asian males.

Sperm head morphology is crucial for male factor infertility diagnosis and assessment of male reproductive potential. Several criteria are available to analyze sperm head morphology, but they are limited by poor methodology comparability and population applicability. This study aimed to explore comprehensive and new normal morphometric reference values for spermatozoa heads in fertile Asian males. An automated sperm morphology analysis system captured 23 152 stained spermatozoa from confirmed fertile males. Of these samples, 1856 sperm head images were annotated by three experienced laboratory technicians as "normal". We employed 14 novel morphometric features to describe sperm head size (head length, head width, length/width ratio, and girth), shape (ellipse intersection over union, girth intersection over union, short-axis symmetry, and long-axis symmetry), area (head, acrosome, postacrosomal areas, and acrosome area ratio), and degrees of acrosome and nuclear uniformity. This straight-forward method for the morphometric analysis of sperm by accurate visual measurements is clinically applicable. The measured parameters present valuable information to establish morphometric reference intervals for normal sperm heads in fertile Asian males. The presented detailed measurement data will be valuable for interlaboratory comparisons and technician training. In vitro fertilization and andrology laboratory technicians can use these parameters to perform objective morphology evaluation when assessing male fertilization potential.

Equilibration time improves the sperm variables of wild ruminant ejaculated and epididymal sperm cryopreserved by ultra-rapid freezing

This work examines the effect of equilibration time with extender on ultra-rapidly frozen-thawed wild ruminant epididymal (origin: Iberian ibex) and ejaculated (origin: mouflon) sperm variables. Sperm samples were prepared either without prior equilibration, or equilibrated for 30 min before freezing. Higher quality (p < 0.05) frozen-thawed spermatozoa were obtained when equilibration was allowed, for ejaculated sperm in terms of sperm motility, acrosome apical ridge integrity, sperm viability, and percentage of normal cells, and for epididymal sperm in terms of linearity and straightness of sperm movement. The sperm head area, head perimeter, head length and head width were smaller (p < 0.01) in the equilibrated than non-equilibrated frozen-thawed epididymal sperm; no such dimensional changes were recorded for ejaculated sperm. In conclusion, equilibration prior to ultra-rapid freezing improves the cryoresistance of sperm cells, although viable sperm cells can be obtained without equilibration. The epididymal sperm showed greater cryoresistance, supporting the idea that it is more resistant to freeze-thawing than ejaculated sperm.

Effects of six kinds of sperm staining methods on human sperm size and evaluation of their staining effects.

Large- and small-headed sperm are common morphological abnormalities. If different sperm staining methods affect sperm size, they will make a difference in the accuracy of sperm morphological analysis results. In this case, the normal reference values of sperm head parameters for different staining methods should be established. Six sperm staining methods, including Papanicolaou, Diff-Quik, Shorr, Hematoxylin-eosin (HE), Wright, and Wright-Giemsa staining, were used to stain the sperm smears of 25 semen samples, respectively. Sperm head parameter's length (L), width (W), area (A), perimeter, acrosomal area (Ac), and the derived values L/W and Ac/A of 2500 sperm (100 for each specimen) per staining method were measured by a computer-aided sperm morphological analysis system. The highest sperm head length and width were observed with the Wright-Giemsa and Wright staining, followed by the Diff-Quik. The lowest sperm head length and width were observed with the Papanicolaou staining, and the sperm head length and width of HE and Shorr staining were between those of Papanicolaou and Diff-Quik staining. There was the same trend in changes in sperm head area and perimeter. Diff-Quik and Shorr staining could clearly distinguish acrosome and nucleus, followed by HE staining, whereas the boundary between acrosome and nucleus was not evident in Papanicolaou, Wright, and Wright-Giemsa staining. Different staining methods influence sperm size, and the normal reference values of sperm head parameters of each staining method should be established. Diff-Quik and Shorr staining may be suitable methods for routine sperm morphological analysis.

