Taurine does not improve the quality of short‐term stored rabbit spermatozoa in vitro

This study examines the impact of taurine on the viability, morphology and acrosome integrity of rabbit spermatozoa in vitro. Semen samples, obtained from four to five sexually mature and healthy New Zealand White rabbits, were pooled in heterospermic semen sample. This was divided and treated with taurine in a concentration of 0 (control), 1.5, 7, 12.5, 50 mM to a final concentration of 10⁸ spermatozoa/ml. The samples were then incubated at 37°C for 4 hr. A combination of fluorescent probes SYBR‐14/propidium iodide/PNA‐Alexa Fluor 647 was used to assess spermatozoa viability and acrosome integrity on a flow cytometer. The sperm morphology was evaluated under a light microscope following fixation in 1.5% paraformaldehyde. The experiment was repeated three times. According to the obtained results, the spermatozoa neither could have benefit from immediate taurine treatment, nor had they after 4‐hr incubation with respect to viability and acrosome integrity. Taurine did not initially alter the total and acrosome morphology of treated spermatozoa nor has it by 4 hr upon treatment. In conclusion, taurine may have no protective effect on the viability, morphology and acrosome integrity of short‐term stored rabbit spermatozoa.     

Stress preconditioning of boar spermatozoa: a new approach to enhance semen quality

Semen preparation and cryopreservation require finely adjusted procedures. Gametes are sensitive to environmental stresses, so in vitro procedures aim to minimize the inevitable harmful conditions. Applying stress to precondition cells has only been investigated recently. Studies demonstrated that by utilizing a well defined and properly applied hydrostatic pressure (HP) stress treatment to spermatozoa before in vitro storage, cryopreservation or insemination, cell survival and fertility improved compared with untreated controls. The birth of healthy piglets from treated fresh or frozen-thawed semen demonstrates the in vivo safety of the procedure. Although the biological mechanism is still unclear, several processes incorporating cellular stress response might explain the observations. This paper summarizes results, background, aspects and considerations of HP treatment for porcine semen. The new principle, i.e. to improve the stress tolerance by a defined sublethal stress may outline a new strategy in assisted reproductive technologies with unique theoretical and practical consequences.     

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

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

Reducing growth rate of broiler chickens with a low energy diet does not improve cortical bone quality

1. Reducing growth rate is often supposed to allow better skeletal development and improve bone quality. In order to test this assumption, flocks of broiler chickens fed on a diet containing 13.31 MJ ME/kg (H) were compared with flocks in which the growth rate was reduced by feeding birds with a low energy diet (L, 9.62 MJ ME/kg). 2. Bone growth was studied in 2 experiments in order to identify any changes in bone tissue of slow-growing chickens that might explain why they are less predisposed to varus-valgus deformities (3.1% vs 19.9%). 3. In experiment 1, the morphology, composition and histomorphometry of the tibiotarsi were analysed in both groups at 1, 12, 26, 42 and 46 d of age. Tibial variables (weight, volume, length, cortex size and structure, dry content and ash percentage) were always reduced in slow-growing birds except at 42 d of age, when tibial weight and cortex areas were similar in both groups. 4. Every variable was similar in both groups, or even higher in H birds, when chickens were compared at similar body weight, suggesting that the low growth rate did not improve bone quantity or quality of the tibiae. 5. In experiment 2, the composition of the tibiotarsi was compared every 2 days during the first week and twice a week until 36 d of age, in H and L birds. Weight and composition of the tibiotarsi did not differ between groups when compared at equal body weight, as also shown in experiment 1. 6. The reduced occurrence of varus-valgus deformities in slow-growing chicks cannot be related to an improvement in the structure and the composition of their bone tissue.     

Red‐light stimulation of boar semen prior to artificial insemination improves field fertility in farms: A worldwide survey

A survey of in vivo fertility data from 31 pig farms distributed worldwide was conducted to determine whether stimulating boar semen with LED‐based red light increases its reproductive performance following artificial insemination (AI). Red‐light stimulation with MaXipig^® was found to increase farrowing rates (mean ± SEM, control: 87.2% ± 0.4% vs. light stimulation 90.3% ± 0.5%) and the number of both total and live newborn piglets. Red‐light stimulation increased farrowing rates in 27 farms, with an increase ranging from 0.2% to 9.1%. Similar results were observed in litter sizes. Suboptimal management after AI was suggested in those farms with no response to red‐light stimulation. Our results indicate that a routine use of red‐light stimulation of boar semen can have a positive effect on the reproductive performance. However, the effectiveness of this system appears to highly rely upon proper management of pig farms.     

