Computer-assisted Semen Analysis Research Articles

20 Effects of a stabilized fish oil on boar semen quality when fed during summer months

Abstract The objective of this study was to investigate the effects of feeding a stabilized fish oil source to different genetic lines on boar semen quality. A total of 127 boars representing three genetic lines (PIC 337, PIC 800, and PIC L03; Hendersonville, TN) at a commercial boar stud were balanced by age, body condition, and previous semen quality and randomly allotted to one of two dietary treatments within genetic line. Boars were assigned to either receive Control; a daily top dress of 32g of corn starch; or Fish oil; a daily top dress of 32g of Salmate DHA+ (The Ballard Group, 2407 Ashland Ave., Cincinnati, OH 45206 USA) to provide 2.38 g of docosahexaenoic acid (DHA). The top-dress supplementation was provided by placing it on top of the daily ration of feed for each individual boar on the floor once each day immediately after their feed has been delivered to them. Supplementation lasted for 16 wk from June-September, 2023 during which weekly semen collection was performed. Semen was evaluated at the stud for concentration and motility parameters using computer assisted semen analysis. Data was analyzed using generalized linear mixed models with the lmer and glmer functions of R. Boar was the experimental unit, with dietary treatment, genetic line, and their interaction tested as fixed effects while age at collection served as a covariate. Boar nested within treatment combinations was used as a random effect to account for repeated measurements within the same boar. There was no evidence (P > 0.10) for dietary treatment by genetic line interaction for any response criteria (Table 1). There was no evidence of fish oil supplementation effects (P > 0.10) on semen volume, concentration, total sperm cells per ejaculate, percentage of accepted ejaculates, or number of doses produced per accepted ejaculate. There was no evidence of fish oil supplementation effects (P > 0.10) on percentage motile sperm cells, or percentage of proximal and distal droplets and abnormal tails. Line 800 boars had the least (P < 0.05) semen volume, but greatest (P < 0.05) sperm concentration, resulting in no evidence for differences (P > 0.10) between the three genetic lines in total sperm cells per ejaculate. Line 03 boars had increased (P < 0.05) percentage of rejected ejaculates as well as decreased number of doses produced per accepted ejaculate (P < 0.05) compared with lines 337 and 800 boars. There was no evidence for differences (P > 0.10) in sperm motility, proximal and distal droplets, or abnormal tails between the three genetic lines. In summary, maternal line boars had an increased proportion of rejected ejaculates per boar and a decreased number of doses produced compared with terminal line boars. Feeding a stabilized fish oil to provide 2.38 g of DHA did not improve semen during summer months.

Potential use of dimethyl sulfoxide (DMSO) in artificial insemination of rabbits

The aim of this work was to analyse the effect of incubation dimethyl sulfoxide (DMSO) with insemination dose (ID) on reproduction parameters (kindling rate, number of live and still-born kits and birth weight of kits). The experiments were carried out under the conditions of two rabbit breeds. An ejaculate was diluted to concentration of 40 to 50 million/0.5 ml/ID. DMSO was added to the ID in concentration of 0.5 M DMSO. Control ID did not contain any DMSO. Kindling rates obtained from 2 different breeds (P1, P2) showed minor improvement (+4.36% P1; +4.06% P2) between the monitored groups of females, however without statistically significant differences. Number of live born kits in litter obtained from breeds P1 and P2 showed minor improvement (+0.61 pcs P1; +0.13 pcs P2) compared to the control groups (p>0.05). The weight of live born kits was higher in experimental groups (+3.85 g P1 = p≤0.001; +1.18 g P2 = p>0.05) compared to the control groups. Monitoring of sperm quality indicators (1, 3 and 6 hours incubation) in vitro conditions (CASA – Computer Assisted Semen Analyzer) showed the best results in the control group of non-affected DMSO. The data obtained from our experiments suggest the possitive effects of DMSO incubation on reproduction parameters especially in vivo.

The Role of Artificial Intelligence in Male Infertility: Evaluation and Treatment: A Narrative Review

Male infertility has affected an increasingly large population over the past few decades, affecting over 186 million people globally. The advent of assisted reproductive technologies (ARTs) and artificial intelligence (AI) has changed the landscape of diagnosis and treatment of male infertility. Through an extensive literature review encompassing the PubMed, Google Scholar, and Scopus databases, various AI techniques such as machine learning (ML), artificial neural networks (ANNs), deep learning (DL), and natural language processing (NLP) were examined in the context of evaluating seminal quality, predicting fertility potential, and improving semen analysis. Research indicates that AI models can accurately estimate the quality of semen, diagnose problems with sperm, and provide guidance on reproductive health decisions. In addition, developments in smartphone-based semen analyzers and computer-assisted semen analysis (CASA) are indicative of initiatives to improve the price, portability, and accuracy of results. Future directions point to possible uses for AI in ultrasonography assessment, microsurgical testicular sperm extraction (microTESE), and home-based semen analysis. Overall, AI holds significant promise in revolutionizing the diagnosis and treatment of male infertility, offering standardized, objective, and efficient approaches to addressing this global health challenge.

