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1.
Reprod Domest Anim ; 59(6): e14588, 2024 Jun.
Article En | MEDLINE | ID: mdl-38822558

Semen cryopreservation is one of the most important reproduction techniques in the livestock and poultry industry. Cryopreservation induces cold stress, generating reactive oxygen species (ROS) and oxidative stress causing structural and biochemical damages in sperm. In this study, we evaluated the effects of the hydroxytyrosol (HT), as an antioxidant, at the concentrations of 0, 25, 50, and 100 µg/mL on post-thaw semen quality metrics in rooster. Semen samples were collected twice a week from 10 roosters (29 weeks), processed and frozen according to experimental groups. Different quality parameters, including total motility, progressive motility, viability, morphology, membrane integrity, and malondialdehyde were measured after thawing. Results showed that 25 and 50 µg/mL of HT produced the highest percentage of total motility (51.01 ± 2.19 and 50.15 ± 2.19, respectively) and progressive motility (35.74 ± 1.34 and 35.15 ± 1.34, respectively), membrane integrity (48.00 ± 2.18 and 46.75 ± 2.18, respectively) as well as viability (53.00 ± 2.17 and 52.50 ± 2.17, respectively) compared with the other groups (p < .05). The group with 25 µg/mL of HT showed the lowest significant (p < .05) MDA concentration (1.81 ± 0.25). Our results showed that the effect of HT was not dose-dependent and optimum concentration of HT could improve functional parameters of rooster sperm after freezing-thawing. These findings suggest that HT may have protective effects on the rooster sperm during the freezing-thawing process.


Antioxidants , Chickens , Cryopreservation , Phenylethyl Alcohol , Semen Preservation , Sperm Motility , Spermatozoa , Animals , Phenylethyl Alcohol/analogs & derivatives , Phenylethyl Alcohol/pharmacology , Male , Cryopreservation/veterinary , Cryopreservation/methods , Semen Preservation/veterinary , Semen Preservation/methods , Spermatozoa/drug effects , Sperm Motility/drug effects , Antioxidants/pharmacology , Semen Analysis/veterinary , Cryoprotective Agents/pharmacology , Malondialdehyde/analysis
2.
Reprod Domest Anim ; 59(5): e14570, 2024 May.
Article En | MEDLINE | ID: mdl-38700367

The cryopreservation process induces alterations in cellular parameters and epigenetic patterns in bull sperm, which can be prevented by adding cryoprotectants in the freezing extenders. The purpose of this study was to compare the protective effects of two extenders based on soybean lecithin (SLE) and egg yolk (EYE) on epigenetic patterns and quality parameters of sperm such as motility parameters, mitochondrial membrane integrity, DNA fragmentation, viability, and apoptotic-like changes of bull sperm after cryopreservation. Results demonstrated that cryopreservation significantly (p < .05) reduced the level of DNA global methylation, H3K9 histone acetylation, and H3K4 histone methylation in both frozen groups compared to the fresh sperm. Also, the level of H3K9 acetylation was lower in the frozen SLE group (21.2 ± 1.86) compared to EYE group (15.2 ± 1.86). In addition, the SLE frozen group had a higher percentage of viability, progressive motility, and linearity (LIN) in SLE frozen group compared to EYE frozen group. However, no difference was observed in mitochondrial membrane integrity and DNA fragmentation between SLE and EYE frozen groups. While soybean-lecithin-based extender showed some initial positive impacts of epigenetics and semen parameters, further investigations can provide useful information for better freezing.


Cryopreservation , Cryoprotective Agents , DNA Fragmentation , DNA Methylation , Epigenesis, Genetic , Semen Preservation , Sperm Motility , Spermatozoa , Male , Cryopreservation/veterinary , Animals , Cattle , Spermatozoa/drug effects , Spermatozoa/physiology , Semen Preservation/veterinary , Semen Preservation/methods , Sperm Motility/drug effects , Cryoprotective Agents/pharmacology , DNA Methylation/drug effects , Egg Yolk/chemistry , Lecithins/pharmacology , Histones/metabolism , Histones/genetics , Glycine max/chemistry , Semen Analysis/veterinary , Acetylation
3.
Reprod Fertil Dev ; 362024 May.
Article En | MEDLINE | ID: mdl-38713807