Relationship of cysteine‐rich secretory protein‐3 gene and protein with semen quality in stallions

Cysteine‐rich secretory protein‐3 (CRISP‐3) and some of its nonsynonymous polymorphism have been related to the fertility and freezability of stallion semen; however, the role of the CRISP‐3 gene and its seminal plasma protein in the raw semen quality is still unknown. The aim of this study was to evaluate the relationship of CRISP‐3 with semen quality in stallions. DNA was obtained from blood samples of 100 stallions, from which 30 stallions were randomly selected to obtain 60 ejaculates. Through PCR amplification and sequencing, the variation of four nonsynonymous SNPs from CRISP‐3 was identified and haplotypes were derived. Semen quality was assessed through the total motility (MOT), sperm vitality (SV), normal morphology (NM), functional integrity of membrane (MI) and a seminal quality index (SQi). CRISP‐3 protein content of seminal plasma (SP) was determined by ELISA. The effect of the genotype, the haplotype and the concentration of the CRISP‐3 protein on the seminal quality were evaluated through generalized linear models and linear regression analyses. Homozygous genotypes for SNP1, SNP2 and SNP3 and the heterozygous genotype for SNP4 showed a positive effect on seminal quality. Different haplotypes with positive effect on MOT, SV, NM, MI and SQi were identified. The allelic substitution analysis resulted in positive regression coefficients for MOT (SNP2) and MI (SNP2 and SNP3). A high level of CRISP‐3 resulted in a higher MOT and SQi. It is concluded that the quality of stallion semen is influenced by the genotype of CRISP‐3 and the concentration of CRISP‐3 protein in SP.     

Fertilizing ability of canine spermatozoa cryopreserved with skim milk‐based extender in a retrospective study

We previously reported that skim milk (SM) is an effective cryoprotectant for cryopreservation of canine spermatozoa instead of egg yolk (EY), which is the conventional cryoprotectant. In this study, the fertilizing ability and practical use of frozen canine spermatozoa prepared with SM were evaluated by transcervical insemination. Frozen‐thawed spermatozoa were inseminated one to four times on days 2–9 after the LH surge. In SM group, a single transcervical insemination (TCI) on Day 5 led to higher delivery rate (83%) than any other days (33%–50%) post‐LH surge. In EY group, delivery rate in double TCI on days 5 and 6 (71%) was higher compared to any other experimental groups (0%–44%). Regardless of single or double, TCI on Day 5 or Day 6 led to higher litter sizes in SM or EY groups, respectively. The breeding efficiency and litter size of single TCI on Day 5 (4.2) and double TCI on Day 5 and Day 6 (3.7) were significantly higher than in the other experimental groups in SM and EY groups, respectively (p < .05). These findings suggest that skim milk is a suitable alternative to egg yolk for cryopreservation of canine spermatozoa, and the suitable timing for insemination might be on Day 5 post‐LH surge.     

Beta‐mercaptoethanol supplementation of in vitro maturation medium does not influence nuclear and cytoplasmic maturation of equine oocytes

In vitro embryo production in the horse is still not as efficient as in other species. Oxidative stress negatively affects oocyte and embryo culture. To attenuate/minimize the oxidative stress, antioxidants such as low‐molecular thiol compounds can be added to culture media. Beta‐mercaptoethanol (BME) has been shown to improve maturation and embryo development in different species. The aim of this study was to investigate whether the addition to maturation medium of BME at common (0.1 mM) and high (0.7 mM) concentration could improve oocyte maturation also in the horse. Equine oocytes recovered from slaughterhouse ovaries were used. Meiotic configuration after in vitro maturation (IVM) and early embryo production after intracytoplasmic sperm injection (ICSI) were considered as criteria for assessing nuclear and cytoplasmic maturation, respectively. A total of 1,076 oocytes were analysed over two experiments: 848 (control n = 293, BME 0.1 n = 270, BME 0.7 n = 285) were stained with Hoechst 33342 and examined for nuclear stage after 26 hr of IVM, and 228 MII oocytes were fertilized by ICSI (control n = 83, BME 0.1 n = 65, BME 0.7 n = 80). Cleavage rates were determined after 60 hr of culture. Unlike results obtained in other species, the addition of BME did not influence maturation rates (51.9% control vs 55.6% BME 0.1 mM and 55.1% BME 0.7 mM), nor cleavage rates after ICSI (38.6% vs 38.5% and 41.3%, respectively). In conclusion, the addition of BME at 0.1 and 0.7 mM to the maturation medium, in our culture conditions, has no effect on nuclear and cytoplasmic maturation of equine oocytes.     