Post-thaw quality of boar spermatozoa is affected by ejaculate fractions and extenders.

The aim of this study was to investigate the effect of different extenders on the post-thaw (PT) quality of sperm originating from the sperm-rich fraction (SRF) and post-sperm-rich fraction (PSRF) of boar ejaculate. Motility and velocity parameters, analyzed using a computer-assisted semen analysis (CASA) system, and membrane integrity parameters were markedly higher in frozen-thawed (FT) spermatozoa of the SRF in both the Belstville Thawing Solution (BTS) and Androhep Plus (AHP) extenders, irrespective of the post-thaw (PT) storage time. Furthermore, reduced cryo-survival was more marked in FT spermatozoa of the PSRF in both extenders following storage for 60 min. It was found that the SRF-stored samples in the AHP extender for 60 min exhibited significantly higher percentages of spermatozoa with total motility, mitochondrial function and acrosome integrity than those stored in the BTS extender. The findings of this study confirm that components of the ejaculate fractions and extender have varying effects on the cryo-survival of boar spermatozoa.

Seminal cell-free DNA as a potential marker for in vitro fertility of Nellore bulls.

This study aimed to identify a marker for freezability and in vitro fertility of sperm samples before freezing. Semen was collected from nine Nelore bulls; half of the ejaculate was used for seminal plasma cell-free DNA (cfDNA) quantification, and the other half was cryopreserved. Evaluation of sperm movement using computer-assisted semen analysis and plasma membrane integrity and stability, acrosomal integrity, apoptosis, and mitochondrial potential using flow cytometry were performed on fresh and frozen/thawed semen at 0, 3, 6, and 12h after thawing. Frozen/thawed sperm was also used for in vitro embryo production. cfDNA was extracted from each bull, and the total DNA and number of cell-free mitochondrial DNA (cfmtDNA) copies were quantified. Semen from each animal was used for IVF, and cleavage, blastocyst formation, and cell counts were evaluated. Two groups were formed and compared based on the concentrations of cfDNA and cfmDNA present: low-cfDNA and high-cfDNA and low-cfmtDNA and high-cfmtDNA. Up to 12h post-thawing, there were no differences between the groups in the majority of the sperm parameters evaluated. Cleavage, day 6 and 7 blastocyst rates, and the number of cells were higher in the high cfDNA group than in the low cfDNA group. Similar results were observed for cfmtDNA, except for the number of cells, which was similar between the groups. The concentration of cfDNA and the relative number of copies of cfmtDNA in seminal plasma cannot predict the freezability of semen but can be used to predict in vitro embryo production.

In vitro characteristics of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and TLR7/8 ligand R848

The quality of the separated fractions in sex-sorted semen is very important for the success of the artificial insemination. This study aimed to evaluate some in vitro characteristics (DNA quantity, kinematic parameters and enzymes activity) of X- and Y-bearing ram spermatozoa sorted by bovine serum albumin (BSA) column and toll-like receptors (TLR)7/8 ligand R848. The ejaculates from six rams were collected by artificial vagina and subjected to a computer-assisted semen analysis (CASA). Total motility and percentage of the sperms with rapid and medium progressivity or non-progressivity in whole ejaculates and in X and Y fractions were analyzed. Activity of the enzymes ALP, GGT, CK, LDH and accumulation of lactate in the seminal plasma of ejaculates and in the environmental fluid of sexed spermatozoa were measured by biochemical analyzer. DNA was isolated from precipitated spermatozoa, and its quantity was measured. For both protocols the DNA mass from X-bearing fractions was higher, than from Y-bearing fractions. The high total motility of X- and Y-bearing spermatozoa as well as greater percent sperms with progressive motility were observed after use of BSA protocol. The application of TLR7/8 ligand R848 protocol led to reducing of Y-sperm motility and enhancement of non-progressivity in both fractions, which corresponded to the determined high amount of the extracellular lactate. For both methods, the significantly reduced activity of enzymes in the X and Y spermatozoa environmental fluids was established. Both protocols produce X- and Y-sperm fractions with satisfactory quality (over 80% total motility and over 50% rapid and medium progressive spermatozoa in each fraction).