Morphometry of Boar Spermatozoa in Semen Stored at 17 °C—The Influence of the Staining Technique

Simple SummaryTo obtain satisfactory results in artificial insemination, it is necessary to use high-quality ejaculates for the production of insemination doses and then maintain the biological value of the sperm during storage. Boar spermatozoa, owing to the specific structure of the cell membrane, are particularly sensitive to temperature changes. For this reason, cryopreservation cannot be used in artificial insemination practice, and there may be limitations to successful storage of semen in a liquid state. The practice of using boar semen for artificial insemination does not include analyses of the effect of storage time of boar semen on sperm dimensions. Therefore, the aim of the study was to analyse the morphometry of sperm during storage of liquid boar semen. An attempt was also made to evaluate the suitability of three staining methods for assessment of boar sperm morphometry. The morphometric dimensions of boar sperm were shown to change during storage of liquid semen. These changes affected the sperm head more than the tail and were due to the staining method used. The analyses are very important as they provide more information about the morphometric dimensions of the sperm during preservation of boar semen. The applied sperm staining techniques allows for a more accurate assessment of male reproductive cells.The aim of the study was to assess the morphometry of sperm during storage of liquid boar semen at 17 °C. An attempt was also made to evaluate the suitability of three staining methods for assessment of boar sperm morphometry. The study was carried out on 20 Landrace boars. Semen was collected from the boars every 5 days by the manual method. Four ejaculates from each boar were analysed (80 ejaculates in total). Analyses were performed five times: at 1 h, 24 h, 48 h, 96 h, and 168 h after semen collection. Blisters with insemination doses were opened immediately before the analyses. From each insemination dose, smears were prepared for morphometric evaluation of sperm, which were stained by three methods (eosin-nigrosin—EN, eosin-gentian—EG, and SpermBlue—SB). Morphometric measurements of 15 randomly selected sperm with normal morphology were performed on each slide. The morphometric measurements included the following parameters: sperm head length, width, area, and perimeter; tail length; and total sperm length. The results of the morphometric measurements were used to calculate the head shape index. The morphometric dimensions of the sperm were shown to change during storage of semen at 17 °C. The extent of these changes, however, depended on the staining method used, as the three methods result in different morphometric dimensions of sperm, in the case of both the head and the tail. In the slides stained by the eosin-nigrosin method, the dimensions of the head and tail were smaller at every time of storage than in the slides stained by the SpermBlue and eosin-gentian methods.

Sperm preparedness and adaptation to osmotic and pH stressors relate to functional competence of sperm in Bos taurus

The adaptive ability of sperm in the female reproductive tract micromilieu signifies the successful fertilization process. The study aimed to analyze the preparedness of sperm to the prevailing osmotic and pH stressors in the female reproductive tract. Fresh bovine sperm were incubated in 290 (isosmotic-control), 355 (hyperosmotic-uterus and oviduct), and 420 (hyperosmotic-control) mOsm/kg and each with pH of 6.8 (uterus) and 7.4 (oviduct). During incubation, the changes in sperm functional attributes were studied. Sperm kinematics and head area decreased significantly (p < 0.05) immediately upon exposure to hyperosmotic stress at both pH. Proportion of sperm capacitated (%) in 355 mOsm/kg at 1 and 2 h of incubation were significantly (p < 0.05) higher than those in 290 mOsm media. The magnitude and duration of recovery of sperm progressive motility in 355 mOsm with pH 7.4 was correlated with the ejaculate rejection rate (R2 = 0.7). Using this information, the bulls were divided into good (n = 5) and poor (n = 5) osmo-adapters. The osmo-responsive genes such as NFAT5, HSP90AB1, SLC9C1, ADAM1B and GAPDH were upregulated (p < 0.05) in the sperm of good osmo-adapters. The study suggests that sperm are prepared for the osmotic and pH challenges in the female reproductive tract and the osmoadaptive ability is associated with ejaculate quality in bulls.