Comparison of in vitro and in vivo fertilizing potential of buffalo bull semen frozen in egg yolk‐, soya bean lecithin‐ and liposome‐based extenders

The objective of this study was to compare different extenders for post‐thaw in vitro sperm function and in vivo fertility of buffalo semen. Accordingly, sperm of 30 ejaculates extended in egg yolk (TRIS with 20% egg yolk; EY), two soya lecithin‐based (SL‐1; AndroMed^® and SL‐2; Bioxcell^®) and a liposome‐based extender (LS; OptiXcell^®) were tested. The post‐thaw semen was evaluated for computer‐assisted sperm analysis (CASA), sperm viability, membrane and acrosome integrity, DNA integrity and acrosome reaction and first service pregnancy rate (FSPR) in a fixed‐time artificial insemination programme. Total motility and VCL were the only CASA‐based parameters that exhibited significantly higher (p < .05) percentage in LS among these extenders. Post‐thaw percentage of acrosome integrity (55.9 ± 1.4, 58.1 ± 2.0, 55.8 ± 2.0, 56.6 ± 2.3) and DNA integrity (68.8 ± 2.0, 69.2 ± 2.3, 71.3 ± 2.1, 69.1 ± 2.1) did not differ (p > .05) in EY, SL‐1, SL‐2 and LS extender, respectively. However, a variable response in terms of efficacy of different extenders for sperm viability and plasma membrane integrity was observed. Assessment of inducibility of acrosome reaction showed significant differences between extenders (51.9 ± 2.1, 44.3 ± 2.4, 46.1 ± 2.3 and 58.1 ± 3.1%, respectively, for EY, SL‐1, SL‐2 and LS). Furthermore, field trials revealed significantly higher (p < .05) FSPR of LS‐extended semen as compared to that for EY, SL‐1 and SL‐2 extender (46.3%, 41.2%, 31.2% and 29.7%, respectively). It is concluded that the liposome‐based extender is more effective than egg yolk‐ and soya lecithin‐based extenders and may be used for cryopreservation of buffalo semen in the future.     

Faecal calcium excretion does not decrease during long‐term feeding of a low‐calcium diet in adult dogs

According to a previous meta‐analysis, adult dogs do not notably increase calcium absorption from the gastrointestinal tract when calcium intake is decreased. This results in a negative calcium balance even with a moderate calcium reduction. In this study we wanted to verify (i) whether a negative calcium balance occurs at a calcium intake equivalent to NRC (2006) (Nutrient requirements of dogs and cats, 2006, The National Academies Press, Washington, DC) minimal requirements, and if so (ii) whether the negative calcium balance will persist for up to 6 months on a low‐calcium diet. After a pre‐feeding period of at least 18 weeks with calcium intake slightly exceeding maintenance requirements (200 mg/kg body weight^0.75), 12 dogs (6 Beagles, 6 Foxhound crossbreds) were fed a low‐calcium diet for 28 weeks. One dog was removed from the trial for reasons unrelated to the study at week 23. Calcium intake amounted to 60 mg/kg body weight^0.75 corresponding to the minimal requirement for maintenance in dogs (NRC, 2006 (Nutrient requirements of dogs and cats, 2006, The National Academies Press, Washington, DC)). Digestion trials were carried out at week 7, 14, 21 and 28 of the low calcium feeding period. At these time points, and at week 18 of the pre‐trial, blood samples were taken and analysed for calcium, ionised calcium, phosphorus, parathyroid hormone, vitamin D, serum crosslaps and bone alkaline phosphatase. Apparent calcium digestibility was negative throughout the study, suggesting a negative calcium balance. There was no systematic decrease in faecal calcium excretion. Serum calcium, ionised calcium and phosphorus remained within the reference range. Serum crosslaps increased continuously from baseline to week 28 of trial, with averages increasing from 0.102 ng/ml to 0.279 ng/ml, suggesting osteoclastic activity, indicative of calcium mobilisation from the skeleton. The study supports the theory of a lack of adaptation of intestinal calcium absorption from diets with relatively low calcium content in dogs. This agrees with clinical findings in dogs eating low‐calcium diet.     