Assessment of Sperm Morphometry in Evaluating Male Infertility

Background: Infertility is a complex issue affecting 15% of couples of reproductive age, with men accounting for 40%-50% of infertility cases. Semen analysis comprises various descriptive measures of sperm and seminal fluid to determine semen quality. Transforming qualitative descriptions of sperm deformities and shape changes into quantitative terms can aid in identifying sub-visual abnormalities. This study aimed to evaluate sperm morphometry parameters in both infertile and fertile men. Methods and Results: The study enrolled a total of 101 participants, divided into three groups: Group A included 38 subfertile patients with varicocele, Group B included 33 patients with idiopathic infertility (23 with asthenozoospermia and 10 with oligozoospermia), and Group C (the control group) included 30 healthy fertile men. The mean age of patients was 31.6±5.81, 31.3±6.0, and 29.47±4.27 years in Groups A, B, and C, respectively. Scrotal duplex examinations were performed to identify the presence of varicocele. Semen samples were collected following WHO Manual (2010). Semen dynamic and morphological analyses were conducted using CASA (Computer-Assisted Semen Analysis, MIRALAB, ISO9001, ISO13485). We found that sperm concentration, total sperm count, sperm progressive motility, and sperm progressive+non-progressive motility were significantly lower in Group A and Group B than in Group C (P=0.000 in all cases); however, there were no differences between Group A and Group B regarding these parameters (Table 5). The sperm morphology index was significantly lower in Group A than in Group C (P=0.0024); no differences were found between Group B and Group C and Group B and Group A. The mean value of the sperm deformity index was significantly lower in Group A than in Group C P=0.004). Conclusion: Our study highlights the significant association between sperm morphology and male infertility in varicocele and idiopathic subfertile males.

Comparative developmental competence of in vitro embryos recovered from Bali cattle with normal and poor sperm motility.

Fertility is crucial for enhancing the efficiency of livestock production, as it directly impacts the reproductive rates. A comprehensive understanding of the relationship between sperm quality and embryo development is key to optimizing reproductive outcomes and improving the quality of livestock. This study analyzed the developmental competence of in vitro embryos recovered from Bali cattle with normal or poor sperm motility. Nine bulls with normal fresh semen (NFS) or poor fresh semen (PFS) motility were ejaculated for semen. Semen ejaculates, including volume, motility, and sperm concentration, were evaluated immediately after collection to measure the quality of the fresh semen. Frozen semen was evaluated using computer-assisted semen analysis (CASA) for motility, progressive sperm motility, distance curve path, distance curve linear, distance straight line, average path velocity, curvilinear velocity, linear velocity, straightness (STR), linearity of forward progression (LIN), wobble, and average lateral head displacement (ALH). Bull groups were used to determine in vitro embryo cleavage ability after fertilization of Bali cattle. Ovaries of Bali cattle were collected by slicing, and only cytoplasmic oocytes with compact cumulus cells were used in this study. The oocytes were matured, and in vitro fertilization was performed using fertilization media with a final sperm concentration of 1.5 × 106 spermatozoa/mL. After 48 h, the embryo cleavage ability of the cultured oocytes was evaluated. There were significant differences in motility values between the NFS and PFS groups; however, there were no significant differences in the volume or sperm concentration. There was a significant difference in the LIN value between the groups but no significant differences in other CASA parameters. There were no significant differences in the cleavage rate and morula between the groups, but a positive correlation was observed between the cleavage rate and the morula and between the morula and ALH. A significant negative correlation was observed between the cleavage rate and STR and between the morula and STR; no significant differences were observed for other variables. Despite variations in sperm characteristics, both normal and poor sperm motility demonstrated comparable in vitro embryonic development competence. These findings provide important insights into the fertility potential of Bali bulls, providing valuable information that can enhance selection strategies to improve the quality of livestock production.