Cryopreservation of dog epididymal spermatozoa by conventional freezing or ultra-rapid freezing with nonpermeable cryoprotectant

This study was aimed to assess the effectiveness of two methods for cryopreservation of dog epididymal spermatozoa, one by conventional freezing (CF) with shortening both equilibration and cooling times, and the other by ultra-rapid freezing (URF) with nonpermeable cryoprotectant. Sixty epididymides were recovered from thirty orchiectomized adult dogs and the sperm samples were retrieved by retrograde flushing using TCG-EY (tris, citric acid, glucose + 20% egg yolk) extender and then 20 pools were conformed. Each pool was divided into 2 aliquots and then cryopreserved by CF and URF methods respectively. The CF method maintained the cooled-pool samples for 2h (1h without and 1h with 5% glycerol) and then were frozen by liquid nitrogen (LN2) vapors for 2 min. The URF method cryopreserved the cooled-pool samples using TCG-EY+250 mM sucrose, equilibrating during 30 min (5 °C) and submerging 30-μL drops directly in LN2. The results showed that the URF method produced a lower percentage of total and progressive motilities and acrosome integrity (P < 0.05) than the CF method. However, the kinetic variables (curvilinear and straight-line velocities, straightness, linearity, wobble, amplitude of lateral head displacement, and beat-cross frequency) and plasma membrane integrity did not differ (P > 0.05) between both cryopreservation methods. Unlike the URF method, the width, area and perimeter of sperm head were reduced after the CF method (P < 0.05). In conclusion, despite the low motility achieved after the ultra-rapid freezing method, the similar values of kinetic, viability and head morphometric dimensions to those obtained after conventional freezing, suggest that ultra-rapid freezing with sucrose may be a useful alternative for the cryopreservation of canine epididymal sperm.

Supraphysiological concentration of urea affects the functional competence of Holstein-Friesian (Bos taurus) sperm

To understand the effects of urea on sperm functional attributes, fresh bull semen (n = 12) was subjected to four different concentrations (mg/mL) of urea to mimic the physiological (0.04 and 0.13), supraphysiological (0.43) concentrations and control (0 mg/mL). Sperm membrane integrity, kinematics, chromatin integrity, and mitochondrial membrane potential were assessed at different time points (before incubation, 0, 1, 2, and 4 h) of incubation. The concentration of urea in serum and seminal plasma was estimated and correlated with the ejaculate rejection rate and sperm functional attributes. The relative expression of urea transporter gene transcripts (UT-A and UT-B) was assessed in sperm and testis (control) using real-time PCR. The supraphysiological concentration of urea affected sperm kinematics, viability, functional membrane integrity, and acrosome integrity within 1 h of incubation (p < 0.05). Sperm head area decreased (p < 0.05) at 0 h and subsequently increased at 1 h of incubation in all media except supraphysiological (0.43 mg/dL) concentration of urea. Seminal plasma urea concentration showed a significant negative correlation with sperm motility, membrane integrity, and mitochondrial membrane potential (p < 0.05), but had a positive correlation with the ejaculate rejection rate (r = 0.69). Relative expression of the urea transporter genes revealed that UT-A was expressed only in the testis. In contrast, UT-B was expressed in both the testis and sperm, suggesting UT-B's role in regulating urea transport in sperm. At a supraphysiological level, urea adversely affected sperm functional attributes, osmoadaptation and may affect fertility.

Sexual selection towards a protamine expression ratio optimum in two rodent groups?

Post-copulatory sexual selection is thought to influence the evolution of genes involved in reproduction. However, the detection of straightforward effects has been proven difficult due to the complexity and diversity of reproductive landscapes found in different taxa. Here, we compare the possible effect of relative testes mass as a sperm competition proxy on protamine genotype (protamine 1/protamine 2 ratio) and the link to sperm head phenotype in two rodent groups, mice, and voles. In mice, protamine expression ratios were found to increase from low values toward a 1:1 ratio in a positive association with testes mass, and relative sperm head area. In contrast, in voles, decreasing protamine expression ratios were found in species with larger testes but, surprisingly, they range from high values, again toward a 1:1 ratio, and showing a negative correlation with relative sperm head area. Altogether, we found differences in the way protamines seem to be selected and involved in adaptations of the sperm head in voles and mice. However, sexual selection driven by sperm competition seems to exhibit a common evolutionary pattern in both groups toward an equilibrium in the expression of the two protamines.