Context Seasonal microclimatic fluctuations can cause changes in sperm quality even in dairy bulls bred under temperate climate. These changes can vary between sires of different age and affect sperm freezability. Aims We aimed to evaluate the modulating effect of bull age and equilibration time before freezing on the seasonal pattern of sperm viability and DNA integrity post-thaw. Methods In the frame of systematic sperm quality control, we assessed the integrity of sperm plasma membrane and acrosome (PMAI) in 15,496 cryopreserved bovine batches, and the percentage of sperm with high DNA fragmentation index (%DFI) after 0h and 3h incubation at 38°C post-thaw (3h) in 3422 batches. Semen was equilibrated for 24h before freezing if collected on Monday or Wednesday and 72h if produced on Friday. We investigated the effect of season, bull age, equilibration, and temperature-humidity index (THI) on the day of semen collection on sperm traits using mixed-effects linear models. Key results PMAI and %DFI (0h and 3h) deteriorated with increasing THI. The effect of THI on %DFI was detected with a 30-day time lag. Seasonal fluctuations of sperm quality were similar between young, mature, and older sires. Prolonged equilibration did not affect PMAI but was linked to elevated %DFI (3h) in summer. Conclusions Extending equilibration from 24 to 72h is compatible with commercial standards of bovine sperm quality post-thaw; however, it could interfere with the seasonal pattern of the latter. Implications Systematic monitoring of bovine sperm quality enables the prompt detection of stress factors related to microclimate and semen processing.


Cryopreservation , DNA Fragmentation , Seasons , Semen Analysis , Semen Preservation , Spermatozoa , Animals , Cattle , Male , Cryopreservation/veterinary , Semen Preservation/veterinary , Semen Preservation/methods , Spermatozoa/drug effects , Spermatozoa/physiology , Semen Analysis/veterinary , DNA Fragmentation/drug effects , Cell Survival/drug effects , Microclimate , Age Factors , Sperm Motility/drug effects
4.
Reprod Domest Anim ; 59(5): e14569, 2024 May.
Article En | MEDLINE | ID: mdl-38715435

The effects of an aqueous extract of Scabiosa atropurpurea L. (AES) on the reproduction potential of Queue Fine de l'Ouest rams were evaluated over 9 weeks. Eighteen mature (4-6 years old) rams (52.8 ± 2.6 kg) were divided into three groups. The control (C) group was fed oat hay ad libitum with 700 g of concentrate and the other two groups were fed the same diet supplemented with AES at 1 and 2 mg/kg body weight (AES1 and AES2, respectively). Ram sperm was collected with an artificial vagina (2 × 2 days/week) to evaluate sperm production and quality, antioxidant activity, the adenosine triphosphate (ATP) and calcium concentrations. Sexual behaviour and plasma testosterone concentrations were also investigated. The administration of AES improved sexual behaviour (the duration of contact and the number of lateral approaches). The addition of AES also improved individual spermatozoa motility (C: 71.7% ± 6.3%; AES1: 78.3% ± 4.9%; AES2: 83.8% ± 4.4%), the sperm concentration (C: 5.6 ± 0.36; AES1: 6.4 ± 0.81; AES2: 6.7 ± 0.52 × 109 spermatozoa/mL), the ATP ratio (C: 1 ± 0.08; AES1: 2.1 ± 0.08; AES2: 3.3 ± 0.08) and the calcium concentration (C: 5.6 ± 0.24; AES1: 7.7 ± 0.21; AES2: 8.1 ± 0.24 mmol/L). AES treatment decreased the percentage of abnormal sperm (C: 18.5% ± 1.2%; AES1: 16.2% ± 1.1%; AES2: 14.8% ± 0.94%) and DNA damage (C: 62%; AES1: 27%; AES2: 33%) and was associated with elevated seminal fluid antioxidant activity (C: 22 ± 0.27; AES1: 27.1 ± 1.08 and AES2: 27.5 ± 0.36 mmol Trolox equivalents/L) and plasma testosterone (C: 8.3 ± 0.7; AES1: 11.7 ± 0.4; AES2: 15 ± 0.7 ng/L). In conclusion, our study suggests that S. atropurpurea may be potentially useful to enhance libido and sperm production and quality in ram.