Post-thaw sperm morphokinetics and mitochondrial genes profile of buck semen supplemented with selenium in two different forms

Background The quality characteristics of semen is a determinant factor for improving fertility of rabbit. Objectives The current investigation was done to evaluate post-thaw sperm morphokinetics and mitochondrial genes expression profile of buck semen supplemented with selenium in two different forms. Materials and methods Sexually mature bucks (n=16) that aged three months on average were used for collecting two ejaculates which were evaluated using computer assisted semen analysis (CASA) program. The semen samples of all experimental animals which recorded progressive motility greater than or equal to 70% were pooled for cryopreservation. The pooled semen was divided into three groups that were supplemented with selenium nanoparticles and selenium in normal form in addition to the control group. Morphological characteristics as well as CASA parameters were assessed after freezing for 1 week. Moreover, enzymatic activity assays were performed to measure the antioxidant capacity of cryopreserved buck semen. Transcriptional profile of mitochondrial activity and antioxidant defense regulating genes was conducted using quantitative real-time polymerase chain reaction (PCR). Results and conclusion The addition of selenium in normal and Nano forms has significantly (P ≤0.05) enhanced some of CASA parameters such as DCL (µm), DAP (µm), DSL (µm), VCL (µm/s), VAP (µm/s), Amplitude of lateral head (ALH) (µ), BCF (Hz), and VSL (µm/s) during prefreezing period compared with control group. Moreover, the data presented in the present study indicated a significant (P ≤0.05) improvement of post-thaw total and progressive sperm motility in the two groups supplemented with normal and Nano compared with control group. The post-thaw level of total antioxidant capacity (TAC) and percentage of live sperm were higher in the two groups supplemented with selenium and Nano selenium than the control group. The expression profile of candidate genes regulating mitochondrial activity (ATP Synthase F1 Subunit Alpha (ATP5A1), NADH dehydrogenase subunit1 (ND1), NADH dehydrogenase subunit2 (ND2), and Carnitine palmitoyltransferase (CPT2)) was increased significantly (P ≤0.05) in semen supplemented with selenium in normal and Nano forms compared with the control group. In conclusion, the data of this investigation demonstrated enhancement of CASA parameters during pre-freezing post thaw total and progressive sperm motility in the two groups supplemented with normal and nano selenium. The proportion of sperm viability and the level of total antioxidant capacity were enhanced in the two groups supplemented with selenium which was coupled with up-regulation of mitochondrial transcripts. It seems the both two forms shared the same mechanism on improving post-thaw physical and molecular qualities of rabbit sperm.

Flagellar pH homeostasis mediated by Na+/H+ exchangers regulates human sperm functions through coupling with CatSper and KSper activation.

Whether and how do Na+/H+ exchangers (NHEs) regulate the physiological functions of human sperm? NHE-mediated flagellar intracellular pH (pHi) homeostasis facilitates the activation of the pH-sensitive, sperm-specific Ca2+ channel (CatSper) and the sperm-specific K+ channel (KSper), which subsequently modulate sperm motility, hyperactivation, flagellar tyrosine phosphorylation, and the progesterone (P4)-induced acrosome reaction. Sperm pHi alkalization is an essential prerequisite for the acquisition of sperm-fertilizing capacity. Different sperm functions are strictly controlled by particular pHi regulatory mechanisms. NHEs are suggested to modulate sperm H+ efflux. This was a laboratory study that used samples from >50 sperm donors over a period of 1 year. To evaluate NHE action on human sperm function, 5-(N,N-dimethyl)-amiloride (DMA), a highly selective inhibitor of NHEs, was utilized. All experiments were repeated at least five times using different individual sperm samples or cells. By utilizing the pH fluorescent indicator pHrodo Red-AM, we detected alterations in single-cell pHi value in human sperm. The currents of CatSper and KSper in human sperm were recorded by the whole-cell patch-clamp technique. Changes in population and single-cell Ca2+ concentrations ([Ca2+]i) of human sperm loaded with Fluo 4-AM were measured. Membrane potential (Vm) and population pHi were quantitatively examined by a multimode plate reader after sperm were loaded with 3,3'-dipropylthiadicarbocyanine iodide and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester, respectively. Sperm motility parameters were assessed by a computer-assisted semen analysis system. Tyrosine phosphorylation was determined by immunofluorescence, and sperm acrosome reaction was evaluated by Pisum sativum agglutinin-FITC staining. DMA-induced NHEs inhibition severely acidified the human sperm flagellar pHi from 7.20 ± 0.04 to 6.38 ± 0.12 (mean ± SEM), while the effect of DMA on acrosomal pHi was less obvious (from 5.90 ± 0.13 to 5.57 ± 0.12, mean ± SEM). The whole-cell patch-clamp recordings revealed that NHE inhibition remarkably suppressed alkalization-induced activation of CatSper and KSper. As a consequence, impairment of [Ca2+]i homeostasis and Vm maintenance were detected in the presence of DMA. During the capacitation process, pre-treatment with DMA for 2 h potently decreased sperm pHi, which in turn decreased sperm motility and kinetic parameters. Sperm capacitation-associated functions, including hyperactivation, tyrosine phosphorylation, and P4-induced acrosome reaction, were also compromised by NHE inhibition. N/A. This was an in vitro study. Caution should be taken when extrapolating these results to in vivo applications. This study revealed that NHEs are important physiological regulators for human CatSper and KSper, which are indispensable for human sperm fertility, suggesting that malfunction of NHEs could be an underlying mechanism for the pathogenesis of male infertility. This work was supported by the National Natural Science Foundation of China (32271167 and 81871202 to X.Z.), Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC20211543 to X.Z.), the Social Development Project of Jiangsu Province (No. BE2022765 to X.Z.), the Society and livelihood Project of Nantong City (No. MS22022087 to X.Z.), and the Natural Science Foundation of Jiangsu Province (BK20220608 to H.K.). The authors have no competing interests to declare.