Seasonal changes in testosterone and thyroxine concentrations in Mediterranean rams and bucks and their relationship with sperm cryoresistance

This study examines the relationship between seasonal changes in plasma testosterone and thyroxine concentrations and the relationship between these changes and the sperm resistance to the freezing-thawing process in Merino x Merino Precoz crossbreed rams and Florida breed bucks. Plasma testosterone and thyroxine concentration, sperm head area, sperm variables and their cryoresistance ratios were analyzed throughout a year. Both species showed a rise in thyroxine preceding testosterone fall. Plasma thyroxine concentrations in rams were higher during autumn than in spring and summer (P<0.05), whereas testosterone concentrations were not influenced by season. Conversely, bucks showed seasonal differences in plasma testosterone levels, with higher concentrations during summer and autumn than winter and spring (P<0.05), whereas thyroxine concentrations were not influenced by season. In bucks, a negative correlation was detected between plasma testosterone concentrations and the cryoresistance ratio for membrane integrity (R=-0.88; P<0.05) from April to September and between plasma thyroxine concentrations and the cryoresistance ratios for total motility (R=-0.66; P<0.05), curvilinear velocity (R=-0.61; P<0.05), the amplitude of lateral head displacement (R=-0.77; P<0.05) and the beat-cross frequency (R=-0.74; P<0.05), throughout the year. The results suggest that the annual variation in sperm cryoresistance may be related to seasonal endocrine changes, at least in goat. Winter and spring are the most appropriate seasons to collect and freeze sperm from Florida goat.

Comparative evaluation of seminal plasma proteins in Holsteiner and East Bulgarian horse breeds in relation to functional parameters of spermatozoa

The present research was focused on the differentiation of specific proteins in the seminal plasma (SP) of two horse breeds - Holsteiner (n=4) and East Bulgarian (n=4) and their relation with indivi­dual or breed characteristics, kinematic parameters of spermatozoa and the sperm head area. After CASA analysis of 8 ejaculates, no statistical differences in the kinematic parameters of the sperms between the two horse breeds were found out with the exception of the sperm head area (P&lt;0.05), which can be considered as a morphometric marker of breed affiliation. The values ​​for rapid sperm in East Bulgarian and Holsteiners were 28.1±0.2 μm2 and 19.9±0.3 μm2 respectively. The chromatographic analysis demonstrated specific quantitative and qualitative protein content of the individual chromatographic peaks (11 for Holsteiner and 15 for the Eastern Bulgarian breed), with similarity to the basic proteins. Three specific proteins with a molecular mass of 76 kDa, 21.6 kDa and 24.3 kDa, were differentiated by SDS PAGE in the Holsteiner breed, whereas in the Eastern Bulgarian horse breed they had a lower protein mass - 30.1 kDa and 14.2 kDa and 12.6 kDa. In conclusion, differences in the specific protein profile of Holsteiner and Eastern Bulgarian horse breeds are individually and naturally determined without significant effect on sperm kinematics. The sperm head area was a breed-specific difference.

Study the Morphometry of Fresh and Cryopreserved Murrah Bull Spermatozoa

Artificial insemination with frozen – thawed spermatozoa was introduced in most of the developing countries more than three decades ago, yet it has not been successfully applied in large scale (Anzar et al., 2003). More than 50% spermatozoa are usually injured by the cryopreservation process (Watson, 1995) injuries due to cryopreservation and more likely due to the increased solute concentration and the formation of intracellular and extracellular ice crystals during cryopreservation (Mazur, 1984) leading to a significant decline in semen quality and alterations in sperm morphometrix. Therefore, the present study was designed to study cryopreservation of buffalo semen and the effect of semen cryopreservation on determinant like semen fertility viz. sperm morphometry. Forty-eight semen samples collected from eight Murrah buffalo bulls maintained at Central semen station, Bhopal were included for the study of physic-morphological characters of neat and cryopreserved semen. Various measurements of the sperm head (length, width, base, ellipticity, elongation, head shape and area) and sperm tail (length of Midpiece and length of tail) were recorded in neat and cryopreserved semen. The average sperm measurements recorded were 7.24 ± 0.06, 4.26 ± 0.05, 2.65 ± 0.79, 11.92 ± 1.53, 43.12 ± 2.79, 61.97 ± 0.50, 25 ± 0.07, 19.13 ± 2.58, 0.59 ± 0.01, and 59.07 ± 0.94, respectively. There was a significant (P<0.05) decline of sperm head length, head width, midpiece length, tail length, total length and head area during the process of cryopreservation. However, no significant difference between width at base, ellipticity, head shape and elongation of the spermatozoa in neat and cryopreserved semen was recorded.

