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A seasonal effect on sperm quality parameters was observed previously. Although identification of the bull semen microbiota by 16S rRNA sequencing was performed previously, it has not been carried out in commercial semen samples from different seasons, and its connection with sperm quality parameters has not been evaluated yet. The objectives in this study were; (i) to evaluate diversity of bull semen microbiota and sperm quality parameters in different seasons, and (ii) to find if specific bacteria were associated with seasonal differences in specific sperm quality parameters. Bull semen microbiota was identified in 54 commercial bull semen samples from 3 seasons (winter, spring, summer). Sperm quality was analysed by Computer Assisted Sperm Analyses (CASA) and Flow Cytometry (FC). From 28 phyla in all samples, six phyla were identified in samples from all seasons, with observed seasonal differences in their distribution. At genus level, 388 genera were identified, of which 22 genera had a relative abundance over 1 % and showed seasonal differences in bacterial diversity, and 9 bacteria genera were present in all seasons. Differences between spring and summer (P < 0.05) were observed for live hydrogen peroxide positive sperm cells. A trend towards significance (0.10 > P > 0.05) was observed for some CASA kinematics (VCL and LIN) and FC parameters (High respiratory activity, and live hydrogen peroxide positive sperm cells) between seasons. Nevertheless, associations between sperm quality parameters and specific bacteria were observed in spring. • Seasonal difference in the relative abundance of the top six phyla were present. • The number of genera and their amount in different seasons vary. • Only small differences in sperm quality between seasons were detected. • Genera were correlated with hydrogen peroxide (H 2 0 2) positive sperm cells in spring. • Genus Cutibacterium was negatively correlated with H 2 0 2 positive sperm cells. [ABSTRACT FROM AUTHOR]

In recent decades, there has been a growing interest in optimizing the protocols intended to sperm cryopreservation in domestic animals. These protocols include initial cooling, freezing, and thawing. While different attempts have been devised to improve sperm cryopreservation, the efficiency of this reproductive biotechnology is still far from being optimal. Furthermore, while much attention in improving cooling/freezing, less emphasis has been made in how thawing can be ameliorated. Despite this, the conditions through which, upon thawing, sperm return to physiological temperatures are much relevant, given that these cells must travel throughout the female genital tract until they reach the utero-tubal junction. Moreover, the composition of the media used for artificial insemination (AI) may also affect sperm survival, which is again something that one should bear because of the long journey that sperm must make. Furthermore, sperm quality and functionality decrease dramatically during post-thawing incubation time. Added to that, the deposition of the thawed sperm suspension devoid of seminal plasma in some species during an AI is accompanied by a leukocyte migration to the uterine lumen and with it the activation of immune mechanisms. Because few reviews have focused on the evidence gathered after sperm thawing, the present one aims to compile and discuss the available information concerning ruminants, pigs and horses. [ABSTRACT FROM AUTHOR]

Intracytoplasmic sperm injection (ICSI) remains inefficient in cattle. One reason could lie in the injection of oocytes with sperm that have not undergone molecular changes associated with in vivo capacitation and fertilizing ability. This study aimed to enhance the efficiency of bovine intracytoplasmic sperm injection (piezo-ICSI) by employing fluorescent-activated cell sorting (FACS) to select the sperm population before injection based on capacitation markers. First, we evaluated the effects of incubating thawed sperm for 2 hours with different capacitating inductors: heparin, methyl-beta-cyclodextrin (MβCD), and dibutyryl cyclic AMP (dbcAMP), alone or in combinations in a basal capacitating (C) medium (Sp-TALP). Sperm capacitation and quality markers were evaluated by flow cytometry, revealing heparin as the most effective inducer of sperm capacitation changes. It, therefore, this treatment was chosen as the sperm pretreatment for FACS-piezo-ICSI. Two cell populations showing high capacitating levels (Heparin-HCL) and low capacitating levels (Heparin-LCL) of the markers associated with sperm capacitation i(Ca2+) levels and acrosome integrity were selected by FACS and used for sperm injection. Pronuclear formation was significantly higher when ICSI was performed with Heparin-HCL sperm than with Heparin-LCL and the control group (Heparin unsorted) groups (50 %, 10 %, and 20 %, respectively). Furthermore, injecting Heparin-HCL sperm resulted in a higher blastocyst rate (22.5 %) than Heparin-LCL (10 %) and the control group (15.2 %). In conclusion, heparin treatment effectively induced changes associated with sperm capacitation. The combination of Heparin-HCL treatment and FACS enabled precise selection of capacitated sperm before ICSI, enhancing the efficiency of this technology in the bovine species. [ABSTRACT FROM AUTHOR]

Semen cryopreservation is a crucial part of assisted reproductive techniques (ART) in animals, and recently it is gaining more and more attention among cat breeders. Even if fresh semen quality is good, sometimes spermatozoa do not survive freezing. The freezability prediction was widely studied in many species, but not in the domestic cat. The aim of this study was to verify the usefulness of osmotic challenge tests and membrane structure markers (Yo-Pro 1 and Merocyanine 540) for the prediction of the quality of post-thawed feline semen. Semen was collected by urethral catheterization from 22 male cats. After a basic evaluation of semen, 20×106 spermatozoa were cryopreserved; the rest were evaluated by flow cytometry for membrane integrity (SYBR-14/PI), acrosome status (lectin PNA/PI), mitochondrial potential (JC-1) and membrane stability (Yo-Pro 1/M540 staining). Hypo- and hyperosmotic challenge tests were also performed. The thawed samples were evaluated as fresh ones. The Pearson correlation between all parameters in fresh semen and all parameters in cryopreserved spermatozoa was assessed. Although some moderate correlations were found between the results of the osmotic tests and markers of sperm membrane stability (Yo-Pro 1 and Merocyanine 540) and post-thaw semen quality parameters, the predictive value of studied markers was rather weak – no cut-off values could be established and, based on regression models, they explained less than 40 % of variability in post-thaw quality. Our results confirm that cryodamage is a complex matter, in which many different factors play a role, affecting sperm motility and membrane integrity differently. ● Cryopreservation differently affects sperm motility and membrane integrity. ● Post-thaw viability is mostly related to initial sperm membrane characteristics. ● Post-thaw motility is moderately correlated with several different parameters. ● Acrosome damage >10 % can be regarded as an unfavorable prognostic factor. [ABSTRACT FROM AUTHOR]

Environmental enrichment is used to provide well-being to the animals, such as fish, in captive conditions, mimicking their natural habitat. It may influence fish behavior, physiology, and survival. In terms of reproduction, however, the relationship between environment enrichment and successful reproduction in captivity is still poorly explored in fish species. Aiming to understand any possible benefits of structural enrichment on fish reproduction, 10-days-hatched larvae of the twospot astyanax Astyanax bimaculatus were raised for 18 weeks in tanks with different elements of structural environmental enrichment (PVC pipes, stones, and artificial plants). In the 5th month of life, those animals were hormonally induced to reproduce to assess gamete formation and offspring quality. Animals raised in a sterile-reared environment (non-enriched) showed earlier spawning than the enriched one, presenting significant quantities of Postovulatory follicle complexes (POCs) and cells in atresia in female ovaries, indicating possible reproductive dysfunction or stress, as well as a greater quantity of empty testicular lumen in males, indicating great release of sperm. On the contrary, animals cultivated in enriched environments showed gonads filled with semen in males and vitellogenic oocytes in females. Furthermore, offspring from the sterile-reared group presented significant rates of larval abnormality compared to the enriched group. In conclusion, the results of this study show that environmental enrichment can interfere with the reproduction of fish in captivity, mainly by preventing early maturation of gametes, which can result in low-quality offspring and, consequently, low production of fish species. • Environmental enrichment improves the reproduction of native fish in captivity. • Offspring from specimens reared in an enriched environment presents fewer abnormalities. • Gametes maturation of specimens reared in an enriched environment is similar to wild animals. [ABSTRACT FROM AUTHOR]

Male reproductive efficiency is primarily defined by the generation of high-quality and viable sperm cells in farm animals. However, the literature shows that male fertility has declined in recent years due various factors including heat stress, which causes the development of free radicals and reactive oxygen species (ROS) which damages sperm cells. This review aimed to examine the potential significance of antioxidants in increasing and preserving sperm quality and viability. Data used to produce this review paper came from recently published articles in peer reviewed journals. Google Scholar, Science Direct, Research Gate, Web of Science, and the Directory of Open Access Journals were used to access the data. Various studies have shown that antioxidants play acritical role in preserving the sperm quality and viability by protecting sperm cells from the potential damage from oxidative stress induced by the development of oxygen species imbalances. However, there is less information on the use of natural or synthetic antioxidants to preserve semen quality through in vivo procedures, despite its growing popularity and promising results. Hence, there is a need for researchers to explore more on this topic, especially in other livestock species than poultry. • Semen preservation enables the successful future use of sperm through AI. • The quality of preserved sperm can be reduced. • ROS cause oxidative stress thus extremely detrimental to spermatozoa. • Antioxidants can neutralise, disposes and inhibit the formation of ROS. [ABSTRACT FROM AUTHOR]