Analysis of Ca2+-mediated sperm motility to evaluate the functional normality of the sperm-specific Ca2+ channel, CatSper.

Ca2+ is a key secondary messenger that modulates sperm motility by tuning flagellar movement in various species. The sperm-specific Ca2+ channel, CatSper, is a primary Ca2+ gate that is essential for male fertility in mammals. CatSper-mediated Ca2+ signaling enables sperm to develop hyperactivated motility and fertilize the eggs in the female tract. Therefore, altered CatSper function compromises the entry of Ca2+ into the sperm, followed by impairing hyperactivation and male fertility. However, methods to evaluate the function of the CatSper channel are limited to patch clamping and functional imaging using Ca2+ dye. Previous studies have revealed that various parameters for sperm motility are highly correlated with intracellular Ca2+ levels in mouse. Here, I cover a step-by-step protocol to analyze the change in Ca2+-mediated sperm motility by using computer-assisted semen analysis (CASA) to evaluate the functional normality of the CatSper channel in sperm. This approach analyzes sperm motility parameters during intracellular Ca2+ chelation followed by in vitro capacitation to recover intracellular Ca2+ via the activated CatSper channel. Thus, this Ca2+-handling method is handy and could be broadly applied in reproductive biology labs and clinics that have CASA equipment to examine the functional normality of the CatSper channel.

Effects of organic and inorganic selenium supplementation on frozen-thawed ram semen at two cooling periods.

The aim of this study was to evaluate the effects of different selenium compounds on the sperm quality of cryopreserved ram semen. Ejaculates from four rams, collected using an artificial vagina heated to 38°C, were individually evaluated. The approved ejaculates were pooled and diluted (1:1 v:v) in Tris-egg yolk extender (20%, v/v) and separated into two control groups, one cooled for 2h and the other for 4h. The pooled ejaculates at the two cooling periods were supplemented with two doses (0.5 and 1μg/mL) of organic selenium (ORG), and inorganic selenium (SeNa), each. The samples were packed in 0.25ml straws, at a concentration of 400 × 106 sperms/mL and stored in liquid nitrogen. The straws were thawed in a water bath at 37°C for 20s, and the samples were subjected to sperm kinetics evaluation by Computer Assisted Semen Analysis software. Sperm membrane integrity, acrosome morphology, and mitochondrial potential were assessed. In addition, oxidative stress markers reactive oxygen species (ROS), ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive species (TBARS), and glutathione peroxidase (GPx) enzyme activity) were also evaluated. No significant improvement was observed in the ram semen quality at the two cooling times. Supplementation of the freezing extender with 0.5μg/mL ORG, subjected to 4h cooling period, increased the sperm motility when compared with the control group at the same cooling time. In addition, the 0.5μg/mL SeNa group, under the 2h cooling period, showed an increase in sperm motility when compared to the control group at the same cooling period. Considering the importance of sperm motility as a fertility parameter, our study indicates that supplementation with ORG and SeNa can help improve the total motility of the cryopreserved ram semen.

The Libido and Semen Quality of Bali Bulls Supplemented with Micro-Nutrients of Zinc, Selenium, Vitamin A and Vitamin E