Plant Extracts , Sexual Behavior, Animal , Spermatozoa , Male , Animals , Spermatozoa/drug effects , Sexual Behavior, Animal/drug effects , Plant Extracts/pharmacology , Plant Extracts/administration & dosage , Testosterone/blood , Semen Analysis/veterinary , Sperm Motility/drug effects , Dietary Supplements , Antioxidants/pharmacology , Diet/veterinary , Sperm Count , Calcium/analysis , Calcium/blood , Sheep, Domestic , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/analysis
5.
Reprod Domest Anim ; 59(5): e14573, 2024 May.
Article En | MEDLINE | ID: mdl-38712666

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.


Caseins , Cryopreservation , Insemination, Artificial , Semen Analysis , Semen Preservation , Sperm Motility , Animals , Semen Preservation/veterinary , Semen Preservation/methods , Male , Female , Cryopreservation/veterinary , Cryopreservation/methods , Insemination, Artificial/veterinary , Caseins/pharmacology , Semen Analysis/veterinary , Pregnancy , Sperm Motility/drug effects , Spermatozoa/drug effects , Spermatozoa/physiology , Cryoprotective Agents/pharmacology , Semen/drug effects , Fertility/drug effects , Sheep , Sheep, Domestic
6.
Reprod Domest Anim ; 59(5): e14585, 2024 May.
Article En | MEDLINE | ID: mdl-38745503

The study investigated midpiece defects in sperm from a 5-year-old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16-month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization.


Cryopreservation , Semen Analysis , Semen Preservation , Spermatozoa , Male , Animals , Cryopreservation/veterinary , Cattle , Semen Preservation/veterinary , Semen Analysis/veterinary , Spermatozoa/abnormalities , Spermatozoa/physiology , Biomechanical Phenomena , Sperm Midpiece , Sperm Motility , Acrosome
7.
Reprod Fertil Dev ; 362024 May.
Article En | MEDLINE | ID: mdl-38753960

Context Several polymorphisms in the melatonin receptor 1A gene (MTNR1A ) have been related to reproductive performance in ovine. Aims To investigate the effect of the Rsa I and Mnl I polymorphisms on ram seminal quality. Methods Eighteen Rasa Aragonesa rams were genotyped for the Rsa I (C/C, C/T, T/T) and Mnl I (G/G, G/A, A/A) allelic variants of the MTNR1A gene. Individual ejaculates were analysed once a month throughout the whole year. Sperm motility, morphology, membrane integrity, levels of reactive oxygen species (ROS), phosphatidylserine (PS) inversion, DNA fragmentation and capacitation status were assessed. The effect of the season and polymorphisms on seminal quality was evaluated by mixed ANOVA. Key results Both polymorphisms had an effect on membrane integrity and viable spermatozoa with low levels of ROS and without PS translocation, and Rsa I also on motile and DNA-intact spermatozoa. An interaction between both polymorphisms was found, pointing to a negative effect on seminal quality of carrying the T or A allele in homozygosity. Differences were higher in the reproductive than in the non-reproductive season. Conclusions Mutations substituting C by T and G by A at Rsa I and Mnl I polymorphic sites, respectively, in the MTNR1A gene in rams could decrease the seminal quality. Implications Genotyping of rams based on melatonin receptor 1A could be a powerful tool in sire selection.


Receptor, Melatonin, MT1 , Sperm Motility , Spermatozoa , Male , Animals , Receptor, Melatonin, MT1/genetics , Receptor, Melatonin, MT1/metabolism , Spermatozoa/metabolism , Sperm Motility/genetics , Sheep/genetics , Genotype , Semen Analysis/veterinary , Polymorphism, Genetic , Reactive Oxygen Species/metabolism , DNA Fragmentation , Polymorphism, Single Nucleotide
8.
Cryo Letters ; 45(4): 257-268, 2024.
Article En | MEDLINE | ID: mdl-38809790