Effect of different concentrations of resveratrol on the quality and in vitro fertilizing ability of ram semen stored at 5 °C for up to 168 h

The aim of the study was to investigate the effects of different concentrations of resveratrol on head morphology, motility characteristics, oxidative state and in vitro fertility of cooled ram spermatozoa. Pooled semen from three Najdi rams was diluted with Triladyl® having different concentrations of resveratrol, zero (control), 200 μM (45.65 μg/mL) and 400 μM (91.30 μg/mL) resveratrol, then stored at 5 °C for 168 h. The head morphometric, sperm kinematic parameters, Malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD) and in vitro fertilizing capability of ram spermatozoa were evaluated after 24, 72, 120 and 168 h of cooling storage. The total motility (TM) of the sperm with resveratrol at 200 μM and 400 μM was significantly (p ≤ 0.05) higher than that in the control group at 72 and 120 h of cooling storage. On the other hand, the progressive motility (PM) of the sperm with resveratrol at 200 μM and 400 μM was significantly (p ≤ 0.05) higher than that in the control group at 168 h of cooling storage period. After 168 h of cooling storage, significantly higher straightness (STR) was observed in 400 μM group than two other groups and in 200 μM group than the control group. Both resveratrol groups had higher linearity (LIN) than control one at 120 and 168 h of cooling storage. The length, width and area of sperm head were lower (P ≤ 0.05) in the control compared to the other treatment groups after 120 and 168 h of storage. There was a significant increase in superoxide dismutase (SOD) concentration in the two resveratrol groups compared with the control one over the seven days of cooling storage and the same result was found in the reduced glutathione (GSH) concentration at 24, 72, and 168 h of storage. There was a significant (p ≤ 0.05) decrease in malondialdehyde (MDA) concentration in the 400 μM resveratrol group than that in two other groups over the seven days of storage period. Cleavage and blastocyst rates following in vitro fertilization were significantly higher (p ≤ 0.05) in 400 μM resveratrol than other groups at 72 h for cooling storage period. In conclusion, addition of resveratrol in the extender can protect sperm head morphology, improve kinematic parameters and in vitro fertility, and reduce oxidative stress of ram spermatozoa during liquid storage at 5 °C.

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.

Aluminum, at an environmental concentration, associated with acidic pH and high water temperature, causes impairment of sperm quality in the freshwater teleost Astyanax altiparanae (Teleostei: Characidae)

Given the toxicity of metals, including aluminum (Al), and the effects of water temperature on ectotherms, we investigated the individual or association effect of these variables (Al + acidic pH + temperature changes) on sperm quality of Astyanax altiparanae. Mature males were divided into nine experimental groups based on the combination of each of three water temperatures (20, 25, and 30 °C) with neutral and acidic pH values (7.0 and 5.5, respectively) with or without 0.5 mg L−1 Al. The fish were subjected to subacute, semi-static exposure and at 24 and 96 h were evaluated for seminal parameters: (1) pH; (2) osmolality; (3) sperm concentration; (4) sperm morphology; (5) sperm kinetics; and (6) sperm ultrastructure. At 30 °C, Al caused a reduction in osmolality (24 and 96 h) and sperm concentration (24 h). When analysing sperm kinetics (30 s post-activation), Al caused a reduction in total motility at all temperatures (24 h), and when this exposure time was longer (96 h), both acidic pH and Al addition to the water caused sperm motility reduction. By analysing curvilinear velocity (VCL) 30 s after sperm activation (24 and 96 h), the acidic pH caused a reduction in sperm movement at 20 and 30 °C, but at 25 °C Al triggered this reduction. Finally, Al in the water caused ultrastructural changes in the sperm head, midpiece, and flagella regardless of water temperature. Also, it was found that the combination of Al at 30 °C caused a reduction in sperm head area while at 20 °C, Al triggered a reduction in the midpiece area. Therefore, acidity influenced some A. altiparanae sperm parameters but Al in the water accentuated these effects on seminal quality, especially seminal osmolality and sperm concentration, kinetics, and ultrastructure. This toxicity was also influenced by changes in water temperature.