Several studies have demonstrated the correlation between Doppler velocimetric parameters of testicular artery and semen quality in domestic species, but in felines data are scarce. This study aimed to correlate the Doppler velocimetry of the testicular artery with sperm kinetics and sperm defects, in sedated and non-sedated cats. Forty tomcats were divided into two groups: sedated (SG; n =20) with dexmedetomidine (10 µm/kg) and ketamine (12 mg/kg), and non-sedated (NSG; n =20). The animals were subjected to ultrasound Doppler velocimetry of the distal supratesticular and marginal region of the testicular artery and subsequently orchiectomized. Epididymal tail spermatozoa were recovered and analyzed using a CASA system for motility, and morphology took place. Animals of SG presented a significantly higher velocity in the marginal region of the cat's testicular artery [peak systolic velocity (PSV) 11.51 cm/s; end-diastolic velocity (EDV) 7.72 cm/s] compared to NSG (PSV 7.72 cm/s, P < 0.001; EDV 4.93 cm/s, P < 0.001). Sedated cats presented higher pulsatility and resistivity indexes than non-sedated cats. The supratesticular PSV of NSG was moderately correlated with major (r s = 0621; P < 0.001) and total sperm defects (r s = 0614; P < 0001). Doppler velocimetry was fairly correlated with minor, major, and total sperm defects. In conclusion, Doppler velocimetric evaluation emerges as an important possibility in the reproductive evaluation of tomcats, once the testicular artery hemodynamics were associated with sperm defects. However, it is advisable to carry out this evaluation in non-sedated animals. If sedation is necessary, peripheral vasoconstriction should be considered. [Display omitted] • Dexmedetomidine promotes vasoconstriction in the testicular artery of cats. • Sperm defects were correlated with testicular artery Doppler velocimetry in non-sedated cats. • Doppler velocimetric evaluation may contribute to the andrological exam of cats. • It is possible to evaluate hemodynamics of two regions of a cat's testicular artery. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation plays an important role in the artificial insemination (AI) industry of small ruminants. It, however the use of frozen-thawed goat semen is limited due to the insufficient number of sperm with good biological functions. Mitochondria are the most sensitive organelles to cryopreservation damage in sperm. This study was conducted to determine the effects of MitoQ, the mitochondrial-targeted antioxidant, in a plant-based extender on the quality parameters of Markhoz goat sperm after the freezing and thawing process. Semen samples were collected and diluted in the extender, divided into five equal aliquots and supplemented with 0, 1, 10, 100 and 1000 nM MitoQ and cryopreserved in liquid nitrogen. After thawing, sperm motility, membrane functionality, abnormal morphology, mitochondrial activity, acrosome integrity, lipid peroxidation (LPO), DNA fragmentation, reactive oxygen species (ROS) concentration, viability and apoptotic-like changes were measured. The use of 10 and 100 nM MitoQ resulted in higher (P ≤0.05) total motility (TM), progressive motility (PM), viability, membrane functionality, mitochondrial activity, and acrosome integrity compared to the other groups. On the other hand, LPO, apoptotic-like changes, DNA fragmentation and ROS concentration were lower (P ≤0.05) in MQ10 and MQ100 groups compared to the other groups. MitoQ has no effect (P >0.05) on sperm abnormal morphology and velocity parameters. In conclusion, MitoQ can reduce oxidative stress by regulating mitochondrial function during the cryopreservation process of buck sperm and could be an effective additive in the cryopreservation media to protect sperm quality. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the effects of replacing egg yolk extender with sesame oil on the quality of sperm in goats following incubation at 37°C, chilling at 4°C, and freezing. Semen samples were collected from four intact male goats. The individual semen sample was divided into six groups consisting of a control group and five treatment groups with different egg yolk-to-sesame oil ratios. Seminal plasma was removed, and the sperm pellet was diluted with experimental semen extenders. The control group contained an extender of 10 % egg yolk (SO0), and the experimental extenders were composed of 8.75 % egg yolk and 1.25 % sesame oil (SO1.25); 7.5 % egg yolk and 2.5 % sesame oil (SO2.5); 5 % egg yolk and 5 % sesame oil (SO5); 2.5 % egg yolk and 7.5 % sesame oil (SO7.5); and 10 % sesame oil (SO10). Each group of semen was divided into three groups, incubated at 37°C for 1 h, chilled at 4°C for 4 h, or frozen for 24 h. Five replicates were performed. Sperm quality was evaluated, including motility, viability, and functional membrane integrity. The SO1.25 group achieved the highest sperm quality rate among the treatment groups, and the extender did not have a negative effect compared to the control. However, the total replacement of egg yolk with sesame oil in an extender resulted in the lowest sperm quality. In conclusion, the ratios of egg yolk and sesame oil that were acceptable for goat semen cryopreservation were 8.75 % and 1.25 %, respectively. • The sperm quality did not differ between the 1.25 % sesame oil and control groups. • The 2.5 % sesame oil initially revealed an adverse effect on goat sperm. • The extenders in groups 5 %, 7.5 %, and 10 % sesame oil separated into two layers. [ABSTRACT FROM AUTHOR]

The study investigated the impact of resveratrol (RES) on bull sperm cryopreservation employing conventional slow (CS) and ultra-rapid (UR) freezing methods on sperm quality and in vitro fertility. Twenty-four ejaculates from four bulls were divided into four groups based on the cryopreservation method and RES addition: CS-RES (n = 80), CS-Co (n = 80), UR-RES (n = 24), and UR-Co (n = 24). The CS freezing involved exposing sperm straws with 5% glycerol to liquid nitrogen (LN 2) vapors, while UR freezing submerged sperm drops with 100 mM sucrose directly into LN 2. Overall, sperm kinematic parameters and integrity of plasma and acrosome membranes significantly decreased (P < 0.001) after cryopreservation. Post-thaw values of motilities (total [TM] and progressive [PSM]), velocities (curvilinear and straight-line), beat cross frequency (BCF), and sperm with intact plasma membrane/intact acrosome (PI-/PNA-) were higher (P < 0.05) with CS-RES and CS-Co treatments compared to UR-RES and UR-Co treatments. CS-RES treatment resulted in greater percentages (P < 0.05) of TM, PSM, P I-/PNA-, and fertility (blastocyst rate) than their control, CS-Co; while UR-RES showed higher BCF values (P < 0.05) than its control, UR-Co. Additionally, UR-RES treatment exhibited lower oxidative stress percentages than UR-Co (P < 0.05). This study presents the following conclusions: (1) the CS freezing resulted in better cryosurvival of bull sperm than UR freezing; (2) the RES supplementation to CS freezing medium improved sperm motility, membrane integrity, and fertility; and (3) despite low cryosurvival sperm and fertility, the RES addition to ultra-rapid freezing medium reduced oxidative stress. • Conventional slow freezing showed higher sperm cryosurvival than ultra-rapid freezing. • Resveratrol improved sperm motility and membrane integrity after slow freezing. • Resveratrol addition to slow freezing medium improved in vitro fertility. • Resveratrol reduced oxidative stress after ultra-rapid freezing. [ABSTRACT FROM AUTHOR]

Cryopreservation is the most efficient procedure for long-term preservation of mammalian sperm; however, its use is not currently dominant for boar sperm before its use for artificial insemination. In fact, freezing and thawing have an extensive detrimental effect on sperm function and lead to impaired fertility. The present work summarises the basis of the structural and functional impact of cryopreservation on pig sperm that have been extensively studied in recent decades, as well as the molecular alterations in sperm that are related to this damage. The wide variety of mechanisms underlying the consequences of alterations in expression levels and structural modifications of sperm proteins with diverse functions is detailed. Moreover, the use of cryotolerance biomarkers as predictors of the potential resilience of a sperm sample to the cryopreservation process is also discussed. Regarding the proteins that have been identified to be relevant during the cryopreservation process, they are classified according to the functions they carry out in sperm, including antioxidant function, plasma membrane protection, sperm motility regulation, chromatin structure, metabolism and mitochondrial function, heat-shock response, premature capacitation and sperm-oocyte binding and fusion. Special reference is made to the relevance of sperm membrane channels, as their function is crucial for boar sperm to withstand osmotic shock during cryopreservation. Finally, potential aims for future research on cryodamage and cryotolerance are proposed, which might be crucial to minimise the side-effects of cryopreservation and to make it a more advantageous strategy for boar sperm preservation., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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. • Coenzyme Q10 preserved quality parameters of buck's spermatozoa during cryopreservation. • Coenzyme Q10 had helpful effects in soybean lecithin-based extender on sperm quality. • Coenzyme Q10 cannot affect sperm morphology and velocity parameters. [ABSTRACT FROM AUTHOR]

To assist in the conservation of collared peccary, it is important to strengthen semen processing protocols. The aim of this study was to compare the effects of different commercial extenders (BTS; NUTRIXcell+ and PRIMXcell Ultra) and TRIS + egg yolk on the functional and morphological aspects of collared peccary semen stored at 17 °C for 48 hours. Ten ejaculates obtained by electroejaculation were divided into 4 aliquots and diluted in the respective extenders, then stored in a biological incubator at 17 °C for 12, 24, 36, and 48 hours. The samples were evaluated for kinetic parameters, membrane functionality, membrane integrity, mitochondrial activity, morphology, and sperm-binding capacity. At the end of storage (48 h), promising results were found for motility parameters, with TRIS + egg yolk (71.0 ± 4.6%) being more efficient than NUTRIXcell+ (38.9 ± 10.9%) (P < 0.05) and similar to BTS (42.9 ± 11.9%) and PRIMXcell Ultra (46.8 ± 10.8%). The results for membrane integrity and mitochondrial activity were around ∼30–50%, with TRIS being the only extender to preserve both parameters (58.9 ± 5.3 and 59.2 ± 5.6%) for up to 48 hours, respectively (P < 0.05). Finally, the extenders could guarantee 60% membrane functionality and ∼ 60–70% normal sperm morphology, as well as similar binding capacity among the groups. In conclusion, TRIS + egg yolk is effective in preserving the sperm parameters of collared peccary semen at 17 °C for 48 hours, while PRIMXcell Ultra and BTS are viable alternatives for this purpose. • Cooling collared peccary semen at 17 °C is a promising approach for this species. • TRIS + egg yolk proved effective in preserving the functional parameters of cooling semen for up to 48 hours. • PRIMXcell Ultra and BTS are viable alternatives for cooling collared peccary semen. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Boar sperm quality deteriorated during freezing and thawing. • After MACS, one third of the original sperm number appeared in flow-through fraction. • Sperm quality was enhanced in the flow-through fraction compared to the original. • Eluate had worse sperm quality than flow-through fraction or original sample. [ABSTRACT FROM AUTHOR]