BACKGROUND: Little is known about the effects of different seasons on the cryopreservation success of buffalo sperm in terms of kinematics and sperm functional parameters. OBJECTIVE: To study the effect of three seasons (winter, comfort and summer) and cryopreservation on sperm kinematics and functional properties in buffalo bulls. MATERIALS AND METHODS: Semen ejaculates (n = 90) collected during three seasons i.e. winter (n = 30), comfort (n = 30), summer (n = 30) were evaluated for sperm kinematics and functional properties. RESULTS: Sperm kinematics with respect to total (TM), progressive (PM) and rapid motility (RM) was higher (P < 0.05) in fresh sperm compared to sperm that had been frozen-thawed. Similarly, all kinematic parameters [viz. average path velocity (VAP), straight linear velocity (VSL), curvilinear velocity (VCL), beats cross frequency (BCF), lateral head displacement (ALH), linearity (LIN) and straightness (STR)] were higher (P < 0.01) at the fresh stage. With respect to season, frozen-thawed semen TM (57.67 ± 115 %), PM (50.2 ± 1.15 %) and RM (51.6 ± 1.19 %) were higher (P < 0.01) when using sperm collected during winter. The stage of cryopreservation (i.e., equilibration and freeze-thawing) also showed significant effects (P < 0.01) on mitochondrial superoxide positive status (MSPS), mitochondrial membrane potential (MMP), acrosome status and intra-cellular calcium status. CONCLUSION: The season of sperm collection and cryopreservation have significant effects on buffalo bull sperm kinematics and functional properties. Doi.org/10.54680/fr24410110612.


Acrosome , Buffaloes , Calcium , Cryopreservation , Mitochondria , Semen Preservation , Sperm Motility , Spermatozoa , Animals , Cryopreservation/veterinary , Cryopreservation/methods , Male , Buffaloes/physiology , Semen Preservation/veterinary , Semen Preservation/methods , Spermatozoa/physiology , Calcium/metabolism , Mitochondria/metabolism , Biomechanical Phenomena , Seasons , Membrane Potential, Mitochondrial , Semen Analysis/veterinary
9.
Cryo Letters ; 45(4): 194-211, 2024.
Article En | MEDLINE | ID: mdl-38809784

This comprehensive review delves into the evolving landscape of assisted reproductive technologies (ARTs) in bovine species, particularly focusing on the pivotal roles of semen additives in the cryopreservation of buffalo and cattle semen. In developing nations, where ARTs are still emerging, these techniques significantly influence bovine reproductive strategies. In contrast, developed regions have embraced them as primary approaches for dairy buffalo and cattle breeding. Semen cryopreservation, while offering advantages like extended storage and genetic propagation, also presents challenges. These include diminished sperm quality due to reactive oxygen species (ROS) production, alterations in sperm structure, and temperature fluctuations. Further, the effect of cryopreservation differs between cattle and buffaloes, with the latter exhibiting poorer semen viability and fertility due to inherent lipid composition susceptibilities. The generation and implications of ROS, especially hydrogen peroxide, contribute significantly to sperm DNA damage and functional impairments. To counteract these challenges, research has intensified on semen additives, aiming to bolster semen quality and protect against oxidative stress-induced damage. As the field advances, the review emphasizes the need for optimized cryopreservation techniques and tailored antioxidant strategies to harness the full potential of ARTs in bovine breeding programs. Doi.org/10.54680/fr24410110112.


Buffaloes , Cryopreservation , Cryoprotective Agents , Semen Preservation , Cattle , Animals , Cryopreservation/methods , Cryopreservation/veterinary , Semen Preservation/methods , Semen Preservation/veterinary , Male , Buffaloes/physiology , Cryoprotective Agents/pharmacology , Semen , Reactive Oxygen Species/metabolism , Semen Analysis/veterinary , Semen Analysis/methods , Spermatozoa/physiology , Oxidative Stress/drug effects , Reproductive Techniques, Assisted/veterinary , DNA Damage/drug effects , Antioxidants/pharmacology , Sperm Motility/drug effects
10.
Theriogenology ; 224: 26-33, 2024 Aug.
Article En | MEDLINE | ID: mdl-38723471

Pigs are usually bred through artificial insemination with liquid semen preserved at 15-20 °C. While this method of preservation brings many benefits, including a greater reproductive performance compared to frozen-thawed sperm, the period of storage is a limiting factor. As the mitochondrion regulates many facets of sperm physiology, modulating its activity could have an impact on their lifespan. Aligned with this hypothesis, the present study sought to investigate whether inhibition of voltage-dependent anion channels (VDACs), which reside in the outer mitochondrial membrane and regulate the flux of ions between mitochondria and the cytosol in somatic cells, influences the resilience of pig sperm to liquid preservation at 17 °C. For this purpose, semen samples (N = 7) were treated with two different concentrations of TRO19622 (5 µM and 50 µM), an inhibitor of VDACs, and stored at 17 °C for 10 days. At days 0, 4 and 10, sperm quality and functionality parameters were evaluated by flow cytometry and computer-assisted sperm analysis (CASA). The effects of inhibiting VDACs depended on the concentration of the inhibitor. On the one hand, the greatest concentration of TRO19622 (50 µM) led to a decrease in sperm motility, viability and mitochondrial membrane potential, which could be related to the observed intracellular Ca2+ increase. In contrast, total sperm motility was higher in samples treated with 5 µM TRO19622 than in the control, suggesting that when VDACs channels are inhibited by the lowest concentration of the blocking agent the resilience of pig sperm to liquid storage increases. In conclusion, the current research indicates that mitochondrial function, as regulated by ion channels in the outer mitochondrial membrane like VDACs, is related to the sperm resilience to liquid preservation and may influence cell lifespan.