The effect of varicocele on semen quality in boars exposed to heat stress.

Semen quality has a dramatic impact on reproductive efficiency in the swine industry, influencing both conception rate and litter size. The objective of this study was to assess whether the presence of varicocele hinders semen quality in both thermoneutral and heat stress (HS) conditions. At approximately 6 mo of age, ultrasonography was used to measure left and right pampiniform plexus area in order to detect varicocele in maternal line boars at the University of Nebraska–Lincoln. Between 10 and 12 mo of age, semen was collected from each boar (n = 28) twice weekly. Boars were collected under thermoneutral conditions, were then heat stressed for 7 d to exacerbate any semen quality issues, and semen was collected post-HS for 6 wk. Sperm characteristics were determined by computer-assisted semen analysis. The presence of varicocele had a significant effect on sperm concentration (P = 0.04) and trended toward significance for mean sperm head area (P = 0.06) throughout the duration of the study. An interaction existed between varicocele and collection time point at weeks 2–5 post-HS for distal droplet percentage, suggesting that boars with varicocele were possibly more susceptible to heat-stress-induced semen quality issues than boars without varicocele. Moreover, semen quality was reduced in boars with versus without varicocele under both thermoneutral and HS conditions. Therefore, detection of varicocele by ultrasound could represent a potential marker of fertility in young boars or as a component trait in selection indices for fertility.

Effectiveness of ultra-rapid cryopreservation of sperm from endangered species, examined by morphometric means

This study compares the effectiveness of the ultra-rapid and conventional freezing of sperm from captive bovids, giraffids, cervids, ursids, a cercopithecid, a delphinid and a phascolarctid. The relationship between sperm head dimensions and cryosurvival was also examined. Compared to conventional freezing, the ultra-rapid freezing of epididymal sperm from the dama gazelle, giraffe and brown bear returned higher cryoresistance ratios (CR, the ratio, in percentage, between the value of the variable after thawing/value before thawing) for sperm viability and motility. In the remaining species, the conventional freezing of epididymal sperm returned better CR values. The conventional freezing method also returned better CR values for ejaculated samples from all species. The head dimensions of both fresh epididymal and ejaculated sperm differed widely among species: for epididymal sperm, dolphin sperm heads were the smallest (7.189 ± 0.049 μm2) and dama gazelle sperm heads the largest (43.746 ± 0.291 μm2), while for ejaculated sperm, giant panda sperm heads were the smallest (15.926 ± 0.150 μm2) and mouflon sperm heads the largest (38.258 ± 0.104 μm2). However, no significant correlations were detected between the CR for motility, viability, membrane functional integrity or acrosome integrity and the sperm head area, either for epididymal or ejaculated sperm. In conclusion, ultra-rapid freezing is especially recommended for the cryopreservation of dama gazelle, giraffe and brown bear epididymal sperm. Sperm head dimensions appear not to be useful predictors of how well sperm might survive freezing.

Seasonal variation in sperm freezability associated with changes in testicular germinal epithelium in domestic (Ovis aries) and wild (Ovis musimon) sheep.