Low quality and quantity of food intake of bulls in the tropics might adversely affect libido and semen quality through their effects on spermatogenesis process. The purpose of this study was to evaluate the effect of micro-minerals (Zn and Se) and vitamins (A and E) on the libido and semen quality of Bali bulls. 2 (two) Bali bulls, aged 4-5 years old with a body weight 300-350 kg. The study was divided into 2 periods and each period was conducted for 8 weeks. At the first period (control), the experimental bulls were fed with natural grasses and concentrates (1% of body weight). Furthermore, at the second period (treatment period), they were fed diet similar to that used in the first period with an additional of a mineral mix (Zn and Se) and vitamin (A and E). Evaluation of libido and semen quality were carried out simultaneously twice a week. Libido was measured by calculating the time required for the bulls from approaching the teaser until they mounted for the first time and from approaching until they ejaculated, respectively. Semen was collected by using an artificial vagina. The volume, colour, pH, and, viscosity, as were recorded. Evaluation of semen quality in the form of the sperm concentration, motility, viability and abnormality were done by using Computer Assisted Semen Analyser (CASA). Fresh semen was diluted by Andromed®. The diluted semen was preserved at the temperature 50C for 144 hrs. Motility, viability and abnormality were observed every 12 hrs. Data were reported as mean and standard error of mean analysed by using the paired student’s t-test. Mean interval between the bulls approaching and mounting the teaser for the first time and between approaching and ejaculation after the supplementation of micro-nutrients were significantly shorter (P<0.05) in compared to those before the supplementation (2.68 vs. 6.52 and 5.09 vs. 10.06 minutes, respectively). The sperm viability after the supplementation of micro-nutrients was significantly higher in compared to that before supplementation. (82.25 vs. 87.50 %). Preservation of diluted bull semen at the temperature of 50C for 144 hrs showed that the sperm viability was significantly higher (P<0.05) and sperm abnormality was significantly lower (P<0.05) after the supplementation of micro-nutrients in compared to those before the supplementation. It can be concluded that the supplementation micro-nutrients in Bali bulls could increase libido, sperm viability and reduce the sperm abnormality.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods

BackgroundThis study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP).ResultsAn addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05).ConclusionsIn conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

Prevalence of Chlamydophila spp. and Canid herpesvirus-1 in Polish dogs.

Chlamydophila spp. affect Leydig and Sertoli cells by dysregulating spermatogenesis, inducing apoptosis and sperm DNA fragmentation, as well as benign prostate hyperplasia. Canid herpes virus 1 (CHV-1) infection in male dogs is manifested by lesions on the base of the penis and foreskin. There is a lack of information on the influence of these microorganisms on the quality of canine semen. Seroprevalence of Chlamydophila spp. (55%-61%) and CHV-1 (22%-81%) in Europe is high. The prevalence of Chlamydophila spp. and CHV-1 has been evaluated using polymerase chain reaction (PCR) only in Sweden and Croatia, respectively. No positive samples were detected in either case. The aim of this study was to evaluate the epidemiological situation in Polish male dogs (PMDs) to provide a solution to limit the spread of these microorganisms using assisted reproduction techniques or elimination from the reproduction of CHV-1 carriers. In addition, we assessed the semen quality of Chlamydophila spp. carriers and CHV-1 carriers. Cotton swabs were collected from prepuce or semen from each dog (n = 130). Real-time PCR for Chlamydophila spp. and CHV-1, as well as semen analysis, was performed using the computer-assisted semen analysis system. To the best of our knowledge, this is the first report of Chlamydophila spp. infection in PMD confirmed by real-time PCR. All parameters, except progressive movement in Chlamydophila semen carriers, were normal. The average velocity values for a dog with Chlamydia are detailed. No CHV-1 was detected. The results achieved should be verified on the basis of a larger number of studies. However, the high prevalence of these pathogens in the PMD population has not been established.

Protocolo de congelamento one-step para sêmen suíno utilizando diferentes crioprotetores

ABSTRACT: The present study evaluated the cryoprotectant efficacy of dimethylacetamide (DMA) and ethylene glycol in a one-step protocol to freeze boar sperm. The sperm-rich portion of the ejaculates from two boars were collected once a week, for 10 weeks. After collection, the ejaculates were diluted (1:1; v/v) in the cooling extender. After determining their spermatozoa concentration, the ejaculates were pooled with the same number of spermatozoa from each boar and stabilized at 20°C for 120 min. Distinct cryoprotectants were added to the cooling extender at 20 °C, at different concentrations, composing six treatments: 1.25% and 2.5% glycerol (control); 1.25% and 2.5% ethylene glycol; 2.5% and 5.0% DMA. The samples were stored in 0.25 mL straws, containing 35 × 106 spermatozoa. After 90 min at 20 °C, the straws were submitted to a cooling curve until 5 °C (0.3 to 0.5 °C/min) and kept at 5°C for 60 min. Freezing was conducted by placing the straws horizontally 5 cm above the liquid nitrogen for 10 min, followed by immersion on liquid nitrogen. After thawing at 37 °C for 30 seconds, sperm quality was evaluated through a computer-assisted semen analysis system and flow cytometry. Sperm motility was greater (P&lt; 0.05) in treatments with 5.0% and 2.5% DMA (22.2 ± 2.6% and 20.0 ± 2.8%, respectively) than in treatment with 2.5% ethylene glycol (8.2 ± 1.0%). The integrity of the plasma membrane (P = 0.08) and mitochondrial membrane potential (P = 0.27) was similar among the treatments. The treatment with 2.5% ethylene glycol was the least efficient to maintain intact acrosome membrane (P&lt; 0.01). Some kinetics parameters (DAP, DCL, DSL, VAP, VCL, VSL e ALH) were positively affected by 5.0% DMA. The one-step freezing protocol resulted in unsatisfactory boar sperm motility after thawing, regardless of the cryoprotectant.