Semen Analysis , Semen Preservation , Spermatozoa , Voltage-Dependent Anion Channels , Animals , Male , Semen Preservation/veterinary , Semen Preservation/methods , Swine/physiology , Spermatozoa/physiology , Spermatozoa/drug effects , Semen Analysis/veterinary , Sperm Motility/drug effects , Membrane Potential, Mitochondrial/drug effects , Cryopreservation/veterinary , Cryopreservation/methods
11.
Anim Reprod Sci ; 265: 107487, 2024 Jun.
Article En | MEDLINE | ID: mdl-38723402

Cryopreservation of small ruminant's semen is an effective strategy for distributing spermatozoa for reproductive programs, but this process decreases the fertility potential of post-thawed spermatozoa. The aim of this research was to assess the effect of different concentrations of CoQ10 in soybean lecithin (SL)-based extender on buck semen quality during cryopreservation process. Semen samples were collected from five bucks, twice a week, then diluted in the SL-based extender containing different concentrations of CoQ10 as follows: extender containing 0 µM (control, Q0), 0.1 µM (Q0.1), 1 µM (Q1), 10 µM (Q10) and 100 µM (Q100) CoQ10. Motion characteristics, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, viability, apoptotic-like changes, lipid peroxidation, DNA fragmentation and ROS concentration were evaluated after freeze-thawing process. The Q10 resulted in greater (P≤0.05) total motility, progressive motility, average path velocity, membrane integrity, mitochondrial activity, acrosome integrity and viability compared to the other groups. Furthermore, supplementation of freezing extender with 10 µM of CoQ10 presented lower (P≤0.05) apoptotic-like changes, lipid peroxidation, DNA fragmentation and ROS concentration compared to the other groups. Regarding to the protective effect of CoQ10 supplement during cryopreservation process, it could be explored as a potent antioxidant for cryopreservation of buck semen as it preserved the post-thawed buck sperm quality.


Cryopreservation , Cryoprotective Agents , Goats , Semen Analysis , Semen Preservation , Spermatozoa , Ubiquinone , Ubiquinone/pharmacology , Ubiquinone/analogs & derivatives , Male , Cryopreservation/veterinary , Cryopreservation/methods , Semen Preservation/veterinary , Semen Preservation/methods , Animals , Semen Analysis/veterinary , Cryoprotective Agents/pharmacology , Spermatozoa/drug effects , Spermatozoa/physiology , Goats/physiology , Sperm Motility/drug effects , Glycine max/chemistry
12.
Anim Reprod Sci ; 265: 107493, 2024 Jun.
Article En | MEDLINE | ID: mdl-38701639

Not all boar sperm samples survive cryopreservation well. A method of eliminating damaged sperm might enable more cryopreserved boar semen to be used for pig breeding. In this study we investigated the use of Magnetic Activated Cell sorting (MACS) to eliminate damaged sperm from thawed boar semen samples. The thawed samples were mixed with Dead cell removal particles and were applied to the column in a SuperMACS II. Different fractions were collected: Original sample (O), Flow-through (FT), and Eluate (E). Sperm membrane integrity, mitochondrial membrane potential and reactive oxygen species were evaluated by flow cytometry after staining with SYBR 14 and propidium iodide, or 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethylbenzimidazolylcarbocyanine iodide, or hydroethidine and dichlorodihydrofluorescein diacetate, respectively. The FT samples had increased membrane integrity, a greater proportion of sperm with high mitochondrial membrane potential and a greater proportion of sperm negative for hydrogen peroxide than O samples (P<0.0001), which in turn had increased membrane integrity than E samples (P <0.0001). However, differences were seen between boars. The FT samples had increased values of live, superoxide positive sperm than O samples (P <0.0001) and O samples had greater values than E samples (P <0.0001), while there was no effect of boar. Sperm quality was best in the FT fraction, comprising approximately 32% of the sperm sample. In conclusion, although there were differences between boars, MACS separation can improve sperm quality in thawed semen samples. It would be interesting to see if this improvement is reflected in fertility outcomes.