Resveratrol beneficially affects meat quality of heat‐stressed broilers which is associated with changes in muscle antioxidant status

This study was conducted to evaluate the effect of dietary resveratrol (Res) supplementation on serum parameters, meat quality and muscle antioxidant status of broilers under heat stress (HS). A total of 270 21‐day‐old male Cobb broilers were randomly assigned to three treatment groups with six replicates of 15 birds each. The three treatment groups were as follows: the control group, in which birds were reared at 22 ± 1°C, and the HS and HS + Res (400 mg/kg) groups, in which birds were reared at 33 ± 1°C for 10 h (08.00–18.00 h) and 22 ± 1°C for the rest of the time. Compared with birds in the control group, birds in the HS group exhibited increased serum corticosterone (CORT) and triacylglycerol contents, L*, drip loss and muscle malondialdehyde content, and decreased serum glucose content, pH_24 h, muscle total antioxidant capacity (T‐AOC), catalase (CAT) and glutathione peroxidase (GSH‐PX) activities (P < 0.05). Compared with birds in the HS group, birds in HS + Res group exhibited increased serum glucose content, a*, pH_24 h, muscle T‐AOC and CAT activities, and decreased serum CORT and triacylglycerol contents, L*_24 h, drip loss and muscle malondialdehyde content (P < 0.05). In conclusion, Res beneficially protects against HS‐impaired meat quality of broilers through regulating muscle antioxidant status.     

Insemination of sows with seminal doses prepared by a two-step hypothermic dilution does not impair the reproductive performance at farm

Reproduction in swine is mostly carried out through artificial insemination (AI). For this purpose, AI studs collect the ejaculates, analyse the sperm quality, dilute and package to produce seminal doses and ship them to sow farms to carry out the AI. Temperature is controlled during the process to avoid sperm damage. Semen is diluted in the extender in a one-step or a two-step process where the second can be isothermic (approximately 32°C) or hypothermic (room temperature 21–22°C). Both techniques are currently performed, and the latter could reduce time and costs, but the literature available comparing the processes is scarce and presents discrepancies. To date, there are no studies about its impact in fertility. This study compared hypothermic two-step dilution (HTSD) and isothermic two-step dilution (ITSD) in laboratory and field trial to elucidate whether HTSD has any effect. Ejaculates from 72 boars in nine AI studs were split and processed with both techniques using a high-performance extender and evaluated in laboratory. Four farms inseminated 345 sows with samples of four of these AI studs, and their fertility and prolificacy were registered. Results show no significant differences between doses prepared by HTSD and ITSD technique, having no impact in laboratory results (percentage of motile sperm, short hypoosmotic swelling test (sHOST) and short osmotic resistance test (sORT), viable sperm, damaged acrosomes, sperm under early apoptosis, high mitochondrial membrane potential (p > .1), fertility (92.2% versus 94.1%, p = .45) or farrowing rate (15.8 ± 0.3 versus 16.1 ± 0.3 p = .46). In conclusion, our results suggest that HTSD of semen on extender could be safely implemented in AI studs under the conditions tested.     

Cryopreservation of Nili‐Ravi buffalo (Bubalus bubalis) semen in AndroMed® extender; in vitro and in vivo evaluation