Boar sperm is highly susceptible to cold damage. When temperature drops to 5°C, the plasmatic membrane is destabilized. The freezing process causes a reduction of the fertility window because frozen/thawed boar sperm has less survivability. The aim of this work was to analyze the effect on sperm characteristics and response to capacitation stimuli of cooling to 5°C using a controlled protocol. Also, we evaluated if the addition of Glycerol 2% or 3% at 5°C was able to modify these parameters. For this purpose, we assessed motility, plasmatic membrane integrity and acrosomal membrane status. Capacitation was induced using Tyrode´s capacitating medium (TCM) and assessed by chlortetracycline stain and induction of acrosomal reaction with Progesterone. Motility patterns were analyzed using a CASA system. These tests were performed at three different points of the freezing curve: 37°C; 17°C and 5°C. Response to TCM vs TBM was only significant at 37°C. While at 37°C and 17°C capacitated sperm was below 20%, at 5°C reached 50% both in the TBM and TCM. CASA analysis showed that spermatozoa exposed to TCM had higher LIN and WOB than those in TBM. All parameters were similar in the Glycerol concentrations studied. These results suggest that the chilling process may be causing an effect similar to cryocapacitation along the cooling curve, starting subtle at 17°C and reaching 50% of the sperm population at 5°C, being independent of Glycerol concentration. • Cryocapacitation of boar sperm starts at 17°C and affects 50% of sperm at 5°C. • Sperm exposed to TCM after cooling to 5°C show high linearity and low oscillation. • Glycerol didn´t modify response to capacitation and induced acrosomal reaction. [ABSTRACT FROM AUTHOR]

Although cryopreservation is a reliable method used in assisted reproduction to preserve genetic materials, it can stimulate the occurrence of oxidative stress, which affects sperm structure and function. This research was conducted to explore the effects of quinoa seed extracts (QSE) on ram sperm quality, oxidative biomarkers, and the gene expression of frozen-thawed ram sperm. Semen samples were diluted in extenders supplemented with 0 (QSE0), 250 (QSE1), 500 (QSE2), 750 (QSE3), and 1000 (QSE4) µg of QSE /mL, and then frozen according to the typical procedure. The findings indicate that the QSE3 and QSE4 groups provided the optimal results in terms of sperm viability and progressive motility. Sperm kinematics were considerably enhanced in the QSE3 group compared to the other groups (P<0.01). QSE (500–1000 µg/mL) significantly decreased the apoptosis-like changes (higher viable and lower apoptotic sperm) in ram sperm (P<0.001). The percentage of live sperm with intact acrosomes was significantly increased, while the percentage of detached and intact acrosomes in live and dead sperm were significantly decreased respectively by the QSE addition (P<0.001). All QSE groups had higher TAC and lower MDA and H 2 O 2 levels than the control group (P<0.001). The expressions of SOD1 , CAT , GABPB1 , and GPX1 genes in sperm samples were significantly increased, while the CASP3 gene was significantly decreased in all QSE-supplemented samples. Our data suggest that QSE has beneficial effects on sperm quality of cryopreserved ram semen, which are achieved by promoting sperm antioxidant-related genes and reducing apoptosis-related gene. • Quinoa seed extracts (QSE) has a robust antioxidant capacity. • QSE improves the cryopreserved ram sperm quality. • QSE reduces the oxidative stress and enhance the antioxidant related gens in ram sperm. [ABSTRACT FROM AUTHOR]

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). • Quality of fractions in sex-sorted semen is very important for successful artificial insemination with sexed semen of ruminants. • DNA quantity, kinematic parameters and enzymes activity of X- and Y- ram spermatozoa are relevant indicators for evaluation of sexing methods. • Sexing by BSA column and TLR7/8 ligand R848 performs close to semen collection time and produces X- and Y-sperm fractions with satisfactory quality. • Both methods provide close percentages of fast and medium progressive motile spermatozoa in each fraction and affect the enzyme activity. [ABSTRACT FROM AUTHOR]

This comprehensive review critically examines the application of proteomics in understanding sperm cryoinjury mechanisms in livestock animals, in the context of the widespread use of semen cryopreservation for genetic conservation. Despite its global adoption, cryopreservation often detrimentally affects sperm quality and fertility due to cryoinjuries. These injuries primarily arise from ice crystal formation, osmotic shifts, oxidative stress, and the reorganization of membrane proteins and lipids during freezing and thawing, leading to premature capacitation-like changes. Moreover, the cryopreservation process induces proteome remodeling in mammalian sperm. Although there have been technological advances in semen cryopreservation, the precise mechanisms of mammalian sperm cryoinjury remain elusive. This review offers an in-depth exploration of how recent advancements in proteomic technologies have enabled a detailed investigation into these molecular disruptions. It presents an analysis of protein-level alterations post-thaw and their impact on sperm viability and functionality. Additionally, it discusses the role of proteomics in refining cryopreservation techniques to mitigate cryoinjury and enhance reproductive outcomes in livestock. This work synthesizes current knowledge, highlights gaps, and suggests directions for future research in animal reproductive science and biotechnology. • Description of the status related to sperm cryoinjury mechanism. • Cryopreservation effect on sperm of livestock animals. • Recent advances on some domestic animals' sperm proteome during cryopreservation. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation technology significantly contributes to the safeguarding of genetic resources, particularly for endangered species, and supports the use of artificial insemination in domestic animals. Therefore, cryopreservation can negatively affect sperm health and function leading to reduce the freezing ability and fertility potential. Therefore, it is essential to prioritize the improvement of cryotolerance in cryopreserved sperm to enhance reproductive efficiency and ensure sustainability in livestock herds. The main reason for sperm dysfunction after thawing may be related to the excessive amount of oxidative stress (OS) produced during cryopreservation. Scientists have different ways for counteracting this OS including the use of plant extracts, enzymes, minerals, anti-freezing proteins, and amino acids. Recently, one such amino acid is L-proline (LP), which has multiple roles such as osmotic and OS defense, nitrogen, and carbon metabolism, as well as cell survival and signaling. LP has been found in seminal plasma and has recently been added to the freezing extender to improve the various post-thaw parameters of sperm. This improvement is related to the ability of LP to reduce the OS, sustain the plasma membrane and to act as an osmoregulatory agent. Moreover, LP can suppress cell apoptosis by modulating intracellular redox in sperm. This review addresses the ongoing research on the addition of L-proline as an osmoregulatory agent in freezing extenders to increase the cryotolerance of animal spermatozoa to freeze-thaw. [Display omitted] • L-proline (LP) improves the sperm motion after sperm cryopreservation in various animals. • This improvement is related to the ability of LP to reduce the OS, sustain the plasma membrane and osmoregulatory agent. • LP can suppress cell apoptosis by modulating intracellular redox in sperm [ABSTRACT FROM AUTHOR]

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM – 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes. [Display omitted] • Heat stress reduced ram testis size, sperm concentration and motility. • Sperm DNA integrity and BSP binding and seminal proteins were critically altered. • Variations in seminal protein expressions coincided with changes in sperm criteria. • Altered seminal proteins were mainly those associated with oxidative stress. • Know animal response to heat stress is vital in the scenario of climate changes. [ABSTRACT FROM AUTHOR]

Short-term sperm storage is a straightforward and cost-effective method of managing logistics in large scale fish hatchery operations but may result in decline in sperm quality. For effective artificial reproduction of fish, use of an appropriate additive to optimize sperm storage conditions is essential. In this study, it was investigated the effect of purified seminal plasma transferrin (Tf) at 10 μg/ml on relevant parameters in common carp Cyprinus carpio sperm during short-term storage. We compared sperm motility and curvilinear velocity, adenosine triphosphate (ATP) content and DNA fragmentation of fresh spermatozoa to that stored for 24, 48, 72, and 144 h with or without Tf. The percentage of motile cells and the curvilinear velocity of spermatozoa in stored samples for 72 h with transferrin supplementation were greater compared to samples with no added protein. The ATP content in samples without added transferrin was reduced (P < 0.05) after 72 h of storage, in contrast to the levels observed in transferrin-supplemented sperm. A time-dependent increase in DNA fragmentation was observed. Significantly lower DNA damage, expressed as percent tail DNA (10.99 ± 1.28) and olive tail moment (0.54 ± 0.12), was recorded in Tf-supplemented samples stored for 48 h compared to that with no Tf. Hence, it is concluded that the beneficial effects of transferrin on common carp sperm could serve as an additional tool for developing and enhancing short-term sperm preservation procedures commonly used in aquaculture. • Treatment with Tf led to an improvement in sperm motility parameters after 72 h. • Tf-supplemented sperm had higher ATP content than those without Tf. • After 48 h, Tf-supplemented samples showed less DNA damage than those without Tf. • Tf addition shows promise for short-term in vitro sperm storage. [ABSTRACT FROM AUTHOR]