Cryopreservation , Semen Preservation , Spermatozoa , Animals , Male , Spermatozoa/physiology , Swine/physiology , Semen Preservation/veterinary , Semen Preservation/methods , Cryopreservation/veterinary , Cryopreservation/methods , Cell Membrane/physiology , Membrane Potential, Mitochondrial/physiology , Cell Separation/veterinary , Cell Separation/methods , Flow Cytometry/veterinary , Reactive Oxygen Species/metabolism , Semen Analysis/veterinary
13.
Theriogenology ; 225: 98-106, 2024 Sep 01.
Article En | MEDLINE | ID: mdl-38801791

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.


Cryopreservation , Goats , Melatonin , Seasons , Semen Preservation , Semen , Spermatozoa , Animals , Male , Melatonin/metabolism , Melatonin/blood , Goats/physiology , Goats/metabolism , Semen/chemistry , Semen/metabolism , Spermatozoa/metabolism , Spermatozoa/physiology , Semen Preservation/veterinary , Cryopreservation/veterinary , Receptor, Melatonin, MT1/metabolism , Receptor, Melatonin, MT1/genetics , Receptor, Melatonin, MT2/metabolism , Receptor, Melatonin, MT2/genetics , Semen Analysis/veterinary , Receptors, Melatonin/metabolism
14.
Theriogenology ; 225: 162-171, 2024 Sep 01.
Article En | MEDLINE | ID: mdl-38805998

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0-5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0-5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (-0.0002 ± 0.07 vs. -0.266 ± 0.09), kurharm0 (-0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0-5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. -0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928.


Buffaloes , Fertility , Semen Analysis , Spermatozoa , Animals , Male , Buffaloes/physiology , Spermatozoa/physiology , Fertility/physiology , Semen Analysis/veterinary , Semen Analysis/methods , Fourier Analysis
15.
Sci Rep ; 14(1): 10214, 2024 05 03.
Article En | MEDLINE | ID: mdl-38702489

Sperm membrane composition and biophysical characteristics play a pivotal role in many physiological processes (i.e. sperm motility, capacitation, acrosome reaction and fusion with the oocyte) as well as in semen processing (e.g. cryopreservation). The aim of this study was to characterize the fatty acid content and biophysical characteristics (anisotropy, generalized polarization) of the cell membrane of domestic cat spermatozoa. Semen was collected from 34 adult male cats by urethral catheterization. After a basic semen evaluation, the fatty acid content of some of the samples (n = 11) was evaluated by gas chromatography. Samples from other individuals (n = 23) were subjected to biophysical analysis: membrane anisotropy (which is inversely proportional to membrane fluidity) and generalized polarization (describing lipid order); both measured by fluorimetry at three temperature points: 38 °C, 25 °C and 5 °C. Spermatozoa from some samples (n = 10) were cryopreserved in TRIS egg yolk-glycerol extender and underwent the same biophysical analysis after thawing. Most fatty acids in feline spermatozoa were saturated (69.76 ± 24.45%), whereas the polyunsaturated fatty acid (PUFA) content was relatively low (6.12 ± 5.80%). Lowering the temperature caused a significant decrease in membrane fluidity and an increase in generalized polarization in fresh spermatozoa, and these effects were even more pronounced following cryopreservation. Anisotropy at 38 °C in fresh samples showed strong positive correlations with viability and motility parameters after thawing. In summary, feline spermatozoa are characterized by a very low PUFA content and a low ratio of unsaturated:saturated fatty acids, which may contribute to low oxidative stress. Cryopreservation alters the structure of the sperm membrane, increasing the fluidity of the hydrophobic portion of the bilayer and the lipid order in the hydrophilic portion. Because lower membrane fluidity in fresh semen was linked with better viability and motility after cryopreservation, this parameter may be considered an important factor in determination of sperm cryoresistance.