Ameliorative effect of crocin on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo (Bubalus bubalis) bull sperm.

Buffalo bull sperm suffer more cryoinjuries due to lipid peroxidation of high structural polyunsaturated fatty acid contents than cattle sperm. Consequently, the post-thaw fertilization potential of buffalo bull sperm is compromised. Crocin is a carotenoid known for its antioxidant potential through scavenging reactive oxygen species. Objectives of the current study were to investigate the effect of crocin addition in the semen extender on post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm. Semen samples (n = 32) from four Nili-Ravi buffalo bulls were extended with tris-citric acid extender containing different concentrations of crocin (0 mM; control, 0.5, 1, 1.5 and 2 mM). The extended semen was packed in 0.5 mL French straws (25 × 106 sperm/straw) and cryopreserved in liquid nitrogen. Computer-assisted semen analysis, hypo-osmotic swelling test, normal apical ridge assay, Rhodamine 123, acridine orange, propidium iodide staining, and thiobarbituric acid reactive substances assay were performed to assess sperm motility parameters, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential, DNA integrity, viability, and lipid peroxidation, respectively. Expression levels of sperm acrosome-associated SPACA3, DNA condensation-related PRM1, anti-apoptotic BCL2, pro-apoptotic BAX, and oxidative stress-associated ROMO1 genes were evaluated through qPCR. The fertility of semen doses containing the most potent concentration of crocin (based on optimum post-thaw semen quality) was compared with control during the breeding season. Buffaloes (n = 400; 200/group) were inseminated 24 h after the onset of oestrus and transrectally palpated for pregnancy at least 60 days post-insemination. Results revealed that 0.5 and 1 mM crocin improved sperm post-thaw total motility, plasma membrane integrity, acrosome integrity, mitochondrial membrane potential and viability, and 1 and 1.5 mM crocin enhanced catalase activity and reduced lipid peroxidation compared to control (p < .05). Moreover, 1 mM crocin improved sperm post-thaw progressive motility, kinematics, and DNA integrity, and 1.5 mM crocin enhanced plasma membrane integrity than control (p < .05). Expression levels of SPACA3, PRM1 and BCL2 genes were higher (p < .05) with 1 mM crocin compared to other groups. In contrast, no difference (p > .05) was noticed in expressions of BAX and ROMO1 genes among all groups. The fertility rate of semen doses containing the most potent concentration (1 mM) of crocin was higher (p = .0465) compared to control (56 ± 0.03% vs. 46 ± 0.04%, respectively). In conclusion, 1 mM crocin in the semen extender improves post-thaw quality, fertility-associated gene expression and fertilization potential of buffalo bull sperm.

Storage of boar semen at 17°C without conventional antibiotics in an extender containing an organic bactericidal substance

IntroductionFacing the global threat of antimicrobial resistance, the reduction of antibiotic use in semen extenders is a main goal in artificial insemination (AI) of pigs. The aim of this study was to investigate the potential of a commercial extender containing an organic bactericidal supplement in the absence of conventional antibiotics to control bacterial growth and to maintain the quality of boar spermatozoa during long-term semen storage for up to 144 h at 17°C.MethodsSemen from 233 boars housed at 16 European AI centers was split and diluted in the long-term extender “Androstar Plus without antibiotics + organic bactericidal supplement” (APlus) and in the control extender Beltsville Thawing Solution (BTS) with gentamicin, which is routinely used in many AI centers. Sperm motility was assessed with computer-assisted semen analysis (CASA) and membrane integrity was evaluated with flow cytometry. The number of bacteria was determined by counting colonies on agar plates.ResultsAt the end of storage, bacterial counts were ≥ 106 CFU/mL in 10.7% of the APlus and in 0.4% of the BTS samples. At the same time, bacterial counts were only weakly correlated with sperm motility (r = −0.23, p &amp;lt; 0.05), and there was no correlation with sperm membrane integrity (p &amp;gt; 0.05). Among the 12 identified bacterial species in APlus samples, loss of sperm quality was exclusively observed in the presence of &amp;gt;106 CFU/mL Serratia marcescens and Klebsiella oxytoca. Both these bacterial species, despite their known multi-drug resistance and the continuous use of gentamicin in Europe, proved sensitive to this antibiotic, thus indicating an efficient quality assurance program and responsible antibiotic use.ConclusionLong-term storage of boar semen at 17°C without conventional antibiotics in an extender containing an organic bactericidal supplement is an option if semen samples are regularly tested for the presence of S. marcescens and K. oxytoca, and the source of contamination is eliminated.