The study was designed to evaluate AndroMed^® for the freezability and fertility of Nili‐Ravi buffalo semen. Semen was collected from four adult Nili‐Ravi buffalo (Bubalus bubalis) bulls for 3 weeks (replicate). Semen ejaculates from each buffalo bull were divided into three aliquots. One aliquot was used for evaluation of motility, plasma membrane integrity, livability, viability, DNA integrity and normal apical ridge. Remaining two aliquots were diluted (37°C; 50 × 10⁶ spermatozoa/ml) in tris‐citric egg yolk or AndroMed^® extender and cryopreserved in 0.5 ml French straws. After thawing, per cent post‐thaw motility (47.9 ± 0.8, 49.2 ± 1.7), plasma membrane integrity (44.4 ± 1.2, 46.8 ± 1.8) and normal apical ridge (81.4 ± 0.3, 83.2 ± 0.3) were recorded similar (p > .05) in tris‐citric egg yolk and AndroMed^® extender. Higher (p < .05) percentage of sperm livability (70.5 ± 1.4 and 64.4 ± 1.0), viability (67.5 ± 1.5 and 61.5 ± 0.6) and DNA integrity (97.0 ± 0.3 and 93.4 ± 0.21) were recorded in AndroMed^® compared to tris‐citric egg yolk post‐thaw. Values for all the aforementioned spermatozoal quality parameters were observed lower (p < .05) in frozen‐thawed compared to fresh semen irrespective of the experimental extenders. Fertility rates of buffalo semen did not differ (p > .05) either cryopreserved in tris‐citric egg yolk or AndroMed^® extender (45.5% vs. 49%). It is concluded that AndroMed^® is capable in protecting the buffalo bull sperm during freeze‐thawing process and can be adopted safely for routine use replacing the tris‐citric egg yolk extender in artificial insemination programme.     

Investigation of in vitro measurable sperm attributes and their influence on electroejaculated bull semen with a fixed‐time artificial insemination protocol in Australian Bos indicus cattle

Increasing use of fixed‐time artificial insemination (FTAI) in beef cattle production has presented an opportunity for the use of fresh or chilled semen as an alternative to standard cryopreserved semen. The objective of this study was to examine in vitro sperm function and pregnancy rate of electroejaculated semen, chilled and stored for 48 hr, compared to conventionally cryopreserved semen with an optimized FTAI protocol in Brahman cattle. Semen from three Brahman bulls was collected, and aliquots were extended in either chilled (at 5°C) or frozen (LN₂) in a Tris‐egg yolk extender base with 2.4% or 7.0% glycerol, respectively. Semen samples were assessed 48 hr after collection or post‐thaw and warming, for sperm motility, in vitro sperm function and fertilizing ability, and used in a FTAI programme. The overall pregnancy rates was significantly different (p < .01) after FTAI with frozen (n = 173; 53.2%) and chilled semen (n = 174; 31.6%). In contrast, the in vitro sperm assessment showed that the chilled semen had significantly faster motility (p < .05), a higher proportion of progressively motile spermatozoa (p < .05), with significantly higher proportions of acrosome intact, viable spermatozoa (p < .01). This study showed that reasonable pregnancy rates in Brahman cattle can be achieved using FTAI with chilled semen collected using electroejaculation and stored for up to 48 hr. However, improvements in semen extenders are required in consideration of semen collection method to improve the longevity of sperm fertilizing ability to significantly increase FTAI output using chilled storage of bull semen.     

Differences between genomic‐based and pedigree‐based relationships in a chicken population,as a function of quality control and pedigree links among individuals

This work studied differences between expected (calculated from pedigree) and realized (genomic, from markers) relationships in a real population, the influence of quality control on these differences, and their fit to current theory. Data included 4940 pure line chickens across five generations genotyped for 57 636 SNP. Pedigrees (5762 animals) were available for the five generations, pedigree starting on the first one. Three levels of quality control were used. With no quality control, mean difference between realized and expected relationships for different type of relationships was ≤ 0.04 with standard deviation ≤ 0.10. With strong quality control (call rate ≥ 0.9, parent‐progeny conflicts, minor allele frequency and use of only autosomal chromosomes), these numbers reduced to ≤ 0.02 and ≤ 0.04, respectively. While the maximum difference was 1.02 with the complete data, it was only 0.18 with the latest three generations of genotypes (but including all pedigrees). Variation of expected minus realized relationships agreed with theoretical developments and suggests an effective number of loci of 70 for this population. When the pedigree is complete and as deep as the genotypes, the standard deviation of difference between the expected and realized relationships is around 0.04, all categories confounded. Standard deviation of differences larger than 0.10 suggests bad quality control, mistakes in pedigree recording or genotype labelling, or insufficient depth of pedigree.     