Cell Membrane , Cryopreservation , Fatty Acids , Membrane Fluidity , Spermatozoa , Animals , Male , Cats , Spermatozoa/metabolism , Spermatozoa/physiology , Fatty Acids/metabolism , Fatty Acids/analysis , Cell Membrane/metabolism , Cryopreservation/methods , Sperm Motility/physiology , Semen Preservation/methods , Semen Preservation/veterinary , Semen Analysis/veterinary
16.
Reprod Fertil Dev ; 362024 May.
Article En | MEDLINE | ID: mdl-38769680

Context Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods Spermatozoa were exposed to either isotonic (300mOsm/kg) or hypotonic (180mOsm/kg) media for 5 and 20min, and were then stained with SYBR14 and propidium iodide (PI). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+ /PI- ) or with different degrees of plasma membrane alteration (SYBR14+ /PI+ and SYBR14- /PI+ ). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P P P Conclusions Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation.


Flow Cytometry , Osmotic Pressure , Semen Analysis , Spermatozoa , Flow Cytometry/methods , Male , Spermatozoa/physiology , Semen Analysis/methods , Semen Analysis/veterinary , Cluster Analysis , Cell Membrane/physiology , Sperm Motility/physiology , Animals
17.
Reprod Domest Anim ; 59(5): e14618, 2024 May.
Article En | MEDLINE | ID: mdl-38798164

This study investigated the effects of storage conditions on the quality of chilled ram semen stored at 4°C for 48 h, comparing aerobic and anaerobic conditions. Ejaculates from INRA180 rams were collected and stored under both conditions, with assessments at 0-, 24-, and 48-h intervals. Various sperm parameters were examined, including motility, velocity, viability, morphology, membrane integrity, and lipid peroxidation. Results showed that storage duration significantly impacted sperm quality, leading to a gradual decline from 0 to 24 h and 24 to 48 h. Notably, after the initial 24 h, progressive motility (PM) and membrane integrity (MI) demonstrated distinct responses to storage conditions. Anaerobic storage consistently improved PM and MI values compared to aerobic storage between 24 and 48 h. Anaerobic conditions also enhanced viability and reduced abnormality at the 48-h mark. Total motility remained stable throughout storage. Velocity parameters (VCL: curvilinear velocity; VSL: straight velocity and VAP: velocity average path) exhibited differences between the 24- and 48-h intervals, with anaerobic storage resulting in higher VAP and VSL values. Moreover, lipid peroxidation exhibited a progressive increase from 0 to 24 h and 24 to 48 h, independent of storage conditions. Remarkably, anaerobic storage consistently yielded lower lipid peroxidation levels compared to aerobic storage, regardless of storage duration. In conclusion, this study highlights that the anaerobic storage proved advantageous for chilled ram semen quality, particularly after the initial 24 h.


Lipid Peroxidation , Oxygen , Semen Analysis , Semen Preservation , Sperm Motility , Spermatozoa , Semen Preservation/veterinary , Semen Preservation/methods , Animals , Male , Semen Analysis/veterinary , Spermatozoa/physiology , Anaerobiosis , Sheep, Domestic , Sheep/physiology , Semen/physiology , Cell Survival
18.
Reprod Domest Anim ; 59(5): e14613, 2024 May.
Article En | MEDLINE | ID: mdl-38812417

Spermatozoa can experience negative changes when subjected to freezing and thawing, including lowered motility, viability and acrosome response. Herein, the effects of different concentrations of soybean lecithin nanoparticles on cryopreserved Holstein bull semen were examined. Semen was collected, cryopreserved and utilized for sperm kinetic parameter analysis following dilution, equilibration and thawing with 0.5% soybean lecithin (E1), the control extender, and 0.75% (E2), 0.5% (E3), 0.25% (E4) and 0.125% (E5) of lecithin nanoparticles. Results revealed that following dilution, the progressive motility (PM) at E3, E4 and E5 of lecithin nanoparticles was higher (p < .05) than it was for E2. After equilibration, compared to the E1, E2, and E3 values, the PM, vitality, normal morphology, membrane integrity and intact acrosome values at the E5 were consistently greater (p < .05). Comparing the percentages of intact acrosome and membrane integrity at E2 and E3 to E4 and E5, a substantial decrease (p < .05) was seen. Following thawing, the percentage of PM improved at E2 and E5, even though their mean PM values were similar (p > .05) compared to E1, E3 and E4. Vigour and progression parameters of sperm (DAP, DCL, DSL, VAP, VCL, VSL and STR) at E5 were higher (p < .05) than those at E1, E2, E3 and E4. In conclusion, the cryopreserved sperm from Holstein bulls revealed outstanding properties both after equilibration and after thawing with 0.125% lecithin nanoparticles, and they were sensitive to high dosages.


Cryopreservation , Cryoprotective Agents , Glycine max , Lecithins , Nanoparticles , Semen Preservation , Sperm Motility , Spermatozoa , Animals , Male , Cattle , Cryopreservation/veterinary , Cryopreservation/methods , Semen Preservation/veterinary , Semen Preservation/methods , Lecithins/pharmacology , Sperm Motility/drug effects , Glycine max/chemistry , Cryoprotective Agents/pharmacology , Spermatozoa/drug effects , Spermatozoa/physiology , Semen Analysis/veterinary , Acrosome/drug effects , Semen/drug effects
19.
Reprod Domest Anim ; 59(4): e14556, 2024 Apr.
Article En | MEDLINE | ID: mdl-38566398

Scrotal surface thermography is a non-invasive method for assessing testicular thermoregulation in stallions; however, few studies have explored the application of this technique concerning the thermal physiology of equine reproductive systems. This study aimed to evaluate the consistency of testicular thermoregulation in stallions over a year using thermography to measure the scrotal surface temperature (SST). Moreover, we assessed the best region for measuring the surface body temperature compared with the SST. Ten light-breed stallions were used in the experiment. Thermographic images of the scrotal and body surfaces (neck and abdomen) were captured. Fresh, cooled and frozen-thawed semen samples were evaluated to verify the impact of thermoregulation on semen quality. Testicular thermoregulation was maintained throughout the year in stallions amidst changes in the external temperature, as evidenced by the weak correlation between the SST and ambient temperature. A lower correlation was observed between the environmental temperature and body surface temperature (BTS) obtained from the abdomen (BTS-A; R = .4772; p < .0001) than with that obtained from the neck (BTS-N; R = .7259; p < .0001). Moreover, both BTS-A and SST were simultaneously captured in a single image. The consistent quality of the fresh, cooled and frozen semen suggests efficient thermoregulation in stallions throughout the year.


Semen Analysis , Thermography , Animals , Horses , Male , Temperature , Thermography/veterinary , Thermography/methods , Semen Analysis/veterinary , Scrotum/physiology , Testis/physiology , Semen/physiology
20.
Reprod Domest Anim ; 59(4): e14562, 2024 Apr.
Article En | MEDLINE | ID: mdl-38591843

Melatonin is an intracellular antioxidant of sperm membrane that protects the cells from lipid peroxidation. Yet, its role as an antioxidant on semen quality of buffalo bulls is still obscure. The present study was undertaken to assess the effect of exogenous melatonin implant (18 mg/50 kg bodyweight) on post-thaw sperm characteristics, oxidative stress, endocrinological profiles and fertility of buffalo bulls. Six apparently healthy breeding Murrah buffalo bulls were randomly selected at bull farm, Guru Angad Dev Veterinary and Animal Sciences University for the present study and divided into two groups viz. control (n = 3) and melatonin implanted group (n = 3). A total of 120 ejaculates were collected from bulls of both groups (n = 60 each) throughout the study period. Most beneficial effects of melatonin implants were observed during post-implantation period. The percentages of post-thaw sperm total and progressive motility, viability and mitochondrial membrane potential were higher (p < .05) in melatonin implanted buffalo bulls compared to controls during post-implantation period. Following melatonin implantation, MDA production in post-thaw semen was lower (p < .05) in melatonin implanted group than in control group. Plasma melatonin and testosterone concentrations were higher (p < .05) in buffalo bulls implanted with melatonin as compared to their control counterparts. No differences (p > .05) in plasma LH concentrations were observed in both groups. First service pregnancy rate was 43.3% using semen of melatonin implanted bulls and 30.0% with semen of controls (p > .05). Thus, melatonin was able to protect sperm membrane against oxidative damage and improve post-thaw semen quality, thereby resulting in higher fertilizing potential of spermatozoa.


Bison , Melatonin , Semen Preservation , Humans , Pregnancy , Female , Male , Animals , Cattle , Semen Analysis/veterinary , Semen , Buffaloes , Melatonin/pharmacology , Antioxidants/pharmacology , Sperm Motility , Cryopreservation/veterinary , Cryopreservation/methods , Semen Preservation/veterinary , Semen Preservation/methods , Spermatozoa
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