Morphometric features, seminal profile and diluters effect on post-thaw semen quality of Munshiganj cattle in Bangladesh

The study investigates morphometric features, seminal profile and post-thaw semen quality of Munshiganj cattle. Morphometric features were measured using measuring tape from 20 Munshiganj cattle while coat color was measured by observing in naked eye. Fresh and post thaw semen quality parameters were analyzed using Computer Assisted semen analyzer (CASA). Coat color of Munshiganj male cattle were creamy white to dull pinkish and female were white to creamy. The mean body weight, body length, hearth girth, height at wither, head length, head width, ear length, ear width, fore leg length, hind leg length, tail length, tail doc circumference, horn length, horn diameter and mouth circumference were 362.80 kg, 137.31 cm, 160.66 cm, 135.21 cm, 50.97 cm, 20.58 cm, 19.75 cm, 9.88 cm, 73.02 cm, 74.84 cm, 106.10 cm, 20.75 cm, 13.60 cm, 16.12 cm and 43.00 cm, respectively. There was significant difference (p < 0.05) between male and female in terms of body weight (418.00 vs 307.60 kg), heart girth (173.74 vs 147.57 cm), head width (22.50 vs 18.67 cm), horn diameter (18.58 vs 13.66 cm) and mouth circumference (46.60 vs 39.40 cm). Average scrotal length was 16.76 cm while scrotal circumference was 32.70 cm. Age had significant effect (p < 0.05) on morphometric characteristics of Munshiganj male and female cattle. On the other hand, season had no significant effect on semen quality. Mean semen volume, sperm concentration, total motile, progressive, static, slow and live spermatozoa were 5.83 ± 0.88 ml, 1510.27 ± 844.07 million/ml, 91.9 ± 2.17 %, 63.80 ± 12.53 %, 8.10 ± 2.17 %, 0.10 ± 0.10 %, 91.38 ± 0.25 %, respectively. On the other hand, sperm head length and width, sperm tail length of Munshiganj cattle were 10.39 ± 0.16 μm, 4.26 ± 0.07 μm, 21.5 ± 0.52 μm, respectively. Individual breeding bull had a significant (p < 0.05) effect on post-thawed motile sperm percentage. Four different diluters (Triladyl, Steridyl, Tris-egg yolk-Citrate and Andromed) were used to compare the effects of diluter on post-thaw semen quality of Munshiganj cattle and found that diluter had no significant effect (p > 0.05) on post thaw semen quality except slow motility and proximal droplet percentages. Munshiganj cattle had a distinctive phenotypic feature with standard quality semen and had no effect of egg yolk free and egg yolk based diluters on post thaw semen quality.

Exploration of semen quality analyzed by casa-mot systems of brahman bulls infected with BLV and BHV-1

Enzootic bovine leukosis virus (BLV) and bovine herpesvirus 1 (BHV-1) are very important infectious agents for the livestock industry worldwide. The present study aimed to explore the association between natural exposure to BLV and BHV-1 with sperm quality analyzed by Computer-Assisted Semen Analysis (CASA) systems. Ten sexually mature Brahman bulls, with sanitary status BLV+/BHV-1+ (n = 2), BLV−/BHV-1+ (n = 6) and BLV-/BHV-1- (n = 2) were evaluated twice, 30 days apart. Results showed that sanitary status of each bull was not associated with semen quality. It was found that the quality of the semen from the second collection was better due to the interruption of sexual rest. The evidence thus revealed that a bull infected with BLV generated good-quality contaminated semen and, therefore, that it is essential to detect contaminated seminal samples to prevent the spread of BLV. A multivariate analysis showed the presence of four sperm subpopulations in Brahman bulls that differ significantly in their kinematic patterns and with respect to sanitary status (P < 0.05), indicating that infection-free and seronegative bulls present the best kinematic parameters, which improved discrimination of sperm quality according to sanitary status. Overall, the analyses indicate that the seropositive-infected bulls with BLV and BHV-1 should be excluded from beef cattle farms.