The aim of this study was to examine ovine sperm cryoresistance during the rutting season (RS) and its association with sperm head area and seminiferous epithelium proliferation. Small ruminants show fluctuating testosterone levels throughout the year, which could interfere with spermatogenesis and sperm cryopreservation. Ejaculates, testicular biopsies and blood were collected during the middle and at the end of the RS (Middle-RS vs End-RS) during periods of high and low testosterone levels in Merino and Mouflon rams. Fresh and frozen-thawed sperm quality, sperm morphometry, seminiferous tubule morphometry and testicular proliferation markers (proliferating cell nuclear antigen, proliferation marker protein Ki-67 and transcription factor GATA-4) were evaluated. Post-thaw sperm viability was higher in the End-RS group in both Merino (69.9±8.2 vs 41.6±7.3%; P=0.020) and Mouflon rams (40.9±3.3 vs 24.2±5.0%; P=0.008). Mouflons had larger sperm head area at the End-RS (38.3±0.2 vs 34.3±0.1µm2; P=0.029), whereas there was no difference between Merino groups (35.7±0.5 vs 34.8±1.0µm2). Seminiferous tubule morphometry and proliferation markers showed higher levels of germinal epithelium proliferation in the Middle-RS of both species. In conclusion, sperm freezability is affected during the RS in domestic and wild rams, which could be correlated with changes that occur during spermatogenesis, since there is an effect of season on cell proliferation in the testis.

Area of ram sperm head modified during the addition of cryoprotectant agents

Area of ram sperm head modified during the addition of cryoprotectant agents

177 Equine Sperm Decondensation and Blastocyst Formation After Conventional versus Piezo-Driven Intracytoplasmic Sperm Injection

Intracytoplasmic sperm injection (ICSI) is currently the most effective method for in vitro fertilization in the horse. There are 2 main techniques, conventional (Conv) ICSI and piezo-driven (Piezo) ICSI. Many laboratories reporting good equine blastocyst rates (&gt;20% per injected oocyte) use Piezo ICSI, but it is not known whether the Piezo confers an advantage. We compared sperm decondensation and blastocyst formation between the 2 techniques. A blunt, 6-µm inner diameter needle, loaded with 10 µL of Fluorinert was used for Piezo ICSI; 1 pulse was used to penetrate the oolemma. Frozen-thawed semen from one stallion was used. In experiment 1, in vitro-matured equine oocytes were randomly assigned to Conv or Piezo ICSI, performed concurrently by separate operators. Blastocyst formation was evaluated on Days 7 to 10 and confirmed by DAPI staining. Data were analysed by Fisher’s exact test. There was no significant difference in blastocyst rates (32/82, 39% for Conv and 35/87, 40% for Piezo; P &gt; 0.1). In experiment 2, equine sperm head decondensation after ICSI was evaluated using porcine oocytes, due to scarcity of equine oocytes. Porcine oocytes were recovered from slaughterhouse-derived ovaries and matured in vitro using a biphasic maturation culture system. Mature oocytes were subjected to Conv or Piezo ICSI, using equine sperm treated with a mitochondrial stain to allow identification of the sperm tail. The oocytes were fixed in 4% paraformaldehyde immediately after injection (0 h) or were cultured for 3, 6, 9, or 18 h after injection, and then fixed. Fixed oocytes (15-22 per treatment per period) were stained with DAPI and the area of the sperm head, in arbitrary units, determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The medians were compared using the Mann-Whitney U-test. Sperm heads in the Piezo treatment increased in area over time, from a median of 1242 at 0 h to 54,991 at 18 h. In contrast, sperm heads in the Conv treatment largely failed to decondense, having median areas of 1275 at 0 h and 1510 at 18 h. Sperm head area was significantly greater for sperm in the Piezo than in the Conv treatment for all time periods except 0 h (P &lt; 0.05). Because this conflicted with the blastocyst results obtained with horse oocytes, we conducted experiment 3 to examine sperm head decondensation after ICSI in horse oocytes. Oocytes (8 to 12 per treatment per period) were fixed 0, 6, or 18 h after ICSI. There was no difference between techniques in sperm head area at any time (median values for 0, 6 and 18 h of 1280, 4323, and 57,185 respectively for Piezo and 1326, 1604, and 62,558 for Conv; P &gt; 0.2). These results indicate that there is a species-specific difference in processing of sperm after ICSI, dependent on injection technique. Further work evaluating sperm from additional stallions, as well as porcine sperm, is necessary to determine whether sperm source affects these results. Research supported by the Clinical Equine ICSI Program, Texas A&amp;M University.