Comparative evaluation of DNA integrity using sperm chromatin structure assay and Sperm‐Ovis‐Halomax during in vitro capacitation of cryopreserved ram spermatozoa

This study aimed to compare the ability of sperm chromatin structure assay (SCSA^®) and Sperm‐Ovis‐Halomax^® to detect DNA fragmentation in frozen‐thawed ram spermatozoa incubated under capacitating conditions in synthetic oviductal fluid (SOF) supplemented with oestrous sheep serum (SOF‐ESS) at multiple time points (0–240 min). Incubation in SOF‐ESS had no significant effects on SCSA^® parameters while the percentage of spermatozoa with fragmented DNA measured by Sperm‐Ovis‐Halomax^® increased after 180 min of incubation. In addition, no correlation or agreement was found between the techniques, suggesting that SCSA^® and Sperm‐Ovis‐Halomax^® may quantify different types of DNA damage in ram spermatozoa under these experimental conditions.     

Development of a fluorescent computer‐assisted spermatozoal quantification method and a comparison of results for manual counting with a haemocytometer and computer‐assisted semen analysis in dogs

The aim of this study was to develop and validate a novel, computer‐assisted spermatozoal quantification (CASQ) method of determining spermatozoal concentration in canine semen. In Experiment A, the spermatozoal concentration was measured (n = 28) with a haemocytometer using light microscopy, CASQ and computer‐assisted semen analysis (CASA; MMC sperm), following three independent dilutions. The limits of agreement between the haemocytometer and CASQ were ?13.1% to 13.8% and ?27.0% to 28.6% between the haemocytometer and CASA. The precision CVs (limits of agreement) were 5.7% (?7.8% to 8.9%) for the haemocytometer, 6.2% (?8.8% to 12.3%) for CASQ and 10.8% (?16.0% to 19.5%) for CASA. In Experiment B, spermatozoa were manually counted (n = 42) with the haemocytometer under fluorescent illumination using the CASQ sample. The limits of agreement between the CASQ and the haemocytometer were satisfactory (?4.6% to 4.6%) and the precision CVs (limits of agreement) were 6.2% (?9.0% to 11.4%) for the haemocytometer and 4.4% (?5.8% to 8.6%) for CASQ. The CASQ method was then clinically applied to compare the haemocytometer (light and fluorescent methods) with CASQ and CASA. Outlying data were removed. These studies demonstrated that CASQ was reliable and that the MMC sperm CASA was unreliable as methods for determining spermatozoal concentration in canine semen. Computer‐assisted spermatozoal quantification was also determined to be more precise than manual counting with the haemocytometer. Using the clinical protocol, the agreement between the haemocytometer and CASQ method was acceptable, but it was worse than in the experiments where duplicate samples and a larger volume of semen were analysed. The CASQ method may be a useful method to measure the membrane status of canine spermatozoa; however, further investigation is required. Counting spermatozoa using fluorescent microscopy and the haemocytometer may improve the efficiency of counting and the accuracy of the method.     

“In vitro” capacitation and further progesterone‐induced acrosome exocytosis are linked to specific changes in the expression and location of threonine phosphorylation of boar spermatozoa

The aim of this study was to determine if the achievement of the “in vitro” capacitation (IVC) status and subsequent progesterone‐induced “in vitro” acrosome exocytosis (IVAE) was accompanied with overall changes in threonine phosphorylation (pThre) of boar spermatozoa. For this purpose, mono‐ and bi‐dimensional Western blot analyses as well as immunocytochemistry studies against pThre were performed in boar sperm subjected to IVC and subsequent IVAE. Mono‐dimensional Western blot in non‐capacitated samples showed that launching of IVC did induce an overall increase in signal intensity in all observed bands that was followed by a subsequent decrease afterwards. Bi‐dimensional Western blot analysis showed the presence of four main signal protein clusters. The attainment of IVC induced an overall decrease in the number and intensity of spots of Clusters A, B and C and a concomitant increase in the intensity of spots of Cluster D. The IVAE launching caused a rapid increase in the intensity of spots of Clusters B, C and D, which was followed by a subsequent decrease of the intensity together with a concomitant pI displacement of Cluster C. Finally, immunocytochemistry showed that the pThre signal of non‐capacitated cells was located at the whole sperm. The IVC did not induce prominent changes in this location. In contrast, the induction of IVAE caused the appearance of an additional an intense acrosome and tail pThre signal that subsequently decreased. In conclusion, our results indicate that IVC and further IVAE induced specific changes in the intensity and appearance of pThre protein phosphorylation which were linked to changes of specific protein characteristics as pI. These results support, thus, the existence of a specific role of pThre in IVC/IVAE of boar sperm.