The commercial swine industry utilizes artificial insemination (AI) in their breeding programs. With this assisted reproductive technology, the process starts by obtaining fresh ejaculates from desirable boars who are housed in a dedicated facility (i.e., stud) that also contains a clean-room laboratory where semen quality is assessed and then ejaculates processed into AI doses. In concert with AI adoption, disruptions in sow herd reproductive performance have been traced back to contributions made from the boar stud. Through field investigations and research, several extrinsic contaminants have been identified that impact semen quality either at the boar or AI-dose level. These contaminants can be categorized as either biological or chemical in origin, eliciting reprotoxic outcomes at the boar level and/or spermatotoxicity at the AI-dose level. Biological contaminants include multiple genera of primarily opportunistic microbes (i.e., bacteria, fungi), along with their secondary metabolites (e.g., endotoxins, exotoxins, mycotoxins). Chemical contaminants appear to originate from products used at the stud, and include cleaning agent/disinfectant residues, leachates from gloves and plastics, semen extender impurities, purified and drinking water impurities, and pesticides (i.e., biocides, fungicides, herbicides, insecticides, wood preservatives). In conclusion, contaminants are a real and constant threat to the health and productivity of a stud, and have caused significant reproductive and economic losses in the swine industry. The knowledge gained in recognizing the types and sources of contaminants provides a solid foundation for the development and implementation of pro-active strategies that mitigate risk to the industry., Competing Interests: Declaration of Competing Interest I have no competing interests to declare., (Copyright © 2024. Published by Elsevier B.V.)

In vitro production of embryos (IVP) is increasingly applied in dairy cattle breeding and promises widespread use of females of superior genetic merits. One of the current challenges with implementation of IVP is the variability in blastocyst rates. Several factors contribute to these variabilities, among which is known to be the bull used for oocytes fertilization. The extent of genetic control of bulls' effect on IVP performances is yet to be investigated. This study estimates genetic parameters for bull effects on IVP performance traits including blastocyst rate, hatching rate and an index trait combining Blastocyst rate, Kinetic Score, and Morphology score (BL&#95;M&#95;K). The IVP experiments were performed using oocytes aspirated from slaughterhouse ovaries from Holstein cows, fertilized with semen from 123 Holstein bulls. A total of 77 in vitro fertilization (IVF) experiments with 163 records (different IVF groups) were available for the analysis. The results indicate low to moderate heritability and moderate to high repeatability estimates for bull effects on IVP performance traits. Our study also showed that some semen quality traits had significant effects on IVP performance. This included strong genetic correlations between pre-cryopreservation sperm viability and blastocyst rate as well as BL&#95;M&#95;K score at days 7 and 8. Despite the generally weak bull effect correlations and the high standard errors of the estimates, our results provide initial evidence of a measurable genetic component in the bull's impact on IVP performance traits. However, the high standard errors underscore the need for further studies with a larger sample size., Competing Interests: Declaration of Competing Interest K. Kupisiewicz is employed by the company Danish Genetics (Randers, Denmark). The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

ProAKAP4 is identified within the flagellum of spermatozoa in various mammalian species, serving as a structural protein associated with motility parameters. This investigation focuses on the presence of proAKAP4 in donkey sperm, elucidating its localization, molecular characteristics, and its correlation with motility descriptors and mitochondrial membrane potential. Twelve ejaculates from Catalan donkeys were analyzed in this study. The initial steps involved proAKAP4 sequencing and detection through Western blotting and immunofluorescence. Post-thaw assessments were conducted at 0, 1, and 3 h, encompassing proAKAP4 levels, sperm motility analyzed via Computer-Assisted Sperm Analysis (CASA), and mitochondrial membrane potential determined by flow cytometry using the JC-1 stain. The findings reveal that proAKAP4 in donkeys exhibits a characteristic localization at the principal piece of the flagellum, consistent with observations in other mammals. The molecular weight of proAKAP4 is determined to be 100 kDa. Significantly, a positive correlation (p ≤ 0.05) is established between proAKAP4 concentration and both total and progressive motility. The presence of cryoprotectant is associated with a lower proAKAP4 concentration. Notably, proAKAP4 experiences a substantial decrease (p ≤ 0.05) during the initial hour post-thawing. In conclusion, proAKAP4 is identified in donkey sperm, akin to its presence in other mammals. It exhibits a positive correlation with total and progressive motility, its concentration is notably affected by the presence of cryoprotectant with significant consumption observed during the initial hour following thawing. These findings contribute to our understanding of proAKAP4 dynamics in donkey sperm, providing insights that may have implications for semen preservation and reproductive technologies in equids. • ProAKAP4 is labeled in donkeys like in other mammals. • In donkey, proAKAP4 correlates with total and progressive motility. • Upon thawing of the semen, higher proAKAP4 concentration was observed removing cryoprotectant [ABSTRACT FROM AUTHOR]

As sperm cryopreservation and other assisted reproductive technologies (ARTs) advance in common amphibian species, focus on applying non-lethal sperm collection methods to the conservation and genetic management of threatened species is imperative. The goal of this study was to examine the application of logistically practical ART protocols in a threatened frog (Litoria aurea). First, we tested the efficacy of various concentrations of human chorionic gonadotropin (hCG) (20, 40 IU/g bodyweight) and Gonadotropin releasing hormone antagonist (0.25 µg/g and 0.5 µg/g body weight GnRH-a) on the induction of spermatozoa. Using the samples obtained from the previous trials, we tested the effect of cold storage and cryopreservation protocols on long-term refrigerated storage and post-thaw sperm recovery. Our major findings include: (1) high quality sperm were induced with 20 and 40 IU/g bodyweight of (hCG); (2) proportions of live, motile sperm post-thaw, were recovered at higher levels than previously reported for L. aurea (>50%) when preserved with 15% v/v DMSO and 1% w/v sucrose; and (3) spermic urine stored at 5 °C retained motility for up to 14 days. Our findings demonstrate that the protocols developed in this study allowed for successful induction and recovery of high-quality spermatozoa from a threatened Australian anuran, L. aurea , providing a prime example of how ARTs can contribute to the conservation of rare and threatened species. • hCG, but not GnRH-a is effective for inducing high numbers of sperm in Litoria aurea. • Post-thaw motility and membrane integrity is not significantly lower 30 min after initial thawing. • Sperm may be stored refrigerated at 5 °C with high levels of motility (>50%) for 5-7 days. [ABSTRACT FROM AUTHOR]

The aim of the present study is to evaluate the effect of Moringa oleifera leave extract (MOLE) on buffalo bull cryopreserved semen quality and fertility. Sixty ejaculates were collected from ten fertile buffalo bulls on a weekly basis for 6 weeks (n = 10 bulls & n = 60) then semen samples were pooled and divided into five groups. The semen of the control group was without additives. The semen of other groups was supplemented with MOLE at doses of 200, 400, 600 and 800 µg/ mL, respectively. One hundred thirty multiparous buffaloes were artificially inseminated with semen supplemented without or with MOLE at dose of 600 µg/ mL. Inclusion of MOLE in semen extender at dose 600 µg/ mL significantly elevated the total motility, progressive motility, membrane integrity and fertilization capacity of the post-thawed spermatozoa, as well as the total antioxidant capacity. However, it significantly decreased acrosomal defects of spermatozoa, and the concentration of malondialdehyde. This study indicated that inclusion of MOLE to semen extender improved the quality and fertility of the post-thawed buffalo bulls' semen through enhancing the activities of the antioxidant enzyme system and decreasing cryodamage of the buffalo bull spermatozoa. • Inclusion of MOLE in semen extender of buffalo bulls enhanced post-thawed semen quality. • MOLE enhanced post-thawed buffalo bulls' sperm acrosomal membrane integrity. • MOLE enhanced post-thawed buffalo bulls' sperm fertilization capacity. • MOLE enhanced post-thawed buffalo bulls' semen total antioxidant capacity. • MOLAE decreased buffalo bulls' sperms acrosomal defects and MDA concentration. [ABSTRACT FROM AUTHOR]

Increases in temperature can affect the reproduction of fish by decreasing the quality of gametes for fertilization. Therefore, this study aimed to evaluate the in vivo effect of temperature on the production and sperm quality of Colossoma macropomum, which is an economically relevant species for Brazil, and other countries. Broodstock were exposed for 10 days at temperatures of 29 °C (n = 4) and 35 °C (n = 4). After exposure, semen was collected and sperm quality was evaluated for kinetic, biochemical, morphological parameters, membrane integrity, and oxygen consumption. The sperm quality of males of specimens of C. macropomum exposed in vivo to the higher temperature was compromised and showed a difference in all the analyses (P < 0.05). Sperm showed decreased motility (51.9 ± 11.6 s) compared to the control (61.3 ± 5.5 s); curvilinear (CLV), straight line (SLV), and average path (APV) velocities decreased, and straightness (STR) and beat cross frequency (BCF) increased; oxygen consumption decreased (32.8 ± 3.4 pmol. (s.ml)−1, picomol per second per ml) compared to the control (46.6 ± 3.5 pmol. (s.ml)−1); and the activity of catalase (CAT) and glutathione S -transferase (GST) enzymes increased and decreased, respectively, in sperm from fish exposed to high temperatures. Furthermore, an increase in non-viable sperm with damaged membranes and sperm with altered morphology was observed. The observed alterations indicate that C. macropomum confined in environments with high temperatures possibly produces more sperm that are unviable for the fertilization process. ● Exposure of tambaqui to high temperature affects sperm quality; ● The increase in temperature causes oxidative stress in tambaqui sperm; ● Tambaqui sperm consume less oxygen when exposed to high temperatures; ● High temperature during cultivation can affect tambaqui reproduction [ABSTRACT FROM AUTHOR]

In small and laboratory animals epididymal spermatozoa can be collected only by the mincing technique however, blood and cell debris contamination may adversely affect the quality of the samples. This study aimed at performing the retrograde flushing technique, for the first time, in the tomcat. In the first experiment attempts to insert a blunted needle into tomcats' ductus deferentes were made and the cannulation time and the success rate were recorded. In the second experiment, insertion of the needle into the ductus was carried out under a stereomicroscope for one testis, while the other was submitted to mincing for sperm recovery and the samples obtained from paired testes were compared. In the first study, only twenty-eight percent of the cannulation attempts were successful whereas in the second experiment, 91% of the deferentes were successfully cannulated: 80% of the ducts were cannulated in less than three minutes. The stereomicroscope-aided retrograde flushing samples showed significantly higher sperm viability, motility and velocities, as compared to those resulted from mincing. The domestic cat serves as a model for more than 36 species of wild felids; the developed stereomicroscope-aided retrograde flushing technique has the potential to become the gold standard method for harvesting spermatozoa of endangered species that have a small ductus deferens. • The tomcat ductus deferens could be cannulated under the stereomicroscope. • The cannulation could be performed in a short time with the stereomicroscope. • Compared to mincing, the retrograde flushing improved the sperm parameters. • The RF could be the gold standard for epididimal sperm collection in small animals. [ABSTRACT FROM AUTHOR]

This study aimed to assess the potential of the centrifuge-free commercial device (MIGLIS®) in selecting functional frozen-thawed bovine sperm by migration-sedimentation, its effect on embryo development, and compare the potential with that of centrifugation-based techniques, including washing and Percoll density gradient centrifugation (DGC). In experiment 1, different dilutions (1.5×, 2×, and 3×) of frozen-thawed spermatozoa were assessed to identify the adequate one for the MIGLIS method. In experiment 2, the recovery rates, quality, and reactive oxygen species (ROS) concentrations of the spermatozoa selected using MIGLIS, washing, and Percoll DGC were compared. In experiment 3, the resultant in vitro fertilised embryos from spermatozoa selected using the three methods were evaluated for blastocyst formation rates and intracellular ROS concentrations at the 2–4 cell stage. The intracellular ROS concentrations were investigated using 2′, 7′-dichlorodihydrofluorescein diacetate staining. Using the MIGLIS device, significantly more spermatozoa were recovered at 2× dilution compared with the other dilution ratio, but the motility was not affected by the dilution ratio. On the selection of spermatozoa using the three methods, employing MIGLIS decreased the recovery rates. However, the MIGLIS method increased motility, viability, and acrosome integrity rates compared to those in spermatozoa from the other methods. The ROS concentration of spermatozoa in the MIGLIS method was significantly lower than that in the washing method. Nevertheless, blastocyst formation rates were similar among the three methods, but the ROS concentration of early-stage embryos produced using MIGLIS was significantly lower than those produced using Percoll DGC. In conclusion, the MIGLIS method has the potential to select functional, high-quality frozen-thawed bovine spermatozoa. • The MIGLIS method without centrifugation decreased the sperm recovery rates. • The MIGLIS method selected sperm with higher quality and lower ROS concentrations. • ROS concentrations of early-stage embryos produced using MIGLIS decreased. [ABSTRACT FROM AUTHOR]

The biological properties of European red deer (Cervus elaphus elaphus) spermatozoa stored in the epididymides and in a liquid state were compared. Spermatozoa were collected from the epididymides harvested post-mortem. In the first variant, spermatozoa were diluted in two extenders (Bovidyl® and Salomon's), and were stored at 5 °C for up to 144 h. In the second variant, spermatozoa were stored in the epididymides at 5 °C for up to 144 h, and then diluted in the same extenders. Biological properties were evaluated after 0, 48, 96, and 144 h of storage. Sperm motility parameters were determined in the CASA system. Plasma and acrosomal membrane integrity, mitochondrial activity, apoptotic changes, and DNA integrity were assessed by the fluorescence method. Most variables were significantly influenced by the storage method and time. At 144 h, differences (P ≤ 0.05) in sperm parameters were observed between storage variants. Total motility (TMOT), plasma membrane integrity, and mitochondrial activity decreased below 50% of baseline values in the spermatozoa stored in the epididymides, but remained above 70% of baseline values in the spermatozoa stored in a liquid state. The compared storage variants did not differ in TMOT, mitochondrial activity, or the percentage of viable spermatozoa without apoptotic-like changes up to 96 h of storage, regardless of the applied extender. The earliest significant changes were noted in sperm motility parameters. In conclusion, European red deer spermatozoa can be stored in the epididymides at 5 °C for up to 96 h, but their biological parameters are more effectively preserved during liquid storage. ● Sperm can be stored in the epididymides in the scrotum at 5 °C for up to 96 h. ● Motility parameters decline earliest during epididymal storage. ● Liquid storage is recommended for short-term preservation of epididymal sperm. ● Bovidyl® has a more positive effect on the progressive movement of sperm than Salomon's. [ABSTRACT FROM AUTHOR]

Cattleyak is a typically male sterile species. The meiosis process is blocked and the scarcity of spermatogenic stems cells are both contributing factors to the inability of male cattleyak to produce sperm. While Glial cell line-derived neurotrophic factor (GDNF) is the first discovered growth factor known to promote the proliferation and self-renewal of spermatogenic stem cells, its relationship to the spermatogenesis arrest of cattleyak remains unclear. In this report, we studied the differential expression of GDNF in the testis of yak and cattleyak, and discussed the optimal concentration of GDNF in the culture medium of undifferentiated spermatogonia (UDSPG) of cattleyak in vitro and the effect of GDNF on the proliferation of cattleyak UDSPG. The results indicated that GDNF expression in the testicular tissue of cattleyak was inferior to that of yak. Moreover, the optimum value for the UDSPG in vitro culture was determined to be 20–30 ng/mL for cattleyak. In vitro, the proliferation activity of UDSPG was observed to increase with additional GDNF due to the up-regulation of proliferation-related genes and the down-regulation of differentiation-related genes. We hereby report that the scarcity of cattleyak UDSPG is due to insufficient expression of GDNF, and that the addition of GDNF in vitro can promote the proliferation of cattleyak UDSPG by regulating the expression of genes related to proliferation and differentiation. This work provides a new insight to solve the issue of spermatogenic arrest in cattleyak. [Display omitted] • Spermatogenesis is impacted by the aberrant expression of GDNF in cattleyak testis. • Abnormal seminiferous tubule is one of the reasons for cattleyak's male sterility. • In vitro proliferation of cattleyak UDSPG can be enhanced by the addition of GDNF. [ABSTRACT FROM AUTHOR]

The objective of this study was to evaluate the effect of the addition of different percentages of seminal plasma (SP) during the cooling at 5 °C of alpaca spermatozoa from vas deferens. Fifteen pools of sperm from vas deferens were evaluated and then divided into four aliquots that were diluted to a final concentration of 30 × 106 sperm/ml with either: (1) Tris with 20% egg yolk (T-EY) (control, 0% SP), (2) T-EY with 10% SP, (3) T-EY with 25% SP, and (4) T-EY with 50% SP. Samples were cooled at 5 °C and the following sperm parameters were evaluated after 24 and 48 h of storage: motility, viability, membrane function, acrosome integrity, morphology, and chromatin condensation. Motility was also evaluated after 72 h of storage. A significant decrease in progressive and total sperm motility was observed in samples cooled with 50% SP with respect to all diluted samples, while these parameters were preserved in samples cooled with 0%, 10%, and 25% SP. The percentages of sperm viability, normal morphology, and highly condensed chromatin did not change after the cooling process and were similar between cooled samples. Although a significant decrease was observed in the percentage of spermatozoa with functional membranes and with an intact acrosome in all refrigerated samples compared to raw sperm, the greatest decrease was observed in samples cooled with 50% SP. No advantage was observed from the addition of SP to alpaca spermatozoa obtained from vas deferens and being cooled. In addition, to preserve the sperm motility of cooled samples for up to 72 h, it should be recommended to include a 10% SP in the extender. • Study evaluates the cryoprotective effect of SP on alpaca sperm from vas deferens. • Seminal plasma addition before cooling did not change alpaca sperm motility pattern. • Samples cooled with 50% seminal plasma showed lower percentages of motile sperm. • Sperm viability did not change after cooling and was similar between cooled samples. • Sperm membrane function and acrosome integrity decreased in all cooled samples. [ABSTRACT FROM AUTHOR]

Transport-related vibrations (TV) compromise the quality of conventionally stored (17 °C) boar semen, but knowledge about TV effects after 5 °C transport is insufficient. This study evaluates the effects of TV after novel 5 °C transport compared to a 17 °C control. Ejaculates of 18 fertile Piétrain boars, diluted in a split sample procedure using Androstar Premium® (AP, 5 °C storage) or Beltsville Thawing Solution (BTS, 17 °C storage), were subjected to transport simulation using a laboratory shaker IKA MTS 4. The timing was set according to the respective processing protocols: for 17 °C BTS samples, TV simulation was performed the day of collection, 5 °C AP samples were subjected to TV the day after collection following completion of the established cooling curve to 5 °C. Six samples per ejaculate were exposed to different TV durations (0 h, 3 h, or 6 h) to evaluate the effect on sperm quality (progressive motility (PM), thermo-resistance test (30 and 300 min incubation at 38 °C (TRT30/TRT300)), mitochondrial activity (MITO), plasma membrane and acrosome integrity (PMAI)). Generalized linear mixed models revealed TV (P = 0.021) and storage time (P < 0.001) dependent declines in PM. Direct, pairwise comparisons revealed that 5 °C samples are not affected by TV (P(3 h vs. 6 h transport) = 1.0; P(0 h vs. 6 h transport) = 1.0). They therefore showed superior quality maintenance after TV compared to 17 °C samples (P(3 h vs. 6 h transport) = 0.025; P(0 h vs. 6 h transport) < 0.001). Concluding, low-temperature transport is possible without significant semen quality loss and with better quality maintenance than standard transport., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hypothermic storage has been proposed as a method to reduce bacterial loads and promoting prudent use of antibiotics. Reducing temperature, however, can lead to cold shock damage and oxidative stress in boar semen. This study verified the effect of L-cysteine on the quality of semen stored at 5 °C for 120 h. Twenty-one normospermic ejaculates were diluted in Beltsville Thawing Solution into five treatments: Positive control (Pos&#95;Cont, storage at 17 °C without L-cysteine) and groups with 0, 0.5, 1, and 2 mmol/L of L-cysteine supplementation stored at 5 °C. Variables were analyzed as repeated measures, considering treatment, storage time, and interaction as main factors. The effects of different L-cysteine concentrations were also evaluated using polynomial orthogonal contrasts. Sperm motility and pH were higher in the Pos&#95;Cont compared to the groups stored at 5 °C (P < 0.05). In polynomial orthogonal contrast models, total motility was affected by the interaction between L-cysteine and storage time (P = 0.04), with a linear increase in motility when increasing the amount of L-cysteine at 72 and 120 h. Progressive motility increased quadratically as the L-cysteine reached 1 mmol/L (P < 0.01). In the thermoresistance test at 120 h, sperm motility increased quadratically up to an L-cysteine dose of 1 mmol/L (P < 0.05). Sulfhydryl content linearly increased with L-cysteine supplementation (P = 0.01), with no effect on intracellular ROS and sperm lipid peroxidation (P ≥ 0.06) in 5ºC-stored doses. In conclusion, L-cysteine supplementation has a positive effect on sperm motility up to 120 h of storage at 5 °C., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The aim of the study was to evaluate the effects of crocin on canine sperm quality parameters during prolonged storage at 4 °C. Ejaculates from 10 dogs were diluted in a TRIS- egg yolk extender supplemented with 0 (control group), 0.5, 1, and 2 mM crocin and stored at 4 °C. Sperm membrane functional integrity, motility, and kinetics were assessed after 3 h, 24 h, 4 days and 7 days of storage. Based on the results, the more efficient concentration of crocin (0.5 mM) was chosen to evaluate sperm intracellular ROS levels, lipid peroxidation, and DNA fragmentation vs. the control. Semen with the addition of 0.5 mM crocin with respect to the control exhibited: i) increased (P < 0.05) sperm membrane functionality at 4 and 7 days of storage; ii) higher (P < 0.05) average path (VAP), straight-line velocities (VSL), and beat cross frequency (BCF) at 4 d of storage at 4 °C; iii) decreased (P < 0.05) intracellular ROS levels after 3 and 24 h storage. No differences in lipid peroxidation and DNA fragmentation were recorded between the control and C0.5 groups at any time point. Lipid peroxidation did not increase over time, while DNA fragmentation increased (P < 0.05) in both groups after 4 days of storage. The results demonstrated that the enrichment of extender with crocin improves to a certain extent canine semen quality, particularly after 4 days of storage at 4 °C. • Crocin supplementation preserves canine sperm viability and kinetics at 4 °C. • Crocin decreases sperm intracellular ROS levels during short-term storage. • Crocin delays the occurrence of sperm DNA fragmentation during prolonged storage. [ABSTRACT FROM AUTHOR]

L -proline has been reported to be useful in semen cryopreservation. However, its use has rarely been reported in the freezing of boar semen. The objective of this study was to evaluate the effects of different concentrations of L -proline (0, 10, 30, 50, and 90 mM) on the quality of boar semen after freezing and thawing. Semen samples from boars (n = 6) were frozen using freezing extenders with added concentrations of L -proline. Total sperm motility, progressive motility, survival time at 37 °C, acrosome integrity, mitochondrial activity, DNA integrity, the content of the lipid peroxidation product malondialdehyde (MDA), total antioxidant capacity (T-AOC) and, expression levels of apoptosis protein (cleaved caspase 3 and Bax) were evaluated after thawing. The results showed that total sperm viability (73.96% vs. 63.58%) and progressive motility (56.88% vs. 47.26%) after thawing were significantly higher in the 10 mM L -proline treatment group than in the control group. The survival time at 37 °C and the total motility of sperm in the 10 mM group within one hour after thawing were significantly higher than in the control group. Acrosome integrity and mitochondrial activity of sperm in the 10 mM group were significantly higher than those in the control, 50 mM, and 90 mM groups. The DNA integrity rate in the 10 mM group was significantly higher than in the control group. The L -proline treatment did not affect sperm MDA content or T-AOC. The expression levels of apoptosis protein (cleaved caspase 3 and Bax) in the 10 mM L -proline supplemented group were lower than those in the control group. In conclusion, the freezing extender containing 10 mM L -proline improved semen quality after freezing and thawing and thus would be a useful reagent for boar semen cryopreservation. • Freezing extender containing 10 mM L -proline improved boar semen quality. • L -proline maintained mitochondrial, acrosome, and DNA integrity, and T-AOC of sperm. • L -proline inhibited boar sperm from apoptosis after freezing and thawing. [ABSTRACT FROM AUTHOR]

Glycerol is a cryoprotectant used widely for the cryopreservation of animal sperm, but it is linked to a decrease in fertility. The mechanism underlying the negative effects of glycerol remains unclear. Therefore, in this study, we aimed to gain a better understanding by using the chicken model. First, we investigated the impact of increasing the concentration of glycerol during insemination on hen fertility. Our findings revealed that 2% glycerol resulted in partial infertility, while 6% glycerol led to complete infertility. Subsequently, we examined the ability of sperm to colonize sperm storage tubules (SST) during in vivo insemination and in vitro incubation. The sperm used in the experiment were stained with Hoechst and contained 0, 2, or 6% glycerol. Furthermore, we conducted perivitelline membrane lysis tests and investigated sperm motility, mitochondrial function, ATP concentration, membrane integrity, and apoptosis after 60 min of incubation with different glycerol concentrations (0%, 1%, 2%, 6%, and 11%) at two temperatures to simulate pre-freezing (4 °C) and post-insemination (41 °C) conditions. Whereas 2% glycerol significantly reduced 50% of sperm containing SST, 6% glycerol completely inhibited SST colonization in vivo. On the other hand, in vitro incubation of sperm with SST revealed no effect of 2% glycerol, and 6% glycerol showed only a 17% reduction in sperm-filled SST. Moreover, glycerol reduced sperm-egg penetration rates and also affected sperm motility, bioenergetic metabolism, and cell death at 4 °C. These effects were observed when the concentration of glycerol exceeded 6%. Furthermore, at 41 °C, glycerol caused even greater damage, particularly in terms of reducing sperm motility. These data altogether reveal important effects of glycerol on sperm biology, sperm migration, SST colonization, and oocyte penetration. This suggests that glycerol plays a role in reducing fertility and presents opportunities for improving sperm cryopreservation. [Display omitted] • Glycerol provides excellent cryoprotection and shows cytotoxicity to sperm cells. • In hens, 2% or 6% glycerol added into semen induce partial or total infertility. • Glycerol affects sperm migration and storage in the oviduct. • Glycerol causes more negative modifications of sperm biology at 41 °C than at 4 °C. • Removing glycerol prior to insemination is the most valuable solution so far to avoid fertility reduction. [ABSTRACT FROM AUTHOR]

Long non-coding RNAs (lncRNAs) act as competing endogenous RNAs (ceRNAs) that play a significant role in bovine embryo development; but the influence of sperm-borne lncRNA on the preimplantation development of bovine embryos has not been reported in detail. In this study, we aimed to clarify how sperm-borne lncRNAs can act to regulate early development of bovine embryos. Utilizing high-throughput sequencing technology and quantitative real-time PCR (qPCR), we found that the lncRNA, loc100847420, was highly enriched in bovine sperm and was carried into the oocyte during fertilization. Introduction of wild-type loc100847420 had no effect on cleavage rate of parthenogenetic embryos compared with injection of mutant loc100847420 (70.58 ± 2.85% vs 70.46 ± 1.98%, p > 0.05), but significantly improved the blastocyst rate (33.67 ± 2.40% vs 28.35 ± 3.06%, p < 0.05), total numbers of cells (p < 0.05), numbers of inner cell mass (ICM) cells (p < 0.05) and numbers of trophoblast (TE) cells (p < 0.05). In summary, the sperm-borne lncRNA, loc100847420, can improve the developmental potential of early bovine embryos. [ABSTRACT FROM AUTHOR]

Plant-based semen extenders, typically derived from soybean lecithin, are easier to modulate more and consistent in their composition than animal-based extenders. As large lecithin particles can, however, reduce effectiveness and solubility in bull semen extenders, sonication was used to create nano-lecithin (NL) particles of soybean lecithin. The objective was to determine the effects of lecithin type and concentration on the quality of frozen-thawed bovine sperm. We hypothesized that reducing the size of lecithin improves its interactions with the sperm and enhances the parameters that favor its motility, viability and fertility. Semen was collected from six mature Holstein bulls and ejaculates meeting minimum standards were pooled. Eight Tris-based extenders that contained 1, 2, 3, or 4 % of either conventional lecithin (L1-L4) or NL (NL1-NL4), plus two control extenders (one animal-based extender containing 20 % egg yolk [EY] and a commercial lecithin-based extender [BioXcell®]) were compared. Among soybean lecithin-based extenders, NL3 had the highest total and progressive sperm motility, and average path, straight-line and curvilinear sperm velocity, and was comparable to EY. Additionally, sperm mitochondrial activity was the highest in NL3, whereas sperm viability was highest in EY, NL3, and L4. Following in vitro fertilization of in vitro-matured bovine oocyes, NL3 had cleavage and hatching rates comparable to BioXcell®, but a lower blastocyst rate than EY. Overall, NL3 performed better than the other extenders for most end points, with efficiency comparable to EY. We, therefore, concluded that reducing lecithin particle size to a nano level improves sperm cryopreservation with optimal performance with 3 % NL. [ABSTRACT FROM AUTHOR]

Cryopreservation is a widely used technique to store spermatozoa for a long time. Some Published articles have identified the cryoprotective effect of nanoparticles on sperm quality after the freeze-thaw process, but others have suggested the opposite results. PubMed, ISI Web of Science, and Scopus were systematically searched in animal studies by ("sperm" OR "spermatozoa") AND ("cryopreservation" OR "cooling storage" OR "freezing" OR "thawing") AND ("nanoparticle (lecithin nanoparticle, selenium nanoparticle, zinc nanoparticle, zinc oxide nanoparticle, nanoliposome, solid lipid nanoparticle (SLN), micelle, hydrogel, nanogel, silica nanoparticle, quantum dot, dendrimer, gold (Au) nanoparticle, silver nanoparticle, nanocomposite and mesoporous)"). Among 154 publications, data on sperm quality were extracted from 11 articles. The meta-analysis results demonstrated that nanoparticles had a positive impact on sperm progressive motility (WMD= 9.72, 95 % CI: 4.70, 14.75, p < 0.0001), total motility (WMD= 6.78, 95 % CI: 0.78, 12.78, p = 0.027), viability (WMD= 14.30, 95 % CI: 9.48, 19.13, p < 0.0001) and plasma membrane integrity (WMD = 13.74, 95 % CI: 8.20, 19.29, p < 0.0001). In conclusion, our results indicated the positive effects of nanoparticles as cryoprotectant agents on post-thawed sperm motility, viability, and membrane integrity. [Display omitted] • The Antioxidant-nanoparticles as novel cryoprotectants system. • Membrane integrity was enhanced after using Antioxidant-nanoparticles. • Lecithin, selenium, zinc oxide nanoparticle, has a cytoprotective effect during freeze-thaw process. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation often reduces sperm quality by forming of intra- and extracellular ice crystals. Various compounds widely used to counteract this effect. The guar gum was considered as an extracellular cryoprotective substance. The present study evaluated the impact of the co-supplementation of guar gum with ethylene glycol or glycerol in the cryopreservation of bull sperm. Four ejaculates from 4 bulls were pooled and divided into ten groups consisting of 4 controls (glycerol 6%, ethylene glycol 6%, glycerol 3.5%, and ethylene glycol 3.5%, and six treatment groups including guar gum in 0.001% and 0.002% alone and or co-supplemented either with 3.5% glycerol or 3.5% ethylene glycol and frozen in liquid nitrogen. The sperm motility, viability, plasma membrane and DNA integrity, apoptotic-like changes, antioxidant capacity (TAC), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities evaluated. The groups contained 3.5% glycerol + 0.001% guar gum, 3.5% ethylene glycol + 0.001% guar gum, and 0.001% guar gum alone showed higher values for live sperm, antioxidant enzymes, membrane integrity, mitochondrial membrane potential (MMP), fertilization, cleavage, and blastocyst rates; and lower values for apoptotic-like changes, H2O2 level, and DNA damage than the control groups. In conclusion, adding guar gum to the bull sperm diluent either alone or combined with glycerol or ethylene glycol ameliorated sperm viability and kinematic parameters and antioxidant capacity while reducing DNA damage and apoptotic-like changes. Guar gum also has improved embryo development. Due to its cost-effectiveness and physicochemical properties, guar gum is a promising supplement for bull sperm cryopreservation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

External factors can affect reproductive traits of breeding boars and especially the sensitive process of spermatogenesis. The aim of this study was to investigate probable influences of bedding materials (chipsy wood shavings (CWS), hemp straw (HS), linen straw (LS), spelt husks (SH), and regional wood shavings (RWS)) on semen traits of 40 randomly selected Piétrain boars (8 boars per group, age: 2.35 ± 1.23 years). After a six-week adaptation period, 40 fresh semen samples were collected weekly for four weeks and diluted in BTS (4 consecutive ejaculates per boar, 32 samples per group, 160 samples in total). Semen samples were analyzed using an extended range of spermatological methods (e.g., computer-assisted sperm analysis and flow cytometry). Generalized linear mixed models for each sperm parameter as well as the area under the curve for total sperm motility and thermo-resistance test were calculated. Materials LS and SH exceeded the standard maximum level for pesticide residues (VO (EG) No. 396/2005). Materials HS and LS presented the highest water-binding capacity of 413 % and 357 %, respectively, while SH showed the lowest value of 250 %. There were no significant (P > 0.05) differences between groups in any sperm characteristic, therefore indicating that bedding material had no influence on sperm quality. For most semen traits, however, we found significant (P ≤ 0.001) differences between sampling weeks. Based on pesticide results, we suggest CWS, RWS, or HS as possible bedding materials for pig production farms in the future. Furthermore, we strongly recommend a quality analysis of any new bedding material before use in swine husbandry., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Artificial insemination (AI) is critical for breeding in the dairy industry. High-merit bulls can present low freezability, hampering genetic dissemination. Thawed semen can be improved using density gradient centrifugation (DGC) with colloids, but little information deals with the pre-freezing application. Thus, the BoviPure colloid (optimized for bull spermatozoa) was tested for pre-freezing application as the usual double-layer (DLC) versus single-layer (SLC, quick and economical). Semen from twelve Holstein-Friesian bulls was extended with OPTIXcell extender, frozen (Control), or processed by SLC or DLC and frozen. Sperm were assessed pre-freezing for motility and viability and post-thawing (directly and after 4 h 38 °C) for apoptosis, capacitation status, acrosomal damage, mitochondrial activity, cytoplasmic and mitochondrial reactive oxygen species (ROS), and chromatin status (SCSA for DNA fragmentation and chromatin compaction and monobromobimane, mBBr, for disulfide bridges evaluation). The DGC improved parameters post-thawing (e.g., 57.5%±10.1 motility vs. control 53.3% ± 11.2) at the cost of sperm loss (sperm recovery of DGC 14.4% ± 2.5 and SLC 17.4% ± 2.5). DNA fragmentation (%DFI) decreased (0.21% ± 0.53 vs. control 1.30% ± 0.10), and SLC reduced chromatin compaction. A clustering procedure separated lesser (LF) and greater freezability (GF) bulls. LF samples were especially benefited by DGC, with SLC providing better post-thawing results for this group. In conclusion, pre-freezing DGC improved sperm parameters post-thawing, potentially improving the cryopreservation of low-freezability semen from high-merit bulls. SLC, quicker and economical, would be preferable since it showed similar or higher performance than DLC., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Nerve growth factor-β (NGF) is a seminal plasma protein associated with improved sperm membrane integrity and motility in mammalian species. The objective of this study was to compare post-thaw semen quality from both ejaculated and pididymal-collected bull sperm incubated with purified NGF prior to cryopreservation. Semen was obtained from Angus × Simmental crossbred bulls (n = 10) collected by electroejaculation, followed by castration and epididymal sperm collections 3 days later. Semen samples were incubated with extender having 0 ng/mL (CONT), 0.5 ng/mL (LOW), 5 ng/mL (MED), or 50 ng/mL (HIGH) of purified NGF prior to cryopreservation. Sperm motility was assessed in each sample prior to treatment and cryopreservation and at post-thaw. Flow cytometry was used for post-thaw assessment of sperm viability (SYBR-14/PI), acrosome integrity (FITC-PNA/PI), and chromatin stability (acridine orange). Values for post-thaw sperm motility and velocity variables were decreased, while linearity was increased in samples of the HIGH compared with CONT group (P < 0.01), but there were no differences in epididymal samples (P > 0.05). Samples from the HIGH group also had a lesser amplitude of lateral head displacement at 2.5 and 3 h post-thaw (P < 0.01). Post-thaw sperm viability, acrosome integrity, and DNA fragmentation index were not affected by NGF treatment in either ejaculated or epididymal sperm (P > 0.05). In conclusion, supplementation of freezing extender with NGF had minimal effects on post-thaw sperm quality in bulls. Results indicate NGF may have a function in preventing premature sperm hyperactivation in ejaculated, but not epididymal-collected spermatozoa. Fertility studies, both in vitro and in vivo , are warranted to ascertain the relevancy of these findings. [ABSTRACT FROM AUTHOR]

The development of analytical methods for the evaluation of crocodilian semen is an important component for the assessment of male breeding soundness and the development of assisted breeding technology in this taxon. Computer-Assisted Semen Analysis (CASA) technology is becoming an increasingly common technique in seminal evaluations for animals but there has been no application of this technique for reptilian spermatozoa. The aim of this study was to analyse sperm kinematic and morphometric variables in Caiman crocodilus fuscus semen samples and to determine whether there were sperm subpopulations. Four ejaculates from four sexually mature captive caimans were used for this study. A CASA-Mot and CASA-Morph system was used with an image acquisition rate of 50 Hz for 2 s of capture. The ISAS®D4C20 counting chambers were used and spermatozoa incubated at 25 °C. Total and progressive motilities did not differ among animals (P > 0.05). There was a significant animal effect in the model with respect to sperm morphometry, and kinematic indices including linearity (LIN) and straightness (STR) (P < 0.05). Results for principal component (PC) analysis indicated variables were grouped into four components: PC 1 related to velocity, PC 2 to progressivity, PC 3 to oscillation and PC 4 to sperm path cross-linking. Subpopulation (SP) structure analysis indicated there were four groups, namely, rapid non-progressive (SP 1), slow non-progressive (SP 2), rapid progressive (SP 3) and medium progressive (SP 4), representing 14.5%, 45.4%, 18.7%, and 21.4% respectively. Findings in the present study indicate the importance of continuing development of reliable protocols regarding the standardisation of computer-based semen analyses in reptilian species. [ABSTRACT FROM AUTHOR]

• Suitability of extenders for liquid storage of dromedary camel semen was examined. • Better semen quality was observed in Triladyl than Green buffer or OPTIXcell. • Semen quality was not affected by storage temperature (4 versus 15 °C). This study was conducted to assess the effects of commercial extenders and storage temperature on dromedary camel sperm quality during liquid preservation. In Experiment 1, ejaculates (n = five males; replicated seven times) were split and diluted with synthetic (OPTIXcell, EquiPlus, INRA96, Bioxcell or AndroMed; Experiment 1a) or egg-yolk based (Biladyl, Green buffer or Triladyl; Experiment 1b) extenders and stored for 48 h at 4 °C. In Experiment 2, split ejaculates (n = five males; replicated six times) were used to directly compare Green buffer, OPTIXcell and Triladyl extenders over 48 h of storage at 4 °C. Ejaculates collected in Experiment 3 (n = five males; replicated five times) were diluted with Green buffer or Triladyl before chilled storage for 48 h at 4 or 15 °C. Sperm kinematics, viability and acrosome integrity were assessed during liquid storage. In Experiment 1a, there was the greatest total sperm motility (TM) in the OPTIXcell group following 24 and 48 h of storage, while in Experiment 1b, there was the greatest TM after 48 h of storage with Triladyl and Green buffer. In Experiment 2, there were greater TM and viable acrosome intact spermatozoa in the Triladyl and Green buffer than with OPTIXcell group. In Experiment 3, there was a greater TM in the Triladyl than Green buffer group at 24 and 48 h of storage regardless of storage temperature (which had no effect on sperm quality). In conclusion, camel sperm have greater viability when preserved in liquid form for 48 h following dilution with Triladyl and storage at either 4 or 15 °C. [ABSTRACT FROM AUTHOR]

Semen cryopreservation is a very important technique for assisted reproduction; however, the cryopreservation process is harmful because it results in a reduction in sperm motility and viability, and leads to premature signals of capacitation, resulting in lesser than desirable fertility rates after artificial insemination. A fraction of seminal plasma, enriched in proteins that contain type II fibronectin domains (FNII) can reverse molecular indicators of cryo-capacitation. The beneficial effects of these proteins, however, depend on the relative abundance in seminal plasma. To create a safe additive for improving frozen sperm functionality, in the present study there was cloning and expression of a recombinant peptide containing four FNII domains (named TrxA-FNIIx4-His 6) and evaluation of its effect after addition to frozen/thawed ram sperm. The cDNA for this protein was expressed in E. coli and after denaturation and re-naturalization of the protein, toxicity and binding capacity were assessed. By fluorescent labelling assessment, there was binding of the protein to the thawed sperm. At the two doses used (0.15 and 0.3 μM), TrxA-FNIIx4-His 6 had the capacity to reverse the molecular indicators of cryo-capacitation as indicated by the reduction on phosphorylated substrates of PKA. Furthermore, the supplementation with this protein resulted in a normal capacitation process as evidenced by the increase in the in vitro fertilization rate when the greatest concentration of the protein was evaluated (73.25 ± 2.95; 40.13 ± 11.82 for 0.3 μM and control, respectively). There was no effect of protein supplementation on sperm objective motility compared to untreated sperm. In conclusion, the use of TrxA-FNIIx4-His 6 is a promising biotechnological approach for cryopreserving ram sperm and maintaining sperm viability. [ABSTRACT FROM AUTHOR]

Sperm vitrification is a rapid freezing method in which carbohydrates are used as cryoprotectants. The aim of this study was to determine the optimal volume, concentration and type of carbohydrates for stallion sperm vitrification using 0.25 ml straws in comparison to conventional freezing. Ejaculates (n = 54) were collected from six stallions. For vitrification, straws were filled with different volumes (30, 70, 100 μl), sperm concentrations (50, 100, 200 × 106 sperm/ml) and extenders containing sucrose (20, 100, 200 mM), trehalose (50, 100, 200 mM) and raffinose (50, 100, 200 mM) and plunged into LN 2. Conventional freezing was performed in 0.5 ml straws frozen in LN 2 vapors. Sperm motility, plasma and acrosome membrane integrities and DNA fragmentation were compared among treatments. The use of straws filled with 100 μl at 100 × 106 sperm/ml with the extender containing 100 mM trehalose resulted in greater values for sperm quality than the other concentrations, volumes and carbohydrates. With vitrification, there were greater values (mean ± SEM; P < 0.05) than freezing for progressive motility (48.2 ± 2.3 compared with 37.3 ± 2.2%), plasma membrane integrity (82.8 ± 1.5 compared with 74.1 ± 1.9%), and intact acrosomes (50.2 ± 1.2 compared with 43.1 ± 1.4%); and less DNA fragmentation (6.4 ± 0.7 compared with 8.2 ± 0.3%). In conclusion, stallion sperm can be vitrified in 0.25 ml straws filled with 100 μl of sperm at 100 x 106 sperm/ml using an extender with 100 mM of trehalose, obtaining better sperm quality after warming than conventional freezing. [ABSTRACT FROM AUTHOR]

The goals of the study were to estimate the heritability and repeatability of boar sperm quality traits during the summer season, estimate phenotypic and genetic correlations between sperm quality traits, and assess the effect of these traits on total number born (TNB). Semen samples were collected and individually processed from Duroc boars (n = 363) from May through October 2017. Single sire litter information was available for 121 of the boars. Heritability, repeatability, genetic correlations, and phenotypic correlations were estimated for and among the following traits: total percentage of motile cells (MOT), total percentage of progressively motile cells (PROG), percentage of cells with a distal droplet (DIST), percentage of cells with a proximal droplet (PROX), percentage of cells with a bent tail (BENT), percentage of cells with a distal midpiece reflex (DMR), mean sperm head elongation (ELON), and total sperm cells per ejaculate (TOTSP). Heritability estimates ranged from 0.08 to 0.24 and repeatability estimates ranged from 0.21 to 0.62. The phenotypic and genetic correlations between sperm motility traits and morphological defects indicated a negative relationship. Sperm morphological defects had positive phenotypic and genetic correlations with each other. Total sperm per ejaculate showed positive genetic correlations with the motility traits. Positive phenotypic and genetic correlations existed between ELON and the motility traits. Total sperm motility (P < 0.001) positively affected TNB while DIST (P < 0.001), PROX (P < 0.001), BENT (P = 0.05) and DMR (P = 0.002) negatively affected TNB. Results indicate genetic selection could enhance sperm quality during the summer season in boars. [ABSTRACT FROM AUTHOR]

Artificial insemination programs for horses usually involve ex vivo handling and transporting of sperm. The present experiment was designed to: (i) assess the effect of transportation on sperm DNA integrity at different time post semen collection, and (ii) evaluate if sperm DNA quality deteriorates rapidly beyond 24 h of cooled storage. After collection, the ejaculates were extended using INRA 96 and semen was prepared for prompt analysis (A0) or 24 h/48 h cooled-shipping (B24 and C48 respectively). Each sample was assessed for sperm DNA fragmentation index (SDFI) at time 0 and after incubation for 2, 6 and 24 h at 37 °C. There was very little difference in SDFI between freshly extended (A0) and 24 h/48 h cooled-transported semen samples (B24/C48) at time 0. After 2 h of incubation at 37 °C, there was an increase in SDFI ranging from 2.7% to 7.5% per hour in freshly extended semen samples (A0: 5.1 ± 1.5), while cooled-transported semen samples had a much greater increase in SDFI, ranging from 5.0% to 20.5% (B24: 14.7 ± 5.6) and from 8.2% to 26.8% (C48: 18.3 ± 7.2) respectively. There were not marked differences in the sperm DNA integrity between 24 and 48 h for transported samples, thus there is the possibility of desirable fertility with use of stallion sperm after 48 h of cooled storage. [ABSTRACT FROM AUTHOR]