Sperm Longevity Research Articles

UTILIZATION OF NANOTECHNOLOGY IN VETERINARY MEDICINE

In veterinary medicine, particularly in diagnostics, drug administration, and reproductive biology, nanotechnology has made significant contributions. By enabling the precise delivery of unstable or insoluble substances, reducing systemic toxicity, and prolonging the action of the active ingredient at the desired site, nanoparticles enhance the effectiveness of treatments. This translates to fewer doses, reduced costs, and less waste from animal-derived products. Furthermore, nanotechnology aids in diagnostics by detecting pathogens and mapping molecular profiles related to diseases. The sensitivity of tests, increased by nanoparticles, allows for more effective detection of contaminants and microbial agents in animal-derived products, which is crucial for food safety. Nanoparticles and nanotubes are also used in reproductive biology to improve fertility, extend sperm longevity, and facilitate semen purification for artificial insemination. Additionally, implanted nanotubes can monitor hormone levels, aiding in the identification of estrus and the selection of breeders. Despite these advancements, it is crucial to monitor the risks and environmental impacts of nanotechnological technologies to ensure their safety and effectiveness in veterinary medicine. The objective of this work is to explore and evaluate current applications of nanotechnology in veterinary medicine, highlighting its benefits, challenges, and potential for future advancements.

Effect of modified smart extender on sperm longevity and frozen-thawed semen quality in Bantam chicken (Gallus gallus)

Effect of modified smart extender on sperm longevity and frozen-thawed semen quality in Bantam chicken (Gallus gallus)

P-042 Enhancing human sperm motility using high-frequency ultrasound

Abstract Study question Is high-frequency ultrasound effective in the treatment of low-motility sperm for use in assisted reproduction? Summary answer Our study reveals that immotile and poorly motile sperm respond to ultrasound treatment and show up to 266% boost to motility parameters upon ultrasound exposure What is known already Sperm motility is central to both natural and assisted reproduction. This crucial importance has led to the utilization of chemical drugs such as pentoxifylline, theophylline, and other phosphodiesterase inhibitors to improve motility. However, these drugs are embryotoxic with a potentially harmful effect on sperm longevity. In a recent study, we showed that sperm cells at the population level showed a 32% boost to motility after 15 seconds of exposure to ultrasound. However, to translate these findings into clinical applications, it is crucial to identify which subset of sperm within a population is most affected by the treatment. Study design, size, duration We conducted single-cell study aimed at comprehensively assessing how different sperm motility grades are affected by ultrasound exposure. We utilized a droplet acoustofluidic system using which sperm cells were isolated in droplets and their responses to ultrasound were monitored. A total of 239 individual sperm from three biologically independent human samples were included in this study and parameters such as sperm motility, viability, DNA integrity, and mitochondrial membrane potential (MMP) upon ultrasound exposure were analysed. Participants/materials, setting, methods Fresh human semen samples were collected from healthy donors with the approval of the Monash University Human Research Ethics Committee. Three human donors contributed to a total of sixteen independent experiments performed in this work. The manual tracking plugin in ImageJ was used to measure sperm motility. The LIVE/DEAD™ Sperm Viability Kit (Invitrogen, USA) was used to evaluate sperm viability and JC-1 (Invitrogen, USA) was used to monitor the MMP. Main results and the role of chance We classified the sperm cells based on their pre-exposure straight-line velocity (VSL) into three categories of grade C (non-progressive, VSL < 5 µm s-1), grade B (slow progressive, 5 ≤ VSL< 25 µm s-1), and grade A (rapid progressive, 25 µm s-1 ≤ VSL) cells. Our single-cell analysis results show that 20 seconds of ultrasound exposure boosts the motility of grade C sperm up to 266% and as a result, 90% of total sperm are graded as progressively motile after exposure. Through ANOVA tests, we showed that non-progressive sperm cells show a boost to all of the motility parameters more than the other grades. In addition, ultrasound exposure renders 34% of live immotile sperm motile with 24% of the cells graded as progressive and the remaining 10% showed a twitching motility after exposure. We also showed that ultrasound exposure induces flagellum deformation to 16% of total live immotile sperm cells, providing opportunities to detect live sperm in testicular samples using this approach. Finally, we explored the non-invasiveness of the ultrasound platform and showed that there is no detrimental effect on sperm DNA integrity and viability post exposure. Limitations, reasons for caution A larger sample size could provide a higher statistical confidence to our study. Treatment of patient samples (testicular or asthenozoospermic) and evaluation of embryo safety must be included in future studies to evaluate the effectiveness of this method for clinical use. Wider implications of the findings Our acoustic mechanotherapy method could enhance sperm motility to potentially benefit assisted reproduction outcomes. It also enables new opportunities for detecting live sperm in testicular samples. Trial registration number not applicable

Sodium caseinate as a strategy for remote equine semen cryopreservation

The objective of this study was to determine whether the addition of sodium caseinate in different concentrations to the freezing extender medium favors sperm viability after a long period of refrigeration. For this, three concentrations of sodium caseinate were used, 0, 1 and 2%, in the cryopreservation medium, and the ejaculates were divided into nine groups 0H/0%, 0H/1%, 0H/2%, 12H/0%, 12H/1%, 12H/2%, 24H/0%, 24H/1% and 24H/2%. The stallions underwent reproductive evaluation, being included only those with sperm motility ≥ 70%. Fluorescent probes, hypoosmotic (HOST) and thermoresistance (TTR) tests were used to evaluate the functionality of the plasma membrane and the longevity of sperm cells, respectively. There was no difference (p>0.05) between groups for membrane integrity. However, for the TTR, it was possible to observe a difference (p<0.05) for the 24H/2% group, with a lower response. The integrity of the plasma membrane, compared to the refrigeration time and the concentration of sodium caseinate, did not change. Therefore, it was concluded that sperm longevity was impaired with the use of a 2% concentration of sodium caseinate, and its use is not recommended for sending refrigerated semen, for insemination or freezing, that exceeds 12 hours.

Sperm competition favours intermediate sperm size in a hermaphrodite1.

Sperm competition is a potent mechanism of postcopulatory sexual selection that has been found to shape reproductive morphologies and behaviours in promiscuous animals. Especially sperm size has been argued to evolve in response to sperm competition through its effect on sperm longevity, sperm motility, the ability to displace competing sperm, and ultimately fertilization success. Additionally, sperm size has been observed to co-evolve with female reproductive morphology. Theoretical work predicts that sperm competition may select for longer sperm but may also favour shorter sperm if sperm size trades-off with number. In this study, we studied the relationship between sperm size and postmating success in the free-living flatworm, Macrostomum lignano. Specifically, we used inbred isolines of M. lignano that varied in sperm size to investigate how sperm size translated into the ability of worms to transfer and deposit sperm in a mating partner. Our results revealed a hump-shaped relationship with individuals producing sperm of intermediate size having the highest sperm competitiveness. This finding broadens our understanding of the evolution of sperm morphology by providing empirical support for stabilizing selection on sperm size under sperm competition.

The effects of oxidative stress and intracellular calcium on mitochondrial permeability transition pore formation in equine spermatozoa

AbstractThe in vitro storage of stallion spermatozoa for use in artificial insemination leads to oxidative stress and imbalances in calcium homeostasis that trigger the formation of the mitochondrial permeability transition pore (mPTP), resulting in premature cell death. However, little is understood about the dynamics and the role of mPTP formation in mammalian spermatozoa. Here, we identify an important role for mPTP in stallion sperm Ca2+ homeostasis. We show that stallion spermatozoa do not exhibit “classical” features of mPTP; specifically, they are resistant to cyclosporin A‐mediated inhibition of mPTP formation, and they do not require exogenous Ca2+ to form the mPTP. However, chelation of endogenous Ca2+ prevented mPTP formation, indicating a role for intracellular Ca2+ in this process. Furthermore, our findings suggest that this cell type can mobilize intracellular Ca2+ stores to form the mPTP in response to low Ca2+ environments and that under oxidative stress conditions, mPTP formation preceded a measurable increase in intracellular Ca2+, and vice versa. Contrary to previous work that identified mitochondrial membrane potential (MMP) as a proxy for mPTP formation, here we show that a loss of MMP can occur independently of mPTP formation, and thus MMP is not an appropriate proxy for the detection of mPTP formation. In conclusion, the mPTP plays a crucial role in maintaining Ca2+ and reactive oxygen species homeostasis in stallion spermatozoa, serving as an important regulatory mechanism for normal sperm function, thereby contraindicating the in vitro pharmacological inhibition of mPTP formation to enhance sperm longevity.

Sperm characteristics of cryopreserved Prochilodus lineatus semen after adding cholesterol-loaded cyclodextrin

The experiment evaluated the effect of adding cholesterol-loaded cyclodextrin (CLC) to Prochilodus lineatus fish (Curimata) semen on post-thaw sperm quality. Twelve adult fish were used for sperm collection after induced spermiation with carp pituitary gland. The semen was diluted and treated with CLC in concentrations of 0 (control), 0.5, 1.0, 2.0, 3.0, and 4.0 mg for 120 × 106 spermatozoa/ml, loaded in 0.5 ml straws, packaged and placed in dry vapor vessel cylinders for 24 h before being submerged in liquid nitrogen for storage. The samples were thawed in a water bath at 60 °C for 8 s, and the sperm parameters evaluated were motility, activation duration, longevity, plasma membrane integrity, and morphology. Data were tested for normal distribution and ANOVA, followed by Friedman test (P < 0.05). Spermatozoa treated with CLC displayed higher motility than the control (P < 0.05). The duration of sperm activation was longer in sperm treated with 0.5, 1.0, and 2.0 mg of CLC than in control (P < 0.05). The membrane integrity was higher in sperm treated with 0.5, 1.0, 2.0, and 3.0 mg of CLC than in control and four mg-treated samples (P < 0.05). The sperm longevity and morphology alterations did not differ between treatments (P > 0.05). Adding 0.5, 1.0, or 2.0 mg of CLC in Prochilodus lineatus semen before cryopreservation improves sperm motility and membrane integrity.

Characterisation of the urinary microbiome of a frog, and the effect of antibiotics on bacterial abundance and sperm viability during refrigerated storage

Reproductive technologies are increasingly being adopted to improve the conservation management of threatened species. The storage and transport of sperm is a vital aspect of the practical implementation of reproductive technologies, however, bacterial contamination during the storage and transport of sperm samples presents a biosecurity risk and can contribute to a reduction in sperm longevity during storage. The present study: 1) characterised the urinary microbiome (bacterial species composition and abundance) using culture-independent 16S rRNA sequencing; 2) quantified the effect of various doses of gentamicin and streptomycin-penicillin on bacterial abundance (colony-forming units; CFUs) and; 3) quantified the effect of antibiotic supplementation on the sperm viability (proportion live/dead) of spermic urine during a 12-day cold-storage period, using the common eastern froglet, Crinia signifera. Overall, urine samples were found to host a diverse array of bacteria, dominated by the phyla Proteobacteria, Firmicutes, and Bacteroidetes. Bacterial abundance was significantly reduced in all antibiotic treatment groups compared to the control group. Antibiotic supplementation had no effect on sperm viability between day 0 and day 6 of storage, however both antibiotic treatments significantly improved sperm viability from days 9 to 12 of storage compared to the control group. Overall, the results of this study provide novel insight into the urinary microbiome, being the first to use a metagenomics approach to characterise the bacterial community present in the urine of an amphibian. Importantly, this study provides evidence that antibiotic supplementation with either gentamicin or streptomycin-penicillin, minimises bacterial proliferation and improves sperm viability during cold storage. These findings will contribute to the development of biosecurity protocols aimed at reducing the risk of disease transmission and cross-infection from unwanted bacteria and infectious agents in amphibian captive breeding programs.

Effect of Antibiotics Metranidazole, Streptomycin, and Gentamicin on the Maintenance of Sperm Motility of the European Common Frog (&lt;i&gt;Rana temporaria&lt;/i&gt;) During Refrigerated Storage

The problem of asynchrony in the production of gametes in amphibians can be solved by increasing the longevity of spermatozoa during the storage of spermic urine with antibiotics. This study evaluated the effect of metranidazole and streptomycin at concentrations of 0.1, 0.2, 0.4, and 1 mg/ml on the maintenance of motility and the longevity of spermatozoa in the spermic urine of the European common frog (Rana temporaria) during refrigerated storage at 4°C. Metranidazole did not improve the lifespan of spermatozoa, and streptomycin had a positive effect at all analyzed concentrations. In addition, the streptomycin and gentamicin at the same concentrations of 0.05, 0.1, and 0.2 mg/ml were compared for their effect on maintaining sperm motility during storage. In spermic urine samples with significant bacterial contamination, no significant differences were found in the effect of streptomycin and gentamicin on sperm motility. In the absence of any visible signs of significant bacterial contamination of spermic urine samples, the motility of spermatozoa with gentamicin remained significantly longer than that with streptomycin for all analyzed concentrations. In addition, in this study, for the first time, a positive correlation was revealed between the initial concentration of spermatozoa and the longevity of spermatozoa during the refrigerated storage of amphibian spermic urine samples with antibiotics (r = 0.899, P 0.0001). However, no relationship was found between the longevity of spermatozoa and the initial osmolality of spermic urine in the range from 62 to 92 mosmol/kg (r = 0.083, P = 0.768).

Effect of seawater temperature and pH on the sperm motility of the European eel.

The current climate change situation could bring critical effects for marine species, especially those already considered endangered. Although some species can adapt fast to the environmental changes, it is necessary to get into the worst scenario and develop tools to anticipatedly assess the physiological effects of such environmental change. With this purpose, our study aims to determine the effect of a range of seawater temperatures and pHs on sperm motility in the European eel (Anguilla anguilla). Low seawater pH (6.5-7.4) decreased the eel sperm motility in comparison to the control (pH = 8.2). We also studied the combined effect of the pH of the artificial seminal plasma (the plasma where the sperm cells are suspended) with the pH of Artificial Sea Water (ASW, pH 7.8 or and 8.2). We did not find statistical differences in sperm motility and kinetic parameters caused by the artificial seminal plasma pH. However, seawater pH induced significantly higher values of total sperm motility, and the percentage of fast spermatozoa with a pH of 8.2 in comparison with a pH of 7.8. In contrast, the seawater temperature did not affect sperm motility parameters or sperm longevity. To study the effect of the interaction between seawater temperature and pH on sperm motility, two temperatures: 4 and 24°C, and two pHs 7.8 and 8.2, were tested. There were significant differences between temperature and pH in several kinetic parameters and, in general, the lowest values were observed in the samples activated at low temperature and low pH (4°C, pH 7.8). This work suggest that eel sperm motility and kinetics will not be affected by the expected changes in pH or temperature due to the climate change.

Mating roles, copulation duration and reproductive output in the hermaphroditic freshwater snail Planorbella trivolvis

ABSTRACT Hermaphrodites exhibit a wide variety of reproductive strategies, yet empirical studies lag behind theories about their evolution. Planorbella trivolvis, a simultaneous hermaphrodite capable of reciprocal copulation and rare self-fertilization, provides a unique model system for studying reproduction. This study assessed sex roles during mating, demonstrating that mating is primarily reciprocal, and investigated the relationship between physical coupling and reproductive output for single copulation events. Snails were isolated before sexual maturity and placed in 10-l aquaria in size-matched groups. Upon copulation, pairs were removed to small cups until they separated; they were then kept isolated and eggs counted until egg production ceased. The frequency of reciprocal copulation (both members of a pair produced egg masses following copulation) was 95% (35/37 productive matings), higher than previously reported. The duration of egg production ranged from 19 to 197 days (mean ± SE = 110.13 ± 6.3 days) with a sharp drop in production of egg masses and eggs per mass occurring between 15 and 20 weeks postmating, consistent with previous estimates of sperm storage. There was no relationship between mating duration and total egg production (a biomarker of sperm availability) or the duration of egg production (a marker of sperm longevity). Further investigation is needed to directly estimate sperm transfer and tease apart the duration of reciprocal physical contact from gamete and accessory protein transfer.

Investigating the safety of antibiotics added to collared peccary (Pecari tajacu) semen extender through a multiparametric thermoresistance test.

The effects of antibiotics on sperm longevity in collared peccary (Pecari tajacu) fresh diluted semen was evaluated. Semen samples from six adult males were collected by electroejaculation and diluted in Tris-citrate-fructose alone (control) and plus streptomycin-penicillin (2 mg/ml-2000 IU/ml) or gentamicin (70 µg/ml). Membrane integrity and functionality, mitochondrial activity and sperm morphology were assessed subjectively. Sperm motility and other kinetic parameters were objectively assessed using CASA (computer-assisted semen analysis). The semen diluted according to the treatments were submitted to the thermoresistance test, incubated at 37 ° C, and the sperm parameters analyzed at 0, 30, 60, 120 and 180 min. The average values of the treatments were compared with each other and between the times. There were no differences (P > 0.05) between treatments until the end of the test. Control and streptomycin-penicillin samples maintained sperm function for up to 180 min (with total motility of 24.3 ± 7.1% and 28 ± 8.7%, respectively). Gentamicin aliquots retained most parameters until the end of the incubation, except for membrane integrity and mitochondrial activity that declined (P < 0.05) at 180 min (53.1 ± 7.1% and 50.7 ± 6.2%, respectively) compared to 0 min (80.5 ± 4.7% and 86.3 ± 3.4%, respectively). In conclusion, a multiparametric thermoresistance test proved that Tris-based extenders used for collared peccary semen can be effectively supplemented by streptomycin-penicillin (2 mg/ml-2000 IU/ml) or gentamicin (70 µg/ml), especially during 180-min incubation at 37 °C.

In Vitro Storage of Functional Sperm at Room Temperature in Zebrafish and Medaka.

The longevity of sperm in teleost such as zebrafish and medaka is short when isolated even in saline-balanced solution at a physiological temperature. In contrast, some internal fertilizers exhibit the long-term storage of sperm, >10 months, in the female reproductive tract. This evidence implies that sperm in teleost possesses the ability to survive for a long time under suitable conditions; however, these conditions are not well understood. In this study, we show that the sperm of zebrafish can survive and maintain fertility in L-15-based storage medium supplemented with bovine serum albumin, fetal bovine serum, glucose, and lactic acid for 28 days at room temperature. The fertilized embryos developed to normal fertile adults. This storage medium was effective in medaka sperm stored for 7 days at room temperature. These results suggest that sperm from external fertilizer zebrafish and medaka has the ability to survive for at least 4 and 1 week, respectively, in the body fluid-like medium at a physiological temperature. This sperm storage method allows researchers to ship sperm by low-cost methods and to investigate key factors for motility and fertile ability in those sperm.

Asparagus racemosus improves seminal antioxidant status and sperm characteristics in buck semen at refrigeration temperature

The present investigation was carried out to evaluate the effect of Asparagus racemosus (Shatavari) aqueous extract on buck semen quality during preservation. In the current study, 8 ejaculates from 8 Jakhrana bucks maintained at Jakhrana unit of ICAR-CIRG (semi-arid region) were collected (total 64 ejaculates) during the period from April to June, 2022. Good quality semen samples were pooled during each collection. Pooled semen samples were then divided into 4 equal parts, and diluted in TRIS buffer containing different concentration of Shatavari aqueous extract (Different groups, i.e. Gr1-5 mg, Gr2-2.5 mg, Gr3-1.25 mg, Gr4-0 mg of Shatavari aqueous extract/ml of semen diluent). All the diluted semen samples were kept at refrigerated temperature (5°C) for seven days, and on each day, diluted semen was evaluated for various sperm characteristics and antioxidant status. Gr3 showed significantly better results in terms of sperm viability, sperm motility, acrosomal integrity and plasma membrane integrity. Along with this, the longevity of sperm was also enhanced in Shatavari supplemented group.

Sperm Longevity and Motility in Ringer’s Lactate Solution with Addition of Egg Yolk among Five Phenotypes of Kokok Balenggek Chicken

The application of cryopreservation to preserve germplasm in such specific breed requires preliminary studies, primarily related to the resistance of spermatozoa to low temperatures (4 – 5 °C) as measured by their motility and longevity. In this study, semen taken from five phenotypes of Kokok Balengeek Chicken (KBC) (Biriang, Jalak, Kinantan, Kuriak, and Taduang) was used to evaluate the effect of Ringer’s Lactate-egg yolk diluent on longevity and motility of spermatozoa. The treatments consisted of Ringer’s Lactate (RL) solution added with egg yolk at a concentration of 1% (RLKT1), 3% (RLKT3), and 5% (RLKT5). Evaluation of fresh semen showed that the spermatozoa of Jalak had the highest motility, namely 75.63 ± 0.5% (P&lt;0.05). Post-dilution longevity and motility observations were carried out at 0, 24, 48, and 72 h, significantly decreasing each time (P&lt;0.05). The lowest range of reduction was found in Jalak spermatozoa diluted with RL with longevity of 7.75 ± 0.70 days. Overall, the RL diluent showed the highest motility after 24 h, namely 41.13 ± 2.27%. Adding egg yolks to Ringer's Lactate solution could not maintain the motility of KBC spermatozoa when stored at 4-5°C for 48-72 h.

An oviduct glycan increases sperm lifespan by diminishing the production of ubiquinone and reactive oxygen species†.

Sperm storage by females after mating for species-dependent periods is used widely among animals with internal fertilization to allow asynchrony between mating and ovulation. Many mammals store sperm in the lower oviduct where specific glycans on oviduct epithelial cells retain sperm to form a reservoir. Binding to oviduct cells suppresses sperm intracellular Ca2+ and increases sperm longevity. We investigated the mechanisms by which a specific oviduct glycan, 3-O-sulfated Lewis X trisaccharide (suLeX), prolongs the lifespan of porcine sperm. Using targeted metabolomics, we found that binding to suLeX diminishes the abundance of 4-hydroxybenzoic acid, the precursor to ubiquinone (also known as Coenzyme Q), 30min after addition. Ubiquinone functions as an electron acceptor in the electron transport chain (ETC). 3-O-sulfated Lewis X trisaccharide also suppressed the formation of fumarate. A component of the citric acid cycle, fumarate is synthesized by succinate-coenzyme Q reductase, which employs ubiquinone and is also known as Complex II in the ETC. Consistent with the reduced activity of the ETC, the production of harmful reactive oxygen species (ROS) was diminished. The enhanced sperm lifespan in the oviduct may be because of suppressed ROS production because high ROS concentrations have toxic effects on sperm.

Effect of FertTalp addition to stallion semen on kinetic parameters and production of intracellular hydrogen peroxide after thawing

Motility stimulants increase sperm energy production and their addition after semen thawing will increase motility (Stephens et al., J. Equine Vet. Sci. 2013;33:615-621), and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al., Anim. Reprod. Sci. 2017;179:27-34). Semen of 12 Mangalarga Marchador stallions was cryopreserved with addition of different concentrations of cholesterol-loaded cyclodextrin (CLC): G1 (control – without cholesterol), G2 (1mg of cholesterol), G3 (1.5mg of cholesterol) and G4 (2mg of cholesterol). To run the experiment, two straws from each stallion per experimental group were thawed and divided into two aliquots of 400µL. This was supplemented with 100µL of Fert Talp, a Tyrode's albumin lactate pyruvate medium with (FT) or without (No-FT) heparin, a motility stimulant. This subdivided the four groups into eight. Sperm kinetics were performed analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®). Sperm longevity was analyzed by using a thermoresistance test at 37°C (TTR). Statistical analysis was done by ANOVA F-test. Complementary to sperm kinetics, the production of intracellular hydrogen peroxide was estimated by flow cytometry using Dichlorodihydrofluorescein diacetate (DCFDA®). Results were classified as: Q1 [Plasma membrane (PM) injured without intracellular hydrogen peroxide]; Q2 (PM injured with intracellularhydrogen peroxide); Q3 (intact MP with intracellular hydrogen peroxide); Q4 (intact PM without intracellular hydrogen peroxide) and Tukey test was performed for statistical analysis. Immediately after thawing, there was no difference between FT and No-FT (p>0.05). After incubation at 37°C, the addition of FT showed higher values for RAP (percentage of rapid sperm, %), VAP (average path velocity, μm/s) and LIN (linearity of the curvilinear trajectory, %) after 80 minutes of incubation and also superior rates of RAP, VSL (straight line velocity, μm/s), VAP and LIN after 120 minutes of incubation (p<0.05), demonstrating satisfactory results both with regard to sperm kinetics and longevity. It can be concluded that the use of FT was beneficial for sperm velocity. These findings suggest that the addition of this motility stimulant after thawing may improve fertility because sperm velocity – VCL (curvilinear velocity, μm/s), VSL and VAP are the most reliable parameters to assess sperm quality (Verstegen et al. Theriogenology, 2002;57:149-179). Significant differences were also observed for Q4 (intact PM, without hydrogen peroxide), where the G2 FT group demonstrated superiority when compared to all groups not supplemented with FT (p>0.05), providing satisfactory plasmatic membrane results against oxidative stress.

The effect of sex-sorting on stallion semen quality

Spermatozoa retain fertilizing potential after sorting, but separation into X and Y populations has deleterious effects on the cell (Samper JC et al. Clinical Theriogenology 2022). The effect of sex-sorting (SS) on stallion sperm was evaluated in single ejaculates from 32 fertile, sexually active stallions. Based on previous work (Samper et al 2022) Botu-Gold was selected to dilute 2 billion cells at 1:3-1:4 ratio. Extender was supplemented with the DNA dye Hoechst 33342 and cooled at 8 ̊C for 18-24 h. Semen was warmed to 20 ̊C, and centrifuged through EquiPure at 200g for 30 min. The pellet was resuspended at 250-300 million/mL using ST Equiship, ST Genetics Texas) and transported at ∼20°C to the sorting facility. Sperm were SS (SexedULTRA™ Genesis III flow cytometer). Purity gate was set at 95% for both genders. Percent morphologically normal (%NORM) (Nomarski 1000X); percent total motility (TMOT), curvilinear velocity (VCL [um/sec]) (IVOS II); percent viability (VIAB; NucleoCounter) were evaluated at times, T1-after cooling, T2- after Equipure, T3- 0 to 6hs after sorting. Semen was cooled to 8°C and TMOT, VCL and VIAB were assessed at 18-24h (T4) and 48h (T5) post-sorting. Treatment means at each treatment/time were analyzed by a repeated measures mixed-model ANOVA. Sort quality measures (% DEAD) Peak Valley Ratio [PVR] and % percent of sperm correctly oriented [ORT]) were correlatedwith %NORM. Although there was a wide variation between stallions, 29/32 (91%) stallions were successfully sorted. The %NORM (mean±sd) was 59±11, 74±9 and 77±9% for T1, T2 and T3 respectively (p<0.05). For TMOT, T1 (67%) was less than T2 (74%; P<0.05) and similar to T3 (71%; P>0.05); for VIA, T1 (74%) was less than T2 and T3 (79 and 80%; P<0.05); for VCL, T1 (157µ/s) was less than T2 (171 µ/s; P<0.05) and similar to T3 (166 µ/s; P>0.05). For sperm longevity (TMOT, VIA, and VCL) T4 (49, 73, 147) and T5 (27, 63, 117) decreased, compared to T3. The initial quality of the semen and the number of morphological abnormalities had a high impact on the sorting and quality parameters ORT and PVR were positively correlated while DEAD was negatively correlated with %Norm cells in T1, T2 and T3. In this study, the SS process (cooling, colloid centrifugation and SS) occurred over an ∼ 24-hour period and the quality of the SS sperm was improved and maintained over that period which is similar to those normally utilized for conventional cool-shipped semen programs. Current sex-sorting technology has improved significantly, resulting in a high number of stallions sorting successfully, allowing broad application of gender selection technology to the equine breeding industry.

Examination of the effects of inbreeding on reproductive capacity in the Pacific oyster, Crassostrea gigas

The Pacific oyster (Crassostrea gigas) has been widely cultured as a commercially important bivalve. Intercrossing of inbred strains is regarded as an efficient method to increase yield (heterosis), so the development of inbred lines is common in the breeding strategies of C. gigas. However, inbreeding usually reduces physiological efficiency or reproductive capacity. The effects of inbreeding on gonadal development and reproductive capacity remain to be explored in oysters. In the present study, the monthly gonadal development of an inbred and a control population was observed. Compared with the control population, the delayed gonadal development in the inbred population may require more time and cost for breeders in the artificial breeding process. We investigated how inbreeding influenced female and male gametic traits, including reproductive effort, egg diameter, fertilization rate, hatching rate, sperm motility, sperm longevity, and sperm curvilinear velocity, as well as sperm competitiveness based on a male's share of paternity. We found no inbreeding depression for egg diameter, fertilization rate, or sperm motility. However, maternal inbreeding decreased reproductive effort and hatching rate, and paternal inbreeding decreased sperm longevity, curvilinear velocity, and competitiveness. Our results show evidence of inbreeding depression in the gonadal development and reproductive capacity of C. gigas, which could help us in a better understanding and management of inbreeding in oyster aquaculture.

Effects of extender type and storage time on sperm quality parameters of Kail ram semen stored at 5 °C

The study was designed to evaluate the effect of different extenders and storage times on sperm quality parameters of extended Kail ram semen. Semen was collected from five adult Kail rams using an artificial vagina. Semen samples with >70% total sperm motility were pooled, diluted with Tris (TR), sodium citrate (SC), and skim milk (SM)-based extenders, and stored at 5 °C. Sperm motility and kinematics, viability, and plasma and acrosomal membrane integrity were assessed every 24 hrs for 120 hrs. Sperm longevity was estimated by placing semen in a water bath at 37°C to assess sperm kinematics. Storage time as the main effect had a negative (P < 0.05) impact on sperm quality parameters. The percentages of total motile (TM), progressive motile (PM), rapid, and medium progressive (MP) motile sperm were similar at 24 hrs of storage for the three extenders. However, semen extended in TR had a higher (P < 0.05) percentage of TM, PM, rapid, and MP sperm compared to SM and SC extenders at 48 hrs of storage. The sperm kinematics (VCL, VSL, VAP, and ALH) of progressive sperm were similar for all three extenders up to 72 hrs of storage. In addition, semen extended in TR had a higher (P < 0.05) percentage of sperm with intact plasma and acrosomal membranes at a storage time of 48 hrs. At 37 °C, the percentage of TM and PM sperm was higher (P < 0.05) in the TR extender than in the SM and SC extenders at 60 minutes and beyond. In conclusion, the Kail ram sperm extended in TR and stored at 5 °C maintained better quality after 48 hrs storage than sperm extended in SM and SC extenders. At 37 °C, sperm extended in TR also retained better quality parameters at 60 min and afterward than sperm extended in SM